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Can prosecutors press a criminal case against a man by suppressing the existence of forensic evidence that shows him to be innocent, without themselves being held accountable? "Yes," says the Supreme Court, by a 5-4 decision (PDF), in a ruling by Justice Clarence Thomas. It's a complicated story, but the core issue is fairly straightforward: prosecutorial accountability for serious misconduct—in this case, misconduct that put an innocent man on death row, and at one point within days of execution.
In 1985, John Thompson was arrested and charged with two separate crimes: a murder and an attempted armed robbery that took place three weeks later in New Orleans. Prosecutors opted for two separate trials, beginning with the robbery. Their thinking was likely tactical. If they got a conviction at the robbery trial, they could use that conviction against him at the murder trial, improving their chances at obtaining a conviction.
Two days before Thompson's robbery trial was slated to begin, however, the prosecutor handling the case got a crime lab report showing that the perpetrator had left blood behind at the scene, and the blood was not Thompson's. The law required the prosecutor to turn this report over to Thompson's attorneys. Instead, he suppressed it and pressed forward with his case. The jury found Thompson guilty of the robbery. That conviction had a decisive impact on the second trial, too. Thompson was warned that if he testified in his own defense, the prosecution would be entitled to inform the jury of the robbery conviction and argue that he was not a truthful or reliable witness. Thompson therefore did not testify. The jury, disbelieving his alibi for the murder, perhaps because he failed to testify in support of it, convicted him and gave him a death sentence.
Just before Thompson was set to be executed in 1999, his attorney learned of the suppressed lab report and the grave misconduct of the prosecutor. Armed with this, Thompson's convictions for both the robbery and the murder were overturned. When he was retried for murder in 2003, a jury, armed with much more testimony and evidence, concluded that he was innocent.
The question is whether Thompson is entitled to be compensated for the imprisonment and anguish to which he was subjected as a result of the gross misconduct of a prosecutor. A Louisiana jury decided that he was, and awarded Thompson $14 million in damages. On appeal, the extremely conservative Fifth Circuit divided evenly, affirming the district court. But Clarence Thomas and four of his colleagues on the bench have overturned that award, leaping to the defense of misbehaving prosecutors and creating a new standard that may render them effectively unaccountable.
The majority wants to make the suppression of the lab report into a momentary failing of a single man. Thomas concludes that the prosecutors are guilty of only a single "Brady violation"—that is, a violation of the duty to turnover exculpatory evidence—and that this is not enough to justify the defendant's case against them. But in fact, the suppression continued over many years and involved faulty judgments by a number of people in the prosecutor's office. Moreover, misconduct of Orleans Parish law enforcement officials has emerged as an embarrassment to the United States on the world stage, figuring even in discussions before monitoring human rights bodies and triggering federal prosecutions. Justice Ginsburg has the better of the argument, pointing to the majority's dodgy presentation of the facts:
What happened here, the Court's opinion obscures, was no momentary oversight, no single incident of a lone officer's misconduct. Instead, the evidence demonstrated that misperception and disregard of Brady's disclosure requirements were pervasive in Orleans Parish.
The majority opinion turns a blind eye to the facts of the case as found by the Louisiana jury. Actually hearing the evidence first hand, they had no difficulty determining that the prosecutor fully intended to do what he did and persisted in doing it.
In America today, violations of Brady are epidemic, and they often produce the conviction of innocent defendants like Mr. Thompson. The court has effectively given a wink and a nod to rogue prosecutors, telling them they can put their thumb on the scale of justice from time to time and get away with it. But bad convictions erode confidence in the justice system, and often as not they let the real culprit run free.
More from Scott Horton:
Conversation — March 30, 2016, 3:44 pm
Joseph Hickman discusses his new book, The Burn Pits, which tells the story of thousands of U.S. soldiers who, after returning from Iraq and Afghanistan, have developed rare cancers and respiratory diseases.
Context, No Comment — August 28, 2015, 12:16 pm
In five easy lessons
From the April 2015 issue
In Havana, the past year has been marked by a parade of bold-faced names from the north — John Kerry reopening the United States Embassy; Andrew Cuomo bringing a delegation of American business leaders; celebrities ranging from Joe Torre, traveling on behalf of Major League Baseball to oversee an exhibition game between the Tampa Bay Rays and the Cuban national team, to Jimmy Buffett, said to be considering opening one of his Margaritaville restaurants there. All this culminated with a three-day trip in March by Barack Obama, the first American president to visit Cuba since Calvin Coolidge in 1928. But to those who know the city well, perhaps nothing said as much about the transformation of political relations between the United States and Cuba that began in December 2014 as a concert in the Tribuna Antiimperialista.
Chances that a Republican man believes that "poor people have hard lives":
Pew Research Center for the People and the Press (Washington)
A school in South Korea was planning to deploy a robot to protect students from unwanted seductions.
Nuremberg's Neues Museum filed a criminal complaint against a 91-year-old woman who completed a crossword puzzle that was in fact a $116,000 piece of avant-garde Danish art.
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"Matt was happy enough to sustain himself on the detritus of a world he saw as careening toward self-destruction, and equally happy to scam a government he despised. 'I'm glad everyone's so wasteful,' he told me. 'It supports my lifestyle.'"


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subset: commoncrawl


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Ratings based on 429 Verified Reviews
They paid and stayed. We double-checked.
4.4 Room cleanliness
4.3 Service & staff
4.2 Room comfort
4.2 Hotel condition
Expedia Verified ReviewsThey paid and stayed. We double-checked.
by A verified traveler from Las Vegas
Not up to par with other MarriotsPosted Jul 23, 2016
Pros: Food was good, and they had starbucks.
Cons: Remodel and refurbish desperately needed. Bigger Gym. Parking was limited.
Location: 17 miles from downtown seattle.
Hotel was run down. Mold in shower between tiles and tub. Staff was fairly nice. The stuck me in a small corner room my first stay there.
Comment from Hotel Management
Jul 25, 2016 by Amanda Guyton, General Manager, Hotel Management
Thank you for feedback. I'm glad you enjoyed our Bistro with Starbucks coffee. I'm disappointed to hear about your room cleanliness. What you experienced is not what we expect and please know that I followed up with our housekeeping manager to ensure it is an isolated issue moving forward. We hope to welcome you back soon.
by A verified traveler from Sacramento
ObservationsPosted Jul 14, 2016
Friendly staff, convenient location, clean facilities
by A verified traveler from Troy, MI
Nice Hotel...the staff goes above and beyond.Posted Jun 13, 2016
Was in town on business. Had a great stay. the staff is very friendly and goes out of their way to make you feel welcome. The Bistro has wonderful assortment of food.
by A verified traveler from Lynnwood WA
Hotel was in a very convenient location.Posted Jun 10, 2016
Pros: Location, cleanliness, the food was great there and they brought it up to our room.
Cons: Size of rooms, ours was a king but not oo my enough.
Location: Location was great. Shopping, eating, Seattle, Silverdale. Easy access to. All the surrounding areas.
This was a great location for shopping, site seeing, eating and all other travel likes. We were able to see Seattle and Tacoma shopping along with surrounding areas such as Port Townsend, Sequim Silverdale, ect. Great Hotel!! Great Time!!
by A verified traveler
Posted May 23, 2016
nettes personal - grosse zimmer - Bistro ist sehr angenehm
by A verified traveler from Spokane
Great hotelPosted May 13, 2016
Close enough to Seattle but far enough to avoid inter-city traffic. Close by to beach access as well as zoos and museums. Near by restaurants as well. The hotel facilities were nice and very clean. Hotel staff was very nice and always smiling.
by A verified traveler
nice comfortablePosted May 4, 2016
Close to shopping and multiple food choices. Away from traffic.
by A verified traveler
Posted Apr 27, 2016
Staff was very nice. The room was nice. A couple of things worth mentioning: There was garbage in the waste bin when we arrived at the room. I have come to find out that "accessible" means a wider door into the bathroom and a grab bar in a bathtub.
Comment from Hotel Management
May 3, 2016 by Amanda Guyton, General Manager, Hotel Management
Thank you for your feedback. I sincerely apologize for the garbage in the waste bin what you arrived. That is not something we expect for our guests; please know that I have your comments with our housekeeping team to ensure it is an isolated issue moving forward.
by Robert
disappointedPosted Apr 26, 2016 on Hotels
Pool was closed. Gave passes to community rec center. Also turned away there. No continental breakfast. Will not choose this hotel again. Not what expected from a Marriot branded location.
by A verified traveler
"Making a reservation doesn't guarantee your room"Posted Apr 26, 2016 on Hotels
I was very disappointed when we walked into our room and there were two beds instead of one King. The receptionist did not tell us this upon check in and when we called to inquire and change rooms she told us they were "all booked and that our reservation only guaranteed us a room but not the type of room." Which is wrong.. I mean that's the whole reason you make a reservation, so you get what size room you need and pay for. She also pushed it aside and told us to come talk to the manager then next morning. The manager however, was very nice and offered to give us a king room and gave us complimentary breakfast the rest of our stay for the inconvenience. He was even surprised that the person from the night before didn't fix the situation.


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Smithfield market will be the museum's new home, but which architectural vision should shape its future: the eye-catching one, the ghostly one, the corporate one … or the one that rings alarm bells?
Little detail has been revealed about the shortlisted schemes, which will go on public exhibition from 10 June to 5 August with a winner chosen by an expert panel later this summer. — theguardian.com
Curb your cultural curiosities with the articles below:Inside Asif Khan's Serpentine Pavilion Summer HouseLondon's Natural History Museum to create outdoor exhibition spacesShortlist for new Museum of London revealed View full entry »
The school collapsed on Tuesday after a heavy rainfall that took over most part of the Lagos including Makoko, a slum and highly populated part of the state [...]
"So as far as that floating school is concerned, it was erected without the permission of the state government.
"The simple answer to the floating school is that it is an illegal structure and it shouldn't be there." — naij.com
Kunlé Adeyemi's floating school was built with the help of the United Nations Development Programme (UNDP) in 2013, to serve 100 elementary school students living in the Makoko slum on Lagos' waterfront. About 300,000 people are estimated to be living in the slum, which before the floating school... View full entry »
Juxtaposed by modern architecture on the western side of the street, the circa 1842 working class terrace facades on the eastern side have been retained and restored in line with strict heritage conditions.
"Kensington Street's integration with the Central Park precinct was of great consideration. We wanted to celebrate its difference in vernacular to the rest of the contemporary precinct but wanted to integrate it with quality landscaping and other infrastructure. — Hospitality Magazine
Until several architectural firms were charged with restoring and revamping it, Kensington Street in Sydney's Chippendale area was a former bustling industrial zone fallen to ruin. Now the street (or at least, the design firms responsible for its transformation, including Turf Design... View full entry »
Last month, the journal Science published a special issue examining the challenges and opportunities of an urbanizing world. Titled "Urban Planet" and featuring an image of clouds wafting across skyscrapers in Dubai, the issue opened with an eye-catching statistic: "More than half of the world's people now live in cities."
Of course, that number would be even more impressive if it were actually true. — UNDark
According to the article, the statistic that half the world's population lives in cities is misleading. Many of these people live in towns and small urban enclaves, not the bustling metropolises conjured by the stat.The author argues that such thinking makes one overlook sprawling fringe... View full entry »
Last year, Greater Manchester's economy outgrew that of inner-city London. Further devolution of powers from Whitehall are about to be realised, and the campaign for the title of first elected mayor of Greater Manchester has picked up pace.
However, Manchester is also about to contend with the capital in other ways. A major housing crisis lurks, and a growing deficit of office space needs to be dealt with. To make amends, Manchester's skyline is heading for dramatic change. — theguardian.com
Read more article concerning the housing crisis spreading across UK cities:To live in London you can't be a LondonerLondon fails to achieve any targets for affordable housingArchitects design 'the house of tomorrow' View full entry »
An approach called Projective Preservation brings speculation about the future into a dialectical relationship with preservation of a city's historic and pre-existing environments. Historic architecture, sites and cities can and should be preserved, but they must also be open to reinterpretation and adaptation to meet the needs of present and future generations. — Strelka Magazine
Ryan Madson (an urban planner and landscape designer who also teaches architecture at SCAD — Savannah College of Art and Design) published an essay digging into authenticity, "memory values" and the "paradox of mainstream preservation ideologies". He also proposes 'Projective Preservation' ... View full entry »
By living above 800 feet, Estis and Enkin are two members of an unexpectedly exclusive group in Manhattan. In my estimation, no more than 40 people currently live above that line, scattered among just three buildings...
As my elevator descended and my ears popped, it occurred to me that I would almost certainly never take in such a view again. And in fact, maybe nobody will, if these apartments wind up becoming empty investments. — The New York Times
In this elegantly observed and exquisitely written piece, Jon Ronson not only takes in the view of Manhattan at 800+ feet with visits to Trump World Tower, One57 and 8 Spruce Street, but looks toward the future of a nation divided by an increasingly intractable wealth gap.Real estate of the... View full entry »
The scheme was designed by EMA Architecture + Design, a local practice specialising in commercial mixed-use masterplans and residential development.
The first phase will be followed by subsequent ones over 20 years, resulting in a £1bn ($1.4bn) "Garden District". As well as the homes, there will be a school, shopping centre, sports facilities and parks.
A quarter of all the homes will be affordable housing, with the remainder split between private homes and apartments. — globalconstructionreview.com
Related on Archinect:Edinburgh's own officials are mucking up the city's historic architecture, says former Daily Mail editorCelebrated Scottish architect Gareth Hoskins dies at 48 from heart attackRed Road towers, built to combat Glasgow's slums in the 1960s, now slated for demolition View full entry »
The Turkish word for Gated Community is site, from the French cité, and they generally resemble the French highrises of the same name, rather than American tract housing. Towers and slabs stand shoulder to shoulder, dancing in a circle around the gardens they surround. — failed architecture
How to Make an Enclosed Paradise:Raze a blighted industrial site or neighborhood close to the city center, preferably along a new highway or metro line.Build an access road around the perimeter. Like a castle moat, this isolates your project from context and gives distance for height... View full entry »
Writer and BLDGBLOG founder Geoff Manaugh's latest book, A Burglar's Guide to the City, isn't just a set of case studies on bank vaults and getaway routes—it's a dialectic for public and private space. It's definitely the first book I've come across classified jointly under... View full entry »
my research shows that longtime residents aren't more likely to move when their neighborhood gentrifies; sometimes they're actually less likely to leave [...]
In a 2009 study, I found that gentrifying neighborhoods are more racially diverse than non-gentrifying ones. [...]
To be sure, market forces help change commerce in gentrifying neighborhoods. But often lurking behind the "invisible hand" are activists and policymakers who wish to nudge the market to produce certain outcomes. — washingtonpost.com
Lance Freeman's research at GSAPP focuses on issues related to gentrification, affordable housing, and race. Watch the Washington Post's video below, summing up the myths:Related on Archinect:A tale of two parks: debate rages over a new plan for a "Maker Park" in BrooklynA telltale sign of... View full entry »
With the new mayor focusing our attention on smart development and social equality, 2016 will be a banner year for the London Festival of Architecture. Election watchers will be familiar with many of this year's hot topics: community spaces, social housing, docklands renewal. But considering the theme this year is 'community', there will be something for every tribe of Londoner. Out of 300 events, we've picked the 10 must-sees. — thespaces.com
See related news here:This week's picks for London architecture and design eventsLondon's Natural History Museum to create outdoor exhibition spacesZaha Hadid's repertoire is a stunning display in Venice's Palazzo Franchetti View full entry »
In any city, space is a commodity. In South African cities space is historical and emotional. A new photo series by an American living in Cape Town captures the dramatic inequality of South Africa's most beloved city. From an aerial view, Cape Town's scenic beauty gives way to a stark reminder of the country's past and the continued racial segregation. [...]
"Looking straight down from a height of several hundred meters, incredible scenes of inequality emerge," he writes on his website. — qz.com
On his website, Unequal Scenes, the creator of the aerial imagery, Johnny Miller, writes:"Discrepancies in how people live are sometimes hard to see from the ground. The beauty of being able to fly is to see things from a new perspective - to see things as they really are. Looking straight down... View full entry »
"If you design for everyone to drive, then what will you get? Congestion." [...]
"We really need to shift now, from a situation like this, where you have a heavy parking load associated with an apartment building in a very urban setting, to way less parking," [...]
"You really have to start with the density and less parking. If you don't, then you've lost your opportunity, because once you've built that infrastructure, it's so difficult to undo that." — news.wabe.org
More on the parking problem and pedestrian infrastructure:Los Angeles County has 3.3 parking spots for every car, taking up 14 percent of its landTrading Parking Lots for Affordable HousingDanish parking garage invites to stay and playOne Woman's Quest to Design Parking Lots People Don't... View full entry »
In a region at once feared and exoticized, we have been witnessing for more than a generation the devastation of old centers and the rise of new ones. Today there is no better context in which to investigate the complexities of global practice in architecture than that of the rapidly changing Arab city. — Places Journal
How does the deeply traditional meet the hypermodern in the older centers of Beirut, Damascus, and Cairo, and in the emerging new cities of Dubai, Doha, and Abu Dhabi? In Amale Andraos' new article on Places, and in the new book, The Arab City: Architecture and Representation, she explores the... View full entry »
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Media Talk; MOMA Shuts Its Doors On 'Sunday Morning'
By KENNETH N. GILPIN
Published: November 23, 1998
This is not an abstract dispute. But it is one that might well bestir the spirit of William S. Paley.
The late Mr. Paley, the founder of CBS and a one-time chairman of the Museum of Modern Art, might be upset to learn that when Morley Safer, co-editor of ''60 Minutes,'' approached officials at MOMA earlier this month about doing a segment for the network's ''Sunday Morning'' show on the widely acclaimed Jackson Pollack retrospective exhibit, he was turned down flat.
MOMA's director, Glenn D. Lowry, explained in an interview why he said no to Mr. Safer, one of the network's most celebrated journalists.
''We get hundreds of requests from serious journalists to do pieces on MOMA and its exhibits,'' Mr. Lowry said. ''We went with them rather than someone who does drive-by shootings.''
He did not elaborate.
Of course, Mr. Safer had once opined on ''60 Minutes'' that most contemporary art was ''worthless junk.''
Still, he characterized his conversation with Mr. Lowry as ''strong but civil.''
But Mr. Safer added: ''I've never been brutalized by a museum director like this. I think it's revenge for my earlier pieces, none of which involved the Museum of Modern Art.
''I explained fairly carefully what I wanted to do with the broadcast,'' he continued. ''Given the fact that Pollack is a difficult artist to understand, I said it would be useful for Kurt Varnedoe, the show's director, to explain the artist's work. He said that's fine, but not with you.''
Mr. Safer, who described MOMA as a ''humorless Comintern of contemporary art,'' was nevertheless amused at Mr. Lowry's characterization of his take on the subject. ''Actually, the first contemporary art piece I did, which aired on '60 Minutes' in 1993, was more like a sniping than a drive-by shooting,'' he said. ''Still, that's a pretty good one for a museum director.''
The ''Sunday Morning'' audience won't be there, but the Pollack show, which closes Feb. 2, won't lack television coverage.
At least 10 shows have already been shown or been shot on the exhibit, including segments on CNN, ''The News Hour With Jim Lehrer,'' ''The Charlie Rose Show'' and the BBC, said a MOMA spokesman, who insisted on anonymity. And discussions with two other networks are in the detail stage, the spokesman added. KENNETH N. GILPIN
Photo: Morley Safer


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Listen to a New Nas Song, 'Accident Murderers,' Featuring Rick Ross
"Have fun, not guns!" — Nas, probably.By Eliot Glazer
"Have fun, not guns!" — Nas, probably.By Eliot Glazer
It belonged to a certain ex-wife.By Amanda Dobbins
Meet Nas's child, Destiny.By Eliot Glazer
Team Nas till the end!By Eliot Glazer
Another Roman Reloaded track.By Amanda Dobbins
It's a different "Don."By Amanda Dobbins
Hear "Like Smoke."By Amanda Dobbins
"Swiss or Cheddar?"By Amanda Dobbins
From the movie soundtrack.By Amanda Dobbins
It's a nice coat.By Amanda Dobbins
Well, there you go.By Willa Paskin
"You're Mouth Got You in It" isn't quite 'Illmatic,' but it's still pretty awesome.
It's scheduled to come out in 2012.By Margaret Lyons
Plus: Donald Glover as Childish Gambino, the Rapture, Nas, and more death!By Ray Rahman
Plus: other bands!By Amos Barshad
Hear the new tracks.By Amos Barshad
"We do not like to go into speculation and rumors, but it seems as though you do not want to release Lost Tapes 2 because you don't want it to count as an official album ... "By Amos Barshad
"With all do [sic] respect to you all, Nas is NOBODY's slave. This is not the 1800's, respect me and I will respect you."By Amos Barshad
"My rap palate has greatly improved."By Amos Barshad
The tennis star and her boyfriend Common have split.


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Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.
Author Summary
Pathogenic Escherichia coli strains and other enterobacteria carry genes acquired from other bacteria by a process known as horizontal gene transfer. Proper regulation of the genes that are expressed in a given moment is crucial for the success of the bacteria. The protein H-NS is a global regulator that binds DNA and maintains a large number of genes silent until they are required, for example, to sustain the bacteria's colonization of a new host. Ler is a member of the H-NS family that competes with H-NS to activate the expression of a group of horizontally acquired genes that encode for a molecular machine used by E. coli to infect human cells. Ler and H-NS share a similar DNA-binding domain and can bind to different DNA sequences. Here, we present the structure of a complex between the DNA-binding domain of Ler and a natural DNA fragment. This structure reveals that Ler recognizes specific DNA shapes, explaining its capacity to regulate genes with different sequences. A single arginine residue is key for the recognition of a DNA narrow minor groove, which is one of, though not the only, hallmarks of the DNA shapes that are recognized by H-NS and Ler.
Citation: Cordeiro TN, Schmidt H, Madrid C, Juárez A, Bernadó P, Griesinger C, et al. (2011) Indirect DNA Readout by an H-NS Related Protein: Structure of the DNA Complex of the C-Terminal Domain of Ler. PLoS Pathog 7(11): e1002380. doi:10.1371/journal.ppat.1002380
Editor: Ralph R. Isberg, Tufts University School of Medicine, United States of America
Received: June 29, 2011; Accepted: September 30, 2011; Published: November 17, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by funds from the Spanish Ministries of Science and Innovation (BIO2010–15683 and CSD2008–00013), EC FP7 BIO-NMR (contract 261863), the Generalitat de Catalunya (SGR2009–1352) and the Max Planck Society (to C.G.). T.N.C. gratefully acknowledges a doctoral fellowship by the Fundação para a Ciêancia e a Tecnologia (FCT). P.B was supported Ramón y Cajal contract that was partially financed by the Spanish Ministry of Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are causal agents of infectious diarrhea. While the former is responsible mainly for infantile diarrhea, EHEC infections are associated with hemorrhagic colitis and may produce a life-threatening complication known as hemolytic uremic syndrome. EPEC and EHEC are non-invasive pathogens that produce characteristic attaching and effacing (A/E) intestinal lesions [1]. The genes required for the formation of A/E lesions are clustered on a pathogenicity island known as the locus of enterocyte effacement (LEE). LEE genes are organized in five major operons (LEE1 to LEE5) and several smaller transcriptional units and they encode the components of a type III secretion system (TTSS), an adhesin (intimin) and its receptor (Tir), effector proteins secreted by the TTSS, chaperones, and several transcription regulators [2]. The first gene of the LEE1 operon encodes the LEE-encoded regulator Ler, which is essential for the formation of A/E lesions in infected cells [3], [4] and for the in vivo virulence of A/E pathogenic E. coli strains [5].
Ler (123 amino acids, 14.3 kDa) is the master regulator of LEE expression and is required to activate LEE genes that are otherwise repressed by the histone-like nucleoid structuring protein H-NS [2].
The H-NS protein, best characterized in E. coli and Salmonella, is a member of a family of transcriptional regulators with affinity for AT-rich DNA sequences that mediate the adaptive response of bacterial cells to changes in multiple environmental factors associated with colonization of different ecological niches, including human hosts. H-NS is usually an environmentally-dependent transcriptional repressor. H-NS-mediated repression (usually termed silencing) is alleviated either by alterations in physicochemical parameters (i.e., a transition from low (25°C) to high (37°C) temperature), by the activity of proteins that displace H-NS from its target DNA sequences, such as Ler, or by a combination of both. H-NS regulation is strongly associated with pathogenicity, thus understanding the basis of the selective regulation of virulence genes could lead to sustainable antimicrobial strategies that are less susceptible to acquiring resistance.
In addition to the LEE genes, Ler is also involved in the regulation of other horizontally acquired virulence genes located outside the LEE loci and scattered throughout the chromosome of A/E pathogenic strains [3], [6], [7]. However, Ler does not regulate other H-NS-silenced operons such as bgl [8] and proU [3]. This observation shows that Ler is not a general antagonist of H-NS, but a specific activator of virulence operons acquired by horizontal transfer (HT). Selective regulation of HT genes has been demonstrated in the plasmid R27 encoded H-NS paralogue (H-NSR27) and in chromosomal H-NS in the presence of a co-regulator of the Hha/YmoA family [9].
The mechanism of Ler-mediated activation has been extensively studied in operons located both within the LEE loci, such as LEE2/LEE3 [10], grlRA [11], [12] and LEE5 [8], and outside, including nleA (for non-LEE-encoded effector A) [13] and the lpf1 fimbrial operon [6], [14]. These studies suggest that Ler counteracts the silencing activity of H-NS by directly binding to DNA and displacing H-NS from specific promoter regions. Ler does not exert dominant negative effects on H-NS function and there is no evidence of a direct interaction between Ler and H-NS [8]. Despite the wealth of biochemical/biophysical data, including the proposal of a DNA sequence consensus motif for H-NS [15], the lack of structural data on the complexes formed between H-NS or H-NS family members and DNA has until now prevented a detailed understanding of the mechanism of DNA recognition and the basis of the selectivity within H-NS family proteins.
All H-NS-related proteins identified to date are predicted to be organized in two structurally different domains. While the oligomerization domains of Ler and H-NS differ greatly, their DNA binding domains are very similar, thereby suggesting that they account for the similar recognition properties of both proteins, and possibly also for their distinct selectivity. While a possible interplay between protein oligomerization and DNA binding cannot be ruled out, a detailed understanding of the recognition mechanism by individual DNA-binding domains is a prerequisite for further studies.
The C-terminal domain of Ler (CT-Ler), exhibits significant amino acid homology with the C-terminal H-NS DNA-binding domain (CT-H-NS; 36.0% identity, 63.8% similarity) and its deletion abolishes DNA binding [16]. CT-Ler contains a sequence (TWSGVGRQP) similar to the consensus core DNA-binding motif found in H-NS-like proteins (TWTGXGRXP) [17]. Here we present the solution structure of a complex formed by CT-Ler bound to a natural occurring DNA sequence of the LEE2/LEE3 regulatory region. This is the first report of a DNA complex that includes a member of the H-NS family characterized at atomic detail. Our results reveal that CT-Ler does not participate in base-specific contacts but recognizes specific structural features in the DNA minor groove. The indirect readout mechanism can be extended to H-NS and other H-NS family members and explains their capacity to modulate the expression of a large number of genes. The CT-Ler/DNA structure provides clues for the mechanism by which HT genes may be specifically recognized by members of the H-NS family and illustrates the general features of DNA minor groove readout.
CT-Ler/DNA complex formation
We used a CT-Ler construct encompassing residues 70–116 (Figure 1A). This construct gave rise to a folded and functional domain (Figure S1) with excellent solubility and long-term stability. Residues 117–123 are part of an extension that is dispensable to counteract H-NS repression [18]. NMR spectra of a construct including these residues showed that they are disordered and have no effect on the structure of the folded domain, as seen by the exact coincidence of the cross-peak position of most residues in HSQC NMR spectra of different constructs (Figure S2).
(A) Sequence alignment of the C-terminal domain of the following proteins: Ler; chromosomal H-NS of E. coli (ecHNS); Shigella flexneri (sfHNS); Salmonella enterica serovar Typhimurium (seHNS); Yersinia enterocolitica (yeHNS); the plasmid R27-encoded H-NS protein (pR27); and E. coli StpA. The secondary structure elements of DNA-bound CT-Ler and free H-NS are shown. Highly conserved residues within the consensus DNA-binding motif are highlighted in red. (B) Analysis of the interaction of CT-Ler with 30 bp DNA fragments (LeeA-G, sequences are listed in Table S1) derived from the DNAse I footprint of Ler in the LEE2/LEE3 regulatory region [10]. Complex formation was followed by the increase of CT-Ler fluorescence anisotropy. (C) Fluorescence anisotropy titrations of CT-Ler with LeeH (black circle) and LeeFG (gray circle). Solid curves are the best fit to a model assuming a 1∶1 complex. The point by point deviations between fitting and experimental points are shown in the top panel.
The sequence of the short DNA fragment used to form the complex was based on the regulatory region of the LEE2/LEE3 operons spanning positions -221 to -101. This region was protected by Ler in footprinting experiments [10]. Seven 30 bp long dsDNA, LeeA-LeeG, with a 15 bp overlap between consecutive fragments (Figure 1B, Table S1) were tested for binding to CT-Ler using fluorescence anisotropy. As positive and negative controls, we used two 30-mer duplexes: an adenine tract that was previously employed to study the DNA-binding properties of CT-H-NS, (GGCAAAAAAC)3 [19] and (GTG)10 (Figure S3). CT-Ler showed the highest affinities for LeeF and LeeG (Figure 1B) and we further analyzed its binding to the 15 bp overlapping region of theses two fragments, namely LeeFG (AAATAATTGATAATA). Fluorescence anisotropy titrations showed small but systematic deviations from the 1∶1 model, suggesting simultaneous multiple binding to this DNA sequence (Figure 1C). Since the consensus binding motif proposed for H-NS is only 10 bp long [15] we designed a new 15 bp DNA, LeeH (GCGATAATTGATAGG), containing the central 10 bp of LeeFG flanked by GC base pairs for thermal stability. LeeH partially matches the proposed H-NS consensus sequence (tCG(t/a)T(a/t)AATT) [15]. A good fit to a 1∶1 model with apparent Kd 1.10±0.05 µM was observed for this duplex (Figure 1C).
Structure of the CT-Ler/DNA complex
The complex of CT-Ler with LeeH was solved by a combination of NMR and small-angle X-ray scattering (SAXS). The structure determination protocol consisted of the independent calculation of the structure of bound CT-Ler and DNA, followed by intermolecular NOE (iNOEs) driven docking and a final scoring including SAXS data. CT-Ler structures were calculated based on 1302 NOE distance restraints, together with torsion angle and experimentally determined hydrogen bonds. The restraint and structural statistics of the 20 lowest energy structures are shown in Table S2. None of the structures contained distance or dihedral angle violations >0.5 Å or 5°, respectively.
The pattern and intensities of bound DNA NOEs were typical of a B-form. The DNA structure was optimized in explicit solvent using experimental restrains determined in the bound form, starting from canonical B-DNA as described in the Materials and Methods section. The absence of major distortions in the DNA structure caused by CT-Ler binding was confirmed by the good agreement between the experimental SAXS curve of free LeeH and the prediction based on the DNA model extracted from the final complex (Figure S4).
The DNA region most affected by CT-Ler binding, identified by the combined chemical shift perturbations of nucleotide protons, is centered in the symmetrical 4 bp AT-tract, AATT (Figure 2A). The largest chemical shift perturbations of CT-Ler (Figure 2B) were observed for residues Val88 to Arg93. The 30 assigned iNOEs involve protein residues located in the region where the chemical shift perturbations were observed. On the basis of these iNOE restraints and the mapped interfaces, 400 CT-Ler/LeeH complex structures were generated as described in Materials and Methods and ranked by energy and NMR intermolecular restraint (irestraint) violations. The quality of the structures was confirmed by comparing the predicted and experimentally determined SAXS curves of the complex. The SAXS profile predicted for the best NMR-derived complex structure is in good agreement with the experimental curve (Figure 3A). The scatter plot in Figure 3B shows that, in general, the best NMR structures also fit SAXS data well. The final ensemble of 20 structures was selected using a scoring function that combined docking energy and measures of the agreement with experimental NMR and SAXS data (red circles). The ensemble is well defined (Figure 3C), with a pairwise RMSD (heavy atoms) of 1.30±0.38 Å and all conformers exhibited good geometry, no violations of iNOE distance restraints >0.5 Å and correctly explained the SAXS data. Most of the protein residues are in the core region of the Ramachandran plot. The small irestraint deviations illustrate that the protein-DNA interface is well defined, allowing us to elucidate a molecular basis for CT-Ler/LeeH recognition.
(A) Mean absolute changes in 1H-NMR chemical shifts caused by the addition of 0.5 equivalents of CT-Ler. The average is over all resolved resonances per nucleotide. The upper and lower LeeH strands are identified by black and gray bars, respectively. (B) Backbone amide chemical shift changes in CT-Ler () upon complex formation with LeeH. The scaling factor corresponds to the ratio of 15N and 1H magnetogyric constants. Resonances that were not observed are denoted by # (Gly87) or * (Pro92).
(A) SAXS intensity in logarithmic scale measured for a CT-Ler/LeeH equimolar sample (open circles) as a function of the momentum transfer , where Å is the X-ray wavelength and is the scattering angle. CRYSOL fit of the SAXS curve using a representative NMR structure (red); the average deviation is 1.16. Only the range 0.018< s <0.4 Å−1 is displayed. The point by point deviations [(I(s)exp−I(s)fit)/], where is the experimental error are shown in the bottom panel. (B) Scatter plot of NMR intermolecular restraint violations versus values for the initial set of 400 complex structures and the final ensemble of 20 low energy structures highlighted in red (inset). The main panel shows a zoom of the best structures. (C) Backbone overlap of the 20 lowest energy complex structures. Protein backbone is coloured in rainbow gradation.
The structure of DNA-bound CT-Ler contains a central helix (residues 93–101) and a triple-stranded antiparallel β-sheet (β1:76–78, β2:84–85, β3:109–110). The β1-β2-hairpin is connected to the α-helix by a loop (Loop2:86–92). A turn and a short 310-helix (105–108) link the helix to the β3 strand. The similarity between the Cα and Cβ secondary chemical shifts of the free and bound forms indicate that the secondary structure is retained upon binding (Figure S5). The overall protein fold is analogous to that previously described for CT-H-NS in the absence of DNA [19].
CT-Ler binds as a monomer inserting Loop2 and the N-terminal end of the α-helix into the DNA minor groove and contacting the central 6 bp region (A6A7T8T9G10A11) (Figure 4). The complex buries 953±55.64 Å2 of surface area and is stabilized by non-specific hydrophobic and polar contacts, involving mainly the sugar-phosphates backbone and residues of the consensus DNA-binding motif found in H-NS-like proteins. Residues Trp85, Gly89, Arg90 and Pro92 (Figure 1A), highly conserved among H-NS-like proteins, are located in the complex interface (Figure 4B), and all gave rise to iNOE restraints with DNA. A summary of the observed intermolecular contacts is shown in Figure 4D.
(A) Structure of CT-Ler/LeeH complex. CT-Ler is shown as a ribbon diagram and transparent surface representation. Interactions involve the DNA minor groove and Loop2 and the α–helix of CT-Ler. (B) Close-up view of the binding interface. CT-Ler residues involved in DNA recognition are shown as stick models. The electrostatic potential of LeeH, calculated with DelPhi in the absence of CT-Ler, is shown. (C) Electrostatic potential of CT-Ler. The orientation of the complex is the same as in A. (D) Schematic representation of the hydrophobic (dashed lines) and polar (solid lines) intermolecular contacts.
The interaction surface of CT-Ler is positively charged and the Arg90 side chain is deeply inserted inside a narrow minor groove (Figure 4B and C). In addition, Arg93 at the N-terminus of the α-helix and the helix-dipole moment itself create a positively charged region that points into the negatively charged minor groove.
The width of the LeeH minor groove varies along the sequence and deviates significantly from the average value of canonical B-DNA (Figure 5). The groove progressively narrows towards the A7pT8 base step, and widens at the T9pG10 base step. The DNA electrostatic potential is modulated by the width of the minor groove. The guanidinium group of Arg90 interacts with the narrowest region of the groove where the electrostatic potential is most negative (Figure 5A and B). The approach of Loop2, where Arg90 is located, is enabled by the adjacent widening of the minor groove.
(A) Stick representation of Arg90 side chain inserted at the floor of the negatively charged LeeH minor groove. The electrostatic potential of LeeH, calculated in the absence of CT-Ler, is plotted on the LeeH surface. (B) Average minor-groove width (blue) and electrostatic potential in the centre of LeeH minor groove (red). The position of the guanidium group of Arg90 is indicated. (C-D) Helical parameters of LeeH in complex with CT-Ler. Roll and helix twist angles are shown. Dashed lines correspond to values typical of canonical B-DNA [56].
Sequence-dependent variations of DNA structure can be described in terms of helical parameters, such as roll and helix twist (Figure 5C and D). The roll angle is most negative (−4.64°±1.38) at the A7pT8 base step and is small or negative for most of the steps in LeeH except for the pyrimidine-purine base steps, which show large positive values. A series of consecutive small/negative roll angles leads to the narrowing of the minor groove [20]. The groove widening at T9pG10 can be traced to a combination of positive roll and a small helix twist of 33.8°±0.8, indicating that the segment is slightly unwound with respect to the standard B-form. The region including the A6A7T8T9 stretch is slightly overwound, with an average helix twist of 37.4°±1.6.
Arg90 is essential for Ler binding
To verify the relevance of Arg90 in the interaction, we replaced this residue by glycine (R90G), glutamine (R90Q) or lysine (R90K) and tested their effects on the affinity of CT-Ler to LeeH. All CT-Ler variants were properly folded, as determined from NMR, and their interaction with LeeH was measured by fluorescence anisotropy (Figure 6A). The mutated domains showed no affinity to LeeH or highly reduced affinity (R90K), thereby confirming that Arg90 is an essential residue.
(A) Fluorescence anisotropy titrations of wild type, R90K, R90Q and R90G CT-Ler with LeeH. (B) EMSA of wild type and mutant Ler proteins. 80 ng of DNA (LEE2 positions −225 to +121) were incubated with the indicated Ler concentrations and analyzed on a 1.5% agarose gel. 1 Kb DNA ladder was included as a reference (lane M).
The effect of these mutations on the binding of Ler(3–116), including the oligomerization domain, to the LEE2 regulatory region (positions −225 to +121) was determined using electrophoretic mobility shift assays (EMSA) (Figure 6B). In agreement with the results obtained with the isolated CT-domain, DNA binding by Ler is abolished by R90Q and R90G mutations and strongly reduced in the case of the R90K variant. These experiments confirm the essential role of Arg90 in the context of the oligomeric Ler protein and for the range of binding sequences present in one of its natural targets.
DNA sequence specificity of Ler binding
The structure of the CT-Ler/LeeH complex does not show base specific contacts. On the contrary, the structure of the complex suggests that CT-Ler recognizes local structural features of the minor groove that may be associated with distinct DNA sequences. In order to gain some insight into the range of DNA sequences that can be recognized by CT-Ler, we measured the dissociation constants of complexes formed by two series of short DNA duplexes related to the LeeH sequence. In the first series we introduced a single base pair replacement in each of the ten central positions of LeeH. Adenines and thymines were replaced by guanines and cytosines, respectively, and guanine in position 10 was mutated to adenine, to preserve the purine-pyrimidine sequence. In the second series, we compared the binding of CT-Ler to several 10-mer duplexes. One of these contained the AT-tract (AATT) that interacts with CT-Ler in the LeeH complex flanked by GC base pairs to ensure thermal stability. Variants were designed to test the effect of interrupting the AT-tract by TpA steps at a number of positions.
Affinity to CT-Ler was measured by fluorescence anisotropy. The results are shown in Figure 7 and the DNA sequences and dissociation constants are listed in Table S3.
(A) CT-Ler binding to DNA variants containing single base-pair substitutions with respect to LeeH (wt). The LeeH minor groove width is also shown to highlight the fact that mutations in the compressed and expanded regions of the minor groove caused the largest effects. (B) Relative Kd values of the complexes formed between CT-Ler and 10-mer duplexes with different AT-rich sequences. The most stable complex, used as reference, has the AATT sequence present in LeeH. Relative Kd values are Kd(mutant)/Kd(reference) determined by fluorescence anisotropy.
Figure 7A shows the relative Kd values of the single base-pair replacements of LeeH. The largest effects were observed when the base pairs of A6 or A7 were replaced. The base pair of G10 resulted to be similarly relevant. A smaller effect was observed at the position of T8. Small non-specific effects were observed in all the remaining sites except that of A4. The most affected base pairs were at the sites where the minor groove width in LeeH is more different from the standard B-DNA and define the features that we hypothesize to be recognized by CT-Ler: the narrow groove where the Arg90 side chain is inserted and the wide adjacent region that enables the approach of Loop2.
Figure 7B show the relative dissociation constants of the complexes formed by the 10-mer duplexes. The presence of TpA steps in CGCAATAGCG, CGCTATAGCG and CGCTTAAGCG results in a decrease in the stability of the complexes. The remaining three sequences (CGCAATTGCG, CGCAAATGCG, and CGCAAAAGCG) show AT-tracts of the same length but their affinity for CT-Ler differs. The complex with the A4 stretch is 2-fold less stable than that containing the AATT motif.
The AT-tract in LeeH is terminated by a TpG pyrimidine-purine step. Replacing it by a TpC pyrimidine-pyrimidine step in a 10 bp duplex had only a minor effect on the affinity for CT-Ler (cf. AATT and AATTC in Table S3). Interestingly, replacement of the T9pG10 step in LeeH by the alternative pyrimidine-purine step, TpA, resulted in a major loss of stability of the complex.
CT-Ler provides insight into DNA binding by H-NS
The DNA binding domains of Ler and H-NS share a high degree of similarity both in sequence and in structure. We carried out experiments to specifically test two key points that are apparent from the analysis of the Ler/LeeH complex, namely the role of the conserved arginine residue (Arg90 in Ler, Arg114 in H-NS) in Loop2 and the requirement for an AT-tract and the effect of interrupting TpA steps.
H-NS Arg114, corresponding to Arg90 in Ler, was mutated to glycine and the affinity towards the −225 to +121 LEE2 region was compared with that of the wild type form by EMSA. As in the case of Ler, replacing the arginine residue in Loop2 results in a substantial loss of affinity (Figure 8A). However, H-NS retains some residual activity even when arginine was replaced by glycine while this drastic mutation caused a complete loss of activity in the case of Ler.
(A) EMSA (1.5% agarose) of the −225 to +121 LEE2 fragment (80 ng) with increasing concentrations of wild type and R114G H-NS proteins. (B) DNA titrations of CT-H-NS followed by NMR. Expansions of 1H-15N HSQC spectra of CT-H-NS in the presence of the 10 bp duplexes AATT (top left, 0, 0.5, 1, 2, 3 and 4.5 equivalents) or TATA (top right, 0, 1, 2, 3, 4.5 and 6 equivalents). The DNA-dependent shifts of selected cross-peaks were fitted to a 1∶1 model (bottom), supported by the strict linear displacement of the cross-peaks during the titration. (C) CT-Ler and CT-H-NS binding to the −225 to +121 LEE2 fragment (20 ng) followed by EMSA on a 7% polyacrylamide gel.
The requirement for a narrow minor groove in the case of Ler can be assessed by the relative affinities towards the AATT and TATA 10-mer duplexes. Titrations of CT-H-NS with both oligonucleotides (Figure 8) provided dissociation constants of circa 41 μM for the AATT complex and 102 μM, 2–3-fold larger, for the TATA complex. CT-Ler showed similar relative affinities for the same oligonucleotides (Table S3), thereby suggesting that these two domains have similar requirements for a narrow minor groove.
As many H-NS and Ler target sequences may overlap, the relative affinity of the DNA-binding domains of these two proteins is relevant. As the CT-Ler complex studied included only the structured domain, we compared CT-Ler with the CT-domain of H-NS including only residues 95 to 137, excluding linker residues. This H-NS construct is properly folded as shown by the observation of well resolved NMR spectra (Figure 8). The same natural DNA fragment (LEE2 positions −225 to +121) used in EMSA assays with Ler (Figure 6B) and H-NS (Figure 8A) was selected to compare the affinities of the CT-domains of these two proteins. The large number of binding sites for Ler and H-NS in this extended DNA fragment, as shown by footprinting experiments, allows the assessment of the relative overall affinities of the two domains for the whole range of sequences present in one of their common natural targets. The affinity of CT-Ler is larger than that of CT-H-NS, which under the conditions of the experiment hardly caused any retardation (Figure 8C). This observation contrasts with the similar affinity towards the same DNA fragment shown by longer constructs of Ler and H-NS that include the oligomerization and linker domains (cf. Figure 6B and 8C) and highlights varying relevance of interactions outside the folded CT-domains of these two proteins. The contribution of residues outside of the structured H-NS DNA-binding domain has been previously described [21], [22].
The structure of the complex between CT-Ler and LeeH shows that DNA shape and electrostatics, rather than base specific contacts, form the basis for the recognition of the CT-Ler binding site. This mechanism is referred to as indirect readout. Arg90 is a key residue for the CT-Ler interaction with DNA. Its side chain is inserted deep into a narrow minor groove. The requirement for Arg90 is strict in the case of CT-Ler and the R90G and R90Q mutants of Ler are totally inactive. The R90K mutant shows some residual binding suggesting that a positive charge is required. Arginine interactions with the DNA minor groove have been described in eukaryote nucleosomes [23], [24] and in DNA interactions by a nucleoid-associated protein of Mycobacterium tuberculosis [25]. These observations suggest that this mechanism may be universal for indirect DNA recognition of AT-rich sequences. A correlation between minor groove width and the electrostatic potential has been demonstrated as well as the preference for arginine binding to the narrowest regions where the electrostatic potential is more negative [23].
For CT-Ler, the narrow minor groove may be provided by a relatively short AT-tract as only the Arg90 side chain has to be inserted. The minimum width in the AATT motif is observed at the ApT step, matching the site where the guanidinium group is inserted. Continuous polyA tracts of 4 (Figure 7) and 6 nucleotides (Figure S3) of length give less stable complexes than sequences combining A and T. However, the presence of highly dynamic TpA steps [26] interrupting the AT-tracts decreases the affinity for CT-Ler. The presence of guanine, with its 2-amino group extending into the minor groove and increasing its width is also predicted to destabilize the insertion of the arginine side chain. We explored the effect of introducing TpG or TpA steps in the sequence recognized by CT-Ler. Figure 7 clearly shows that an uninterrupted AT-tract is needed for an efficient interaction with CT-Ler. However, a narrow AT-tract is not the only requirement for CT-Ler interaction. The lower affinity of the G10A variant of LeeH shows that, next to the narrow region, a rigid wide minor groove is also required to enable the access of Loop2 delivering the side chain of Arg90 into the narrowest region of the minor groove. Both sequences, T9pG10 in LeeH and T9pA10 in the mutated duplex, could adopt wide minor grooves. However, while the former is expected to provide a permanently wide groove, the flexible TpA step may switch between expanded and compressed forms, interfering with the approach of Loop2 directly or indirectly through the entropic penalty associated to stiffening of the DNA in the complex.
The structure of the complex as well as the affinity data with DNA sequence variants show that CT-Ler recognizes a pattern in the minor groove of DNA formed by two consecutive regions that are narrower and wider, respectively, with respect to standard B-DNA and show the optimal shape and electrostatic potential distribution for binding.
This structural pattern is present in the free LeeH DNA fragment as shown by the observation of diagnostic inter-strand NOES between AdeH2 and ThyH1' protons of A7/A23 and T25/T9, respectively supporting minor groove narrowing both in the free and bound forms of LeeH. Moreover, the SAXS data of free LeeH is better explained by the structure of LeeH in the complex than the structure of a canonical B-DNA LeeH (Figure S4). Therefore, at least in the case of LeeH, CT-Ler recognizes pre-existing DNA structural features following an indirect readout mechanism.
The molecular basis of the preference that H-NS displays for some promoter regions has been extensively studied. AT-tracts were initially postulated to be high affinity sites for H-NS and related to the presence of a narrow minor groove [27]. More recently, two short high affinity H-NS sites with an identical sequence, 5'-TCGATATATT-3' were identified in the E. coli proU promoter [28]. Lang et al. proposed that a 10 bp long consensus sequence (tCG(t/a)T(a/t)AATT) [15] acts as a nucleation site for cooperative binding to more extensive regions. In a recent study, a shorter segment of 5–6 nucleotides comprising only A/T nucleotides was found to be over-represented in genomic loci bound by H-NS in E. coli [29]. The interaction of the H-NS CT-domain, including a few residues from the linker region, with a short oligonucleotide was studied by NMR [22]. The authors concluded that a structural anomaly in the DNA associated with a TpA step was crucial for H-NS recognition.
Our results suggest that AT-tracts and wide TpA steps may be simultaneously required by H-NS family proteins. The correct positioning of a compressed and widened minor groove is the specific recognition signal for CT-Ler. Pyrimidine-purine steps tend to widen the minor groove and TpA steps may contribute to its widening, which is required after the AT-tract. However, in the case of Ler, a TpG step was preferred to the TpA step, suggesting that a wide narrow groove after the AT-tract is the true structural requirement.
CT-Ler and CT-H-NS showed similar structural requirements: mutation of Arg114 reduced the affinity of the complex, and introduction of TpA steps in the AT-tract caused a similar decrease in stability. This result is consistent with the fact that Ler targets can also be occupied by H-NS. Ler and H-NS bind to multiple sites. An indirect readout mechanism allows recognition of multiple sequences, if they adopt similar minor groove patterns.
The absence of structural changes between the free and bound forms of CT-Ler (Figure S5) supports a lock and key model for interactions involving the structured CT-domain and may account for the relatively high specificity of Ler, as compared with H-NS where additional interactions outside the CT-domain are comparatively more important. Comparison of constructs containing exclusively the structured region of the CT-domains of Ler and H-NS show that the former has higher affinity for the range of sequences present in a natural segment where both proteins bind. Several features, not present in CT-H-NS, may contribute to the higher stability of the CT-Ler complex. An additional arginine residue (Arg93) combined with the helix dipole provides additional electrostatic interactions, thus stabilizing the CT-Ler complex. While both Ler and H-NS have a conserved tryptophan residue that, in the case of Ler, forms hydrophobic interactions with DNA, CT-Ler presents an additional tryptophan residue in close contact with DNA. The dipoles of both indole rings are oriented with their positive end towards the negatively charged DNA backbone and the side chain NH of Trp94 forms a hydrogen bond with the DNA backbone.
We have determined for the first time the structure of a complex formed by the DNA-binding domain of a member of the H-NS family. Our results highlight the similarities in the DNA recognition mechanisms used by CT-Ler and CT-H-NS but also evidence some differences that may contribute to the differential recognition of some genes by Ler and H-NS.
Materials and Methods
Samples preparation
DNA fragments containing the coding sequence of Ler residues 65–123, 70–116 (CT-Ler) and 3–116 fused to an N-terminal His6-tag were amplified by PCR from EHEC strain 0157:H7 and subcloned into the pHAT2 vector. To overexpress CT-H-NS, DNA encoding this fragment (amino acids 95–137) with six histidine residues tagged at its N terminus was amplified by PCR using the full length H-NS construction [30] as template and then subcloned into the pHAT2 vector. Point mutations were generated using the QuikChange site-directed mutagenesis kit (Stratagene).
Ler fragments 65–123, 70–116 and 3–116 and CT-H-NS were overexpressed in BL21(DE3) cells with overnight incubation at 15°C by induction with 0.5 mM IPTG when an O.D.600 of 0.7 was reached. For 15N and/or 13C isotopic labeling, cells were grown in M9 minimal media containing 15NH4Cl and/or 13C-glucose. For 10% 13C enrichment we used a carbon source consisting of a 1∶10 mixture of 12C-glucose/13C-glucose [31], [32]. Cells were harvested by centrifugation, frozen and resuspended in 20 mM HEPES (pH 8.0), 1 M NaCl, 5 mM imidazol, 5% (v/v) glycerol, treated for 30 min with lysozyme and DNAse and sonicated (6×10 s on ice). After centrifugation, the His-tagged fusion proteins were isolated with Ni-NTA beads (Qiagen) and further purified by size exclusion chromatography on a Superdex 75 column in 20 mM sodium phosphate, 150 mM NaCl, 0.2 mM EDTA, 0.01% (w/v) NaN3 pH 5.7 or 20 mM sodium phosphate, 300 mM NaCl, 0.01% (w/v) NaN3 pH 7.5. The expression and purification procedure for full length H-NS has been previously described [30].
DNA samples were prepared by hybridization of complementary oligonucleotides purchased from Sigma-Aldrich. Quality control was assessed by MALDI-TOF mass spectrometry. Oligonucleotides were mixed in equimolar amounts and annealed by heating to 92°C for 4 min and slowly cooled to room temperature.
Fluorescence anisotropy measurements
Changes in CT-Ler intrinsic fluorescence anisotropy were monitored upon DNA addition. All measurements were recorded on a PTI QuantaMaster spectrophotometer equipped with a peltier cell, using an excitation wavelength of 295 nm to selectively excite CT-Ler tryptophans and emission detection at 344 nm. Fluorescence measurements were performed in 40 mM HEPES (pH 7.5), 60 mM potassium glutamate, 0.01% (w/v) NaN3 at 20°C. More details on data acquisition and equipment settings were previously described [33]. For the initial screening of the -221 to -101 regulatory region of LEE2, the apparent fraction saturation of CT-Ler was used to infer about DNA binding preferences. To measure the affinity of CT-Ler for 15 bp and 10 bp DNA fragments, titrations were performed at least in duplicate. The fitting was performed assuming a 1∶1 binding using the following equations [34]:(1)(2)where A is the observed anisotropy, Af and Ab are the anisotropies of free CT-Ler and the complex respectively, fb is the fraction of bound CT-Ler and Q is the ratio of quantum yields of bound and free forms. Equations 1 and 2 were solved iteratively until the theoretical binding isotherm matched the experimental data. Kd and Ab were considered to be adjustable parameters.
NMR spectroscopy
All spectra were acquired at 25°C on 600, 700, 800 or 900 MHz Bruker spectrometers. Data processing and analysis were carried out with NMRPipe [35], NMRViewJ [36], and CARA [37].
NMR spectra for structure determination were recorded on a ∼1 mM sample containing a 1∶1 complex of uniformly 13C- and 15N-labeled CT-Ler and unlabeled DNA in 20 mM sodium phosphate (pH 5.7), 150 mM NaCl, 0.2 mM EDTA and 0.01% (w/v) NaN3. Backbone and aliphatic assignments of free and DNA-bound CT-Ler were obtained by standard methods. Aromatic resonances were assigned using 2D 1H-13C-edited-NOESY optimized for aromatic resonances. Stereospecific assignments of Val and Leu methyl groups were obtained from a constant time 1H-13C-HSQC on a 10% 13C-labeled protein sample [31]. Non-exchangeable protons of the LeeH duplex bound to CT-Ler were assigned using 2D F1,F2-13C-filtered TOCSY and NOESY spectra in D2O [38]. Exchangeable protons and H2 protons were assigned from 2D F1,F2-15N/13C-filtered NOESY spectrum in H2O [39]. Free DNA resonances were assigned using 2D DQF-COSY, TOCSY and 2D NOESY spectra. Proton chemical shifts were referenced using 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as an internal standard, whereas 15N and 13C chemical shifts were indirectly referenced. Chemical shift assignments have been deposited in the BioMagResBank database under BMRB accession number 17729.
Protein distance restraints were obtained from 2D 1H-13C-edited NOESY (aromatic optimized in D2O), 3D 1H-15N-edited NOESY-HSQC and two 3D 1H-13C-edited NOESY-HSQC (in H2O and in D2O) experiments with a mixing time of 120 ms. Data were automatically assigned and the NOE distance restraints were obtained iteratively using the Unio'08/CYANA 2.1 suite program [40], [41] and manually inspected. The distance restraints for the DNA in complex with CT-Ler were obtained measuring initial NOE build-up rates from 2D F1,F2-15N/13C-filtered NOESY spectra recorded with mixing time of 50, 75, 100 and 150 ms. Intermolecular NOEs were detected using a combination of 2D NOESY, 2D F1,F2-13C-filtered NOESY and 2D F2-13C-filtered NOESY experiments, together with 3D F1-13C,15N-filtered, [F2] 13C-edited 3D NOESY spectrum [42]. Additional intermolecular NOEs were obtained by analyzing the 3D 15N-edited and 13C-edited NOESY spectra.
Protein backbone dihedral angle restraints were derived using a combination of TALOS [43] and quantitative analysis of 3JHNHα obtained from a 3D HNHA spectrum [44]. Restraints on side chain angle and stereospecific assignments of Hβ proton resonances were based on 3JNHβ couplings, obtained from a 3D HNHB spectrum, in combination with observed intraresidual NOEs using the HABAS routine of the CYANA 2.1 program [45].
1H-15N HSQC spectra for analysis of the interaction of 15N-labeled CT-H-NS (100 µM) with dsDNA were obtained at 25°C in 20 mM sodium phosphate (pH 5.7), 150 mM NaCl, 0.2 mM EDTA and 0.01% (w/v) NaN3.
Structure calculation and refinement
The structure of CT-Ler was determined by simulated annealing using the torsion angle dynamic simulation program CYANA 2.1 [45] and further water refinement with CNS 1.2.1 [46], [47]. Protein structure calculation was based on Unio'08/CYANA-generated upper distances, 3JHNHα/3JNHHβ couplings, and TALOS-driven dihedral angle restraints. Based on H/D exchange experiments, backbone NOE pattern and 13Cα/13Cβ chemical shifts, hydrogen bond restraints were also used in the structure calculation. An ensemble of 100 protein structures was generated and the 20 lowest energy conformers were docked onto a B-DNA.
The observed overlap and broadening of DNA resonances hampered the complete quantitative analysis of NOESY spectra for bound DNA. Only a set of 282 well resolved cross-peaks were converted into distances using initial build-up rates and reference to the cytosine H5-H6 cross-peaks. Upper and lower limits were defined as ± 20% of the calculated distances. The structure of LeeH was fixed as B-DNA and further energy-refined using miniCarlo [48] followed by a 20 ps molecular dynamics refinement in explicit solvent using the Amber force field [49] and including NOE-derived distance restraints. To preserve the helical conformation of DNA, weak planarity restraints were also introduced. The DNA backbone was constrained to a range typical of B-form and all glycosidic angles were restrained as anti. Hydrogen bond restraints were used for all base pairs in which the imino proton was observed. The complex structure was generated employing 30 iNOEs, supplemented with highly ambiguous intermolecular restraints (AIRs) that were driven from the mapped binding interfaces. A total of 22 intermolecular NOE restraints were simultaneously assigned to the two symmetry-related protons in the AATT central region of the DNA and used as ambiguous restraints. HADDOCK 2.0 [50] was used to generated 2000 structures by rigid docking energy minimization, and 400 structures with the lowest energy were selected for semi-flexible refinement process. These 400 structures were finally refined in explicit water including all experimental restraints. Structures were then ranked using the energy-based HADDOCK scoring function (sum of intermolecular electrostatic, van der Waals, desolvation and AIR energies) and NOE energy term. The quality of these structures was evaluated in terms of the violations to the NOE data and the value defining the agreement to SAXS curve. A final ensemble of 20 structures was obtained by re-scoring the pool of 400 structures using the following scoring function.(3)(4)where and correspond to the root mean squared deviations with respect to the best possible value in and Ei respectively. Coordinates of the final ensemble were deposited in the Brookhaven Protein Data Bank under the accession number 2lev.
Minor groove geometry and helical parameters were analyzed using w3DNA [51]. Electrostatic potentials were obtained at physiological ionic strength using DelPhi [52].
SAXS data collection and analysis
SAXS data for LeeH and the CT-Ler/LeeH complex were collected on a MAR345 image plate detector at the X33 European Molecular Biology Laboratory (DESY, Hamburg, Germany) [53]. The scattering patterns were measured at 25°C for 2 min at sample concentrations of 4.6 and 2.7 mg/ml and 6.6 and 3.3 mg/ml for LeeH and CT-Ler/LeeH, respectively. A momentum transfer range of 0.018< s <0.62 Å−1 was measured. Repetitive measurements indicated that samples did not present radiation damage. Buffer subtraction and the estimation of the radius of gyration, Rg, and the forward scattering, I(0), through Guinier's approach were performed with PRIMUS [54]. The scattering profile of LeeH was obtained from merging curves at both concentrations. For CT-Ler/LeeH, SAXS profiles at both concentrations were virtually equivalent and only data from the highest concentrated sample were used for further analysis. Using Guinier's approach, the radii of gyration of LeeH and CT-Ler/LeeH were estimated to be 15.6±0.1 and 18.2±0.1 Å, respectively. All data manipulations were performed with the program PRIMUS. Using a bovine serum albumin sample (3.3 mg/ml), an estimated molecular weight of 18 kDa was obtained for CT-Ler/LeeH (theoretical MW of 16.3 kDa), thereby indicating the presence of a monomeric particle in solution. The agreement of the SAXS curve to various three-dimensional models was quantified with the program CRYSOL [55] using a momentum transfer range of 0.018< s <0.40 Å−1.
Electrophoretic mobility shift assays
The DNA fragment used in this assay (LEE2 positions −225 to +121) was obtained by PCR amplification from EHEC strain 0157:H7. The indicated concentrations of PCR-generated DNA and H-NS or Ler proteins were mixed in a total volume of 20 μl of 15 mM sodium phosphate, 100 mM NaCl, 0.01% (w/v) NaN3 pH 7.5. 1 mM tris(2-carboxyethyl)-phosphine (TCEP) was included for samples containing full length H-NS. After 20 min of incubation at room temperature, glycerol was added to 10% (w/v) final concentration and the reaction mixtures were electrophoresed on either 1.5% agarose or 7% polyacrylamide gels in 0.5x Tris-borate-EDTA buffer. The DNA bands were stained with ethidium bromide.
Supporting Information
Figure S1.
Interaction between CT-Ler and the LeeH dsDNA fragment. 1H-15N-HSQC NMR spectra of Ler70–116 (CT-Ler) recorded in the absence (black contours) and upon equimolar addition of LeeH (red contours). Side-chain NH groups are indicated by 'sc' after the residue number. The excellent chemical shift dispersion observed in the 1H-15N-HSQC NMR spectra indicates that the domain is properly folded.
Figure S2.
1H-15N-HSQC spectra of Ler fragments. Overlap of 1H-15N-HSQC NMR spectra of Ler65–123 (green contours) and Ler70–116 (CT-Ler) (black contours) at pH 7.0 and 25°C. Most of the cross-peaks from CT-Ler coincide exactly with a cross-peak from Ler65–123. Additional residues from Ler65–123 show chemical shifts typical of unstructured residues.
Figure S3.
CT-Ler binds preferentially to AT-rich DNA sequences. Fluorescence anisotropy titrations of CT-Ler with the following 30-mer duplexes: LeeG, (GGCAAAAAAC)3 and (GTG)10.
Figure S4.
Small-angle X-ray Scattering analysis of the 15 bp LeeH duplex. SAXS intensity in logarithmic scale measured for LeeH sample (open circles) as a function of the momentum transfer s = 4πsin(θ)/λ, where λ = 1.5 Å is the X-ray wavelength and 2θ is the scattering angle. Best CRYSOL fits to the curve using the structure of LeeH in the complex with CT-Ler (red) or the structure of a canonical B-DNA LeeH generated with w3dna (cyan); and the average deviations, χ, are 0.85, 1.27 respectively. Only the range 0.016< s <0.5 Å−1 is displayed. The point by point deviations for each fitting [(I(s)exp−I(s)fit)/σ(s)], where σ(s) is the experimental error are shown in the bottom panel with the same colour code.
Figure S5.
The CT-Ler secondary structure is not affected by DNA binding. (A) Ribbon structure of a representative conformer of LeeH-bound CT-Ler. Elements of secondary structure are labeled on the structure. (B) Differences between the 13Cα (top panel) and 13Cβ (bottom panel) chemical shifts observed for residues 70–116 of Ler and those expected for a random coil are plotted against the residue number. White and black bars correspond to the free and LeeH-bound forms, respectively.
Table S1.
DNA fragments used in the initial optimization of the CT-Ler/DNA complex. DNA fragments span the Ler-footprint within the LEE2/LEE3 regulatory region. Only the sequence of one of the complementary strands is shown.
Table S2.
NMR and refinement statistics. Refinement statistics including the number and type of experimental restraints and the results of quality controls performed using PROCHECK [57] and CRYSOL [55].
Table S3.
DNA sequence effect on CT-Ler complex stability. Sequences of the LeeH variants designed to test CT-Ler binding specificity and the corresponding dissociation constants. Only the sequence of one of the complementary strands is shown.
We thank D. Lee for useful comments on NMR data acquisition, members of Griesinger group for stimulating discussion, H. Memni (U. Tunis El Manar) for help in measuring some fluorescence titrations, and N. Ulyanov and T. James (UCSF) for miniCarlo-minimized DNA structure. We acknowledge the support of the European Community – Research Infrastructure Action to the EMBL Hamburg Outstation for SAXS measurements, the NMR Instalación Científico Tecnológica Singular of the Universitat de Barcelona, supported by MICINN and the EMBL Protein Expression and Purification Core Facility for providing the pHAT2 expression vector.
Author Contributions
Conceived and designed the experiments: TNC JG MP. Performed the experiments: TNC HS CM PB JG. Analyzed the data: TNC HS PB JG. Contributed reagents/materials/analysis tools: AJ CG MP. Wrote the paper: TNC JG MP.
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Tuesday, July 12th, 2011 AT 12:35 AM


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iHerb 顾客评论
节省: ¥16.24 (12 %折扣)
unbelievably EFFECTIVE for my dog's warts
Reviewer2256362发表于 Feb 04, 2011
my dog has had warts growing all over her back for the last 6 years. desperate to figure out how to help her from what is clearly a viral problem I recently found that camu camu is the MOST ABSORBABLE form of Vitamin C and potent for herpes so I gave her (she weighs 50 pounds) 1/2 teaspoon in the AM and then at night...and THEY HAVE DISAPPEARED. in 3 weeks many of them were gone. NO JOKE. after years of them growing they are NOW disappearing. absolutely amazing. I now tell everyone about it. Remember these are warts that are disappearing and NOT skin tags.
Tangy sour tart ... but I like that!
Reviewer1399339发表于 Nov 03, 2011
Taste great with stevia and mixed with Emergen-C too.
The taste is not very good
Reviewer1817474发表于 Mar 23, 2011
Not the best tasting vit c I have tried. I prefer Madre-C from Madre Labs, which is a blend of different natural sources of vit c, also with camu-camu in it.
Ghastly bitter taste
4839964790182216380发表于 Sep 05, 2014
Love the fact that this has 1100 times the daily dose of vitamin C and use it sparingly. However, this is one of THE worst tasting powders I've ever had. Half a teaspoon in 20mls of fizzy mineral water is just tolerable if you drink it faster than the speed of light.
Dissolves easily, raw, organic and affordable
Reviewer2647272发表于 Nov 08, 2011
I had purchased a very small jar of Camu Camu at a health food store and paid more for it than I did this bag. This dissolves more easily too. Another great product by Navitas!
Raw camu camu
holahola发表于 Dec 23, 2010
Super! A healthbomb against illness! Taste VERY bitter, so I use to fill it in veggie capsules.
5154170390377322146发表于 Sep 13, 2014
Well,I was told to use a naturally derived vitamin C to help lower my high Copper levels..had also ordered Rosehip powder BUT unfortunately its a histamine Liberator So reacted badly to it ,so switched to this Instead.. Very happy I did! Feel like it is helping to lower my histamine levels So I can recover from my histamine probs and lower my copper at the same time! Its a win win!
like the taste
4655756283823407200发表于 Jun 02, 2014
I put 4 stars as still use it and get deffently said the result for health. Camu powder is an excellent food-based source of Vitamin C. I liked it in a fruit and vegetable smoothies, salads, and put it on the top of cereals. The taste is not very sour for me very pleasant. The package is quite big and I think it will be enough for 3-4 months.
pas d'amélioration
5711994911375105219发表于 Oct 16, 2014
Après utilisation de cette poudre je n'ai constaté aucun changement ni effet bénéfique sur mes pathologies inflammatoires.
bought this because of the extremely high content of vit C
Reviewer2778088发表于 Sep 24, 2010
- more than 5 times the amount than the other organic Camu form the same brand - it´s the only camu that advertises to be raw so guess it is because of that? anyway - it seems like the best value C-vit wise, it doesn´t actually taste good but have no experience with other camu powders to compare and since you need so little it´s of less importance. I mix it with other powders for an all natural vitamin cocktail that doesn´t taste good anyway (brewers yeast, spirulina, amla) but is concentrated, in this mixture it dissolves easily, alone not.


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Gooey Jack Burger
Total Time
Prep 30 mins
Cook 0 mins
Looking for an out of this world burger? Maybe something different than the usual burger? Try this.........Cook time - would be to your desired taste and however you like your burger!
Ingredients Nutrition
- 226.79 g package monterey jack cheese
- 907.18 g ground beef
- 118.29 ml barbecue sauce, divided
- red onion, sliced (optional)
- tomatoes, sliced (optional)
- 1 leaf lettuce (optional)
- Cut cheese into 16- 18 thin slices.
- In a bowl, combine ground beef and 1/4 C of the barbecue sauce.
- Mix until well combined.
- Divide the mixture into 8 or 10 equal amounts.
- Shape into patties.
- Place slices of cheese on the patties then place patties together so the cheese is in the center and pinch the edges shut. You should have 4 – 5 patties.
- These burgers can be cooked on the grill, under the broiler, baked or pan-fried.
- Once your burgers are done top with cheese.
- Serve on toasted hamburger buns with your choice of condiments and the remaining barbecue sauce.
Most Helpful
I love barbecue sauce on burgers sometimes and this was great with the mj cheese. Thanks KimmieOH for a nice meal. Made for All You Can Cook Buffet special.
Kimmie, I have made these several times now, and my family really loves them. Thank you for a keeper.