Chlamydia trachomatis CT771 (nudH)\nis an asymmetric Ap4A hydrolase
Asymmetric diadenosine 5\xe2\x80\xb2,5\xe2\x80\xb2\xe2\x80\xb3-P1,P4-tetraphosphate (Ap4A) hydrolases are members of the Nudix superfamily that asymmetrically cleave the metabolite Ap4A into ATP and AMP while facilitating homeostasis. The obligate intracellular mammalian pathogen Chlamydia trachomatis possesses a single Nudix family protein, CT771. As pathogens that rely on a host for replication and dissemination typically have one or zero Nudix family proteins, this suggests that CT771 could be critical for chlamydial biology and pathogenesis. We identified orthologs to CT771 within environmental Chlamydiales that share active site residues suggesting a common function. Crystal structures of both apo- and ligand-bound CT771 were determined to 2.6 \xc3\x85 and 1.9 \xc3\x85 resolution, respectively. The structure of CT771 shows a \xce\xb1\xce\xb2\xce\xb1-sandwich motif with many conserved elements lining the putative Nudix active site. Numerous aspects of the ligand-bound CT771 structure mirror those observed in the ligand-bound structure of the Ap4A hydrolase from Caenorhabditis elegans. These structures represent only the second Ap4A hydrolase enzyme member determined from eubacteria and suggest that mammalian and bacterial Ap4A hydrolases might be more similar than previously thought. The aforementioned structural similarities, in tandem with molecular docking, guided the enzymatic characterization of CT771. Together, these studies provide the molecular details for substrate binding and specificity, supporting the analysis that CT771 is an Ap4A hydrolase (nudH).
<!>Cloning, Overexpression, and Purification of Recombinant CT771<!>Crystallization<!>Diffraction Data Collection, Structure Determination, Refinement and\nAnalysis<!>Kinetic Characterization of CT771<!>Ligand Docking<!>Multiple Sequence Alignments and Figure Modeling<!>CT771 Crystal Structure<!>Structural Comparison with Ap4A Hydrolases<!>Kinetic Characterization of Ap4A Hydrolase Activity of\nCT771<!>Structural Basis for Substrate Recognition of CT771<!>Substrate Induced Structural Changes within CT771<!>Bioinformatic Analysis of CT771 and Orthologs within Chlamydia<!>DISCUSSION
<p>Found throughout all kingdoms of life, Nudix hydrolases are a superfamily of
metal-dependent (typically Mg2+) enzymes that catalyze the cleavage of
nucleoside diphosphates linked to any
other moiety X (Nudix) 1. These proteins serve to maintain
physiological homeostasis by modulating levels of signaling molecules and potentially
toxic metabolic intermediates 2.
Enzymes of this family are characterized by a structurally conserved metal-and-substrate
binding catalytic loop-helix-loop 'Nudix' motif (comprising 23 amino
acids,
GX5EX7REUXEEXGU
where U is preferably a hydrophobic residue), which forms one edge of the putative
active site 1. Despite a diverse range
of substrates, a conserved αβα-sandwich scaffold is present within
all structurally studied Nudix hydrolases. Substrate specificity is thus determined by
side chains outside the Nudix motif, which further subdivides this superfamily, and
includes dinucleotide polyphosphatases (e.g. diadenosine tetraphosphate
(Ap4A) hydrolases), nucleotide sugar pyrophosphatases, ribo- and
deoxyribonucleotide triphosphatases, among others 2.</p><p>Ap4A hydrolase subfamily members are found throughout all kingdoms of
life with previous phylogenetic analyses suggesting the subdivision of plant/bacterial
and mammalian/archeal enzymes into two distinct classes 2, 3. Studies
detailing structural insights of substrate binding and enzyme mechanics have been
revealed within plant 4, 5, bacterial 6, 7 and mammalian
8 enzymes. Asymmetric cleavage
of the substrate, Ap4A, results in the generation of ATP and AMP (EC
3.6.1.17). While the biological role of Ap4A is not completely understood, it
is found at micromolar intracellular concentrations in both eukaryotes and eubacteria
9 and typically accumulates as
a result of protein synthesis when aminoacyl-tRNA synthetases are charged with their
cognate amino acid 10. Ap4A
levels have also been implicated in a variety of signaling pathways including apoptosis
11, DNA repair 12, pathogenesis 13 and as a link between transcription
and protein synthesis 10, among
others.</p><p>Chlamydia trachomatis is an obligate intracellular bacterium
that replicates within a parasitophorus vacuole, termed the inclusion. A distinguishing
feature of Chlamydia is its virulence-defining developmental cycle that
involves an infectious, metabolically inactive form (elementary body or EB) and
noninfectious, replicative form (reticulate body or RB) 14. After host cell entry and formation of the inclusion
by an EB, a burst of nascent bacterial metabolism is initiated that coincides with the
EB-to-RB morphological transition. As Chlamydia have coevolved with
their human host, reductive evolution has resulted in the loss of numerous genes
involved in substrate and oxidative phosphorylation, resulting in a limited ability to
manufacture ATP and an energetically parasite-like existence 15, 16. Thus, the
ability to salvage and utilize nucleotide intermediates (such as Ap4A) within
the inclusion, whether chlamydial-generated or imported from the host cell, is likely
crucial to basic bacterial biology as well as pathogenesis. BLAST analysis of the
Chlamydia trachomatis genome reveals the presence of a single ORF
containing a Nudix motif, CT771 (Chlamydia
trachomatis ORF 771), which is annotated as a
17.4 kDa MutT/Nudix family protein and is conserved across all sequenced
Chlamydiaceae members. Typically, pathogens that rely on a host for
replication and dissemination (e.g. Rickettsia,
Borrelia, mycoplasmas, etc.) lack or have a single Nudix family
enzyme 1, suggesting the singular
presence of a Nudix protein could be of critical importance to
Chlamydia. In order to further characterize CT771 as a Nudix
subfamily member, a crystal structure of CT771 was determined to 2.60 Å.
Structural homology with Ap4A hydrolase members as well as examination of the
putative active site through molecular docking guided the subsequent enzymatic
characterization of CT771. Finally, a co-crystal structure of CT771 bound to products of
Ap4A hydrolysis was determined to 1.9 Å, which ultimately provided
the mechanistic details of substrate binding and specificity for CT771, as well as
confirmed its functional role as an Ap4A hydrolase
(nudH).</p><!><p>A gene fragment encoding the entire open reading frame (residues
1–150) of CT771 was amplified from C. trachomatis
(serovar 434/Bu) genomic DNA via PCR and subcloned into
BamHI-digested expression plasmid pTBSG through ligation
independent cloning 17. Upon
DNA sequence confirmation, the vector was transformed into BL21 (DE3) E.
coli cells. This strain was grown to an OD600 of 0.8 at
37°C in Terrific Broth supplemented with Ampicillin (100 μg/ml),
and protein expression was induced overnight at 17 °C by the addition of
isopropyl 1-thio-β-D-galactopyranoside (IPTG) to a 1 mM final
concentration. Bacterial cells were harvested by centrifugation, resuspended in
lysis buffer (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and 10 mM imidazole), and
then lysed by microfluidization. The soluble tagged protein was collected in the
supernatant following centrifugation of the cell homogenate and purified on a
Ni2+-NTA-Sepharose column according to published protocols
18. Recombinant
tobacco etch virus (TEV) protease was used to digest the fusion affinity tag
from the target protein. After desalting into 20 mM Tris-HCl (pH 8.0), final
purification was achieved by ResourceQ anion-exchange and gel filtration
chromatography (GE Healthcare). The purified protein was concentrated to 10
mg/ml and buffer exchanged by ultrafiltration into 10 mM Tris-HCl (pH 7.5), 50
mM NaCl, and stored at 4 °C for further use.</p><!><p>Recombinant C. trachomatis CT771 was crystallized by
vapor diffusion in Compact Jr. (Emerald Biosystems) sitting drop plates at 20
°C. Specifically, 0.5 μl of protein solution (10 mg/ml in 10 mM
Tris-HCl (pH 7.5), 50 mM NaCl) was mixed with 0.5 Î¼l of reservoir
solution containing 100 mM Tris-HCl (pH 8.5) and 1.5 M ammonium sulfate, from
the Salt Rx HT screen condition F3 (Hampton Research), and equilibrated against
75 Î¼l of the reservoir solution. Single bipyramidal-shaped crystals
appeared after 1 day and continued to grow for ~3 days. Crystals were
flash-cooled through serial dilution in a cryoprotectant solution consisting of
2.0 M ammonium sulfate with an increasing concentration of glycerol (5%, 10%,
20% (v/v) final).</p><p>Prior to cocrystallization, CT771 (10 mg/ml in 10 mM Tris-HCl (pH 7.5),
50 mM NaCl) was incubated on ice with 10 mM Ap4A (Sigma,
reconstituted in 10 mM MgCl2) for 15 minutes. Upon mixing 0.5
Î¼L of the protein/Ap4A solution with 0.5 uL reservoir solution
containing 200 mM MgCl2, 100 mM Tris-HCl (pH 8.5) and 25% (w/v) PEG
3350, from the Index HT screen condition H1 (Hampton Research), single block
shaped crystals appeared after 1 day and continued to grow in size for up to 5
days. Crystals were harvested by flash-cooling in a cryoprotectant solution
consisting of 80% mother liquor and 20% (v/v) glycerol and prepared for X-ray
diffraction.</p><!><p>Monochromatic X-ray diffraction data were collected at 100K using a
Dectris Pilatus 6M pixel array detector at beamline 17ID at the APS IMCA-CAT
(Table I). Individual reflections
were integrated with XDS 19
and scaled with Aimless 20,
which suggested the Laue class was 6/mmm with a likely space
group of P6122 or P6522
for the apo-CT771 crystals and C2 for the
CT771/AMP-PO4 cocrystals.</p><p>Initial phase information was obtained for the apo-CT771 structure by
maximum-likelihood molecular replacement using BALBES 21. Both of the likely space groups were
analyzed, with only P6522 producing a potential
solution. Specifically, 134 residues of chain B from PDB entry 3I7V
(Aquifex aeolicus Ap4A hydrolase) were altered
to reflect the sequence of CT771 (residues 13 – 148), and the resulting
hypothetical structure was used as a search model. The top solution contained a
single copy of CT771 in the asymmetric unit, which corresponded to a
Matthew's coefficient 22 of 4.66 Å3/Da and a solvent content of
73.6%. The final refined CT771 structure was used as a search model for the
CT771/AMP-PO4 dataset, and the top solution contained eight
copies of CT771 in the asymmetric unit with a Matthew's coefficient of
2.35 Å3/Da and a solvent content of 47.7%.</p><p>Structure refinement for the apo-CT771 structure was carried out using
Phenix 23, 24. One round of individual coordinates and
isotropic atomic displacement factor refinement was conducted, and the refined
model was used to calculate both
2Fo-Fc and
Fo-Fc difference
maps. These maps were used to iteratively improve the model by manual building
with Coot 25, 26 followed by subsequent refinement cycles. TLS
refinement 27 was incorporated
in the final stages to model anisotropic atomic displacement parameters.
Hydrogen atoms were included during all rounds of refinement. Ordered solvent
molecules were added according to the default criteria of Phenix and inspected
manually using 2Fo-Fc
weighted feature enhanced (FEM) electron density maps (contoured at 1.0
σ) within Coot prior to model completion. Residues 1-3 were not modeled
as a result of poor map quality.</p><p>The CT771/AMP-PO4 cocrystal structure was refined in a
similar manner as described above. Regions of poor map quality with the
CT771/AMP-PO4 cocrystal structure prevented modeling of residues
1-4 and 149-150 in all chains (with the exception of chain B, where residues 1-4
were modeled and chain C, where residue 4 was modeled). The following residues
were not modeled: chain F, 147-148; chain G, 18-21, 84-88. Disordered side
chains were truncated to the point where electron density could be observed.
Additional information and refinement statistics are presented in Table 1.</p><!><p>A luciferase-based bioluminescence assay was used to characterize the
kinetic constants of wild-type CT771 enzyme via the detection of liberated ATP.
Each 100 Î¼L assay mixture was comprised of 100 mM Tris-HCl (pH 7.5), 1.5
– 5.0 mM MgCl2 or MnCl2, 10 μL of
reconstituted rL/L Reagent (Promega), 10 uL of Ap4A at varying
concentrations (Sigma) and 10 ng of purified CT771, and was incubated at room
temperature for 5 minutes before measurements were taken. Light output was
measured on a Biotek Synergy 2 luminometer and converted into units of enzyme
activity (Î¼M/min) using an ATP standard calibration curve. All reactions
were measured in triplicate and data interpretation was carried out using
GraphPad (GraphPad Software). Additional information is presented in Table 4.</p><!><p>A model for diadenosine tetraphosphate (Ap4A), 8-deoxy-dGTP,
adenosine diphosphate ribose and GDP-mannose were generated using the PRODRG
server 28. Polar hydrogens
were added to ligands prior to docking. Docking calculations were carried out
with the refined crystal structure of CT771 using AutoDock Vina, a comprehensive
program for molecular docking and virtual screening 29. As grid maps are calculated automatically in
AutoDock Vina, only the size and location of search space is required. The grid
box was set to 60 × 60 × 60 Å with grid points spaced every
0.375 Å and centered within the putative Nudix family active site cleft.
Docking simulations were performed at physiological conditions (37° C).
Theoretical dissociation constants were calculated for the lowest energy site
using the following equation: ΔG = RT*ln(Kd). Only ligands
docked within the putative active site cleft were analyzed. Additional
information is presented in Table 3.</p><!><p>Multiple sequence alignments were carried out using ClustalW 30 and aligned with secondary
structure elements using ESPRIPT 31. The Ap4A hydrolase sequences used in
alignments, along with their respective GenBank™ accession numbers, are
as follows: C trachomatis serovar 434/Bu (166154113);
Parachlamydia acanthamoebae (282890520); Candidatus
Protochlamydia (46446871); Simkania negevensis
(338733601); Waddlia chondrophila (297621593); Homo
sapiens (4502125); Caenorhabditis elegans
(17509897); Aquifex aeolicus (15605731). Three-dimensional
structures were superimposed using the Local-Global Alignment method (LGA)
32. The
Ap4A hydrolase structures obtained from the PDB 33 are as follows: Homo
sapiens (3U53); Caenorhabditis elegans (1KTG);
Aquifex aeolicus (3I7V). Representations of all structures
were generated using PyMol 34.
Calculations of electrostatic potentials at the molecular surface were carried
out using DELPHI 35.</p><!><p>The structure of CT771 was solved by molecular replacement and refined
to 2.60 Å (Table 1). CT771 is
characterized by an αβα-sandwich fold (Fig. 1) with dimensions approximately 50 × 25
× 30 Å, typical of Nudix family proteins, with 3 α-helices
(residues Pro52-Thr64, Phe120-Arg125 and Arg133-Tyr146, respectively)
surrounding 7 β-sheets (residues Lys5-Phe18, Ser24-His33, Lys37-Gly40,
Gly67-Phe71, Phe76-Asn83, Phe89-Lys102 and Cys113-Leu118, respectively)*. The overall topology of CT771 is
β1-β2-β3-β4-α1-β5-β6-β7-α2-α3
(Fig. 1). The conserved Nudix motif
(GX5EX7RELXEEXGL,
residues 44-66) is found within α1 and the loop regions surrounding this
helix (represented as ball-and-sticks and colored magenta in Fig. 1, right panel). The
protein interfaces and assemblies (PISA) 35 server was used to assess potential modes of
oligomerization. This analysis failed to identify any interfaces that were
judged to be thermodynamically favorable, consistent with the monomeric elution
profile of CT771 in analytical gel filtration experiments (data not shown).</p><!><p>In order to better understand the function of CT771, the refined crystal
structure was used to search for structurally-related enzymes within the PDB via
the DALI server 36, with the
top 5 hits listed in Table 2. Not
surprisingly, over 100 of the highest scoring matches are enzymes from the Nudix
family. Structures displaying the highest homology to CT771 are diadenosine
tetraphosphate
(adenosine-P1-P2-P3-P4-adenosine
or Ap4A) hydrolases. Furthermore, both mammalian and eubacterial
enzymes are found within the top 5 hits, suggesting that they are structurally
more alike than previously thought2,
3. Indeed, structural
alignment of CT771 and the top 3 DALI hits, Ap4A hydrolases from
H. sapiens
8, C. elegans
37 and A.
aeolicus
7, (Fig. 2A) further underscores the conserved nature of the
αβα-sandwich fold found within this class of enzymes. All 4
structures align with RMSD values better than 2.2 Å, with large-scale
variations limited to the loops connecting β1-β2 and
β5-β6. Neither of these regions are expected to contribute to
substrate binding or catalysis and likely reflect crystallographic differences
stabilized by packing.</p><!><p>In order to better understand the function of CT771 and provide support
for the structural similarities to Ap4A hydrolases revealed by both
the DALI search 36 and
molecular docking (described in Supporting Information) 29, purified protein was enzymatically
characterized using a Luciferase assay as described in the Materials and
Methods section. This assay allows the continuous monitoring of
cleaved Ap4A via released ATP upon substrate hydrolysis, and has been
extensively used to characterize this specific Nudix enzyme class 3, 38. The results of this analysis support the conclusion
that CT771 is a bona fide Ap4A hydrolase (Table 4). Intriguingly, the largest
observed kinetic activity was in the presence of 5 mM Mg2+ with
kcat = 3.2 ± 0.1 (s−1),
while the lowest observed binding affinity was in the presence of 5 mM
Mn2+ with a Km = 5.0 ± 0.5
Î¼M (Fig. 3).
Km measurements assumed that the rate of product
formation was slower than enzyme-substrate dissociation. Despite its ability to
increase substrate affinity and enzyme efficiency
(kcat/Km),
increasing concentrations of Mn2+ had an adverse affect on the
catalytic rate of CT771 in the presence of Ap4A. These values are in
agreement with previously reported values, using a bioluminescent assay, for
H. sapiens Ap4A, among others 38.</p><!><p>Crystals of CT771 grown in the presence of Ap4A diffracted
X-rays to 1.90 Å resolution in the space group C2 (Table 1). Upon modeling and refinement of
eight polypeptides in the asymmetric unit, two regions of strong contiguous
electron density (visible when contoured up to 5.0 σ) were apparent
within each putative active site (Fig. 4A).
While the electron density was clearly not large enough to accommodate an entire
Ap4A molecule (Fig. 4B), an
AMP moiety was modeled near the loop region connecting α2 and α3
(Fig. 4C). This phosphate will be
designated the P1-phosphate in further discussions, in accordance
with Guranowski et al.
39, as it is proximal to the
tighter binding adenosine group within Ap4A. Additionally, a large
cluster of electron density was apparent near α1, where a single
phosphate, three magnesium ions, and a network of waters were modeled (Fig. 4D). As wild-type CT771 was used in the
crystallization experiments with Ap4A, it was not unexpected for the
substrate to undergo hydrolysis. Likewise, previous structural studies on
C. elegans Ap4A hydrolase 37 with the substrate analog
AppCH2ppA yielded nearly the same interpretable regions of
electron density as described here (Fig. S3B). The authors speculated that AppCH2ppA
had either been catalytically processed by the hydrolase during crystallization
or that flexible regions within the substrate (e.g. P2- and
P3-phosphates and the second adenosine group) subsequently lacked
interpretable electron density. Given the extreme similarities in bound active
site ligands between those studies and ours, despite the use of different
substrate molecules, we speculate that both compounds have been processed by the
crystallized hydrolase in each structure, resulting in a bound AMP molecule and
metal-phosphate complex.</p><p>Analysis of the bound AMP molecule within each CT771/AMP-PO4
subunit active site revealed that it resides in a deep groove (~11
Å as measured from the Tyr96 hydroxyl to the N3 atom of the bound AMP
molecule) created by the loop regions connecting β2-β3 and
β5-β6, as well as the β sheets themselves (Fig. 5). The adenine ring of AMP adopts the
anti conformation, where it is stabilized by a hydrogen
bond interaction with the carboxylic acid moiety of Glu131 (Fig. 4C). Additional protein contacts with the adenine ring
involve an extensive set of π-π stacking interactions created by
the aromatic side chains Phe129 and Tyr80. All three side chains are highly
conserved across examined Ap4A hydrolases (red
circles, Fig. 2B), suggesting
a common mechanism of substrate binding and orientation. The ribose sugar
hydroxyl groups of the bound AMP molecule adopt the
C2′-endo and C3′-exo
conformations, and the P1-phosphate is oriented towards the catalytic
α1 Nudix helix (Fig. 4C). While the
ribose sugar moiety lacks any hydrogen bonding with CT771, the
P1-phosphate is stabilized by an extensive network of positively
charged side chains (His38, Lys43 and Lys92), along with Tyr80 (Fig. 4C, S1C). As with the
adenosine moiety, all four of these residues interacting with the
P1-phosphate are highly conserved, with both Lysine residues being
invariant (red and blue circles, Fig. 2B). While bond catalysis occurs at the
P3-P4-phosphate interface 39, 40,
proper orientation of Ap4A clearly begins in this ~11
Å-deep groove.</p><p>Consistent with the previously reported Ap4A hydrolase binary
complex from C. elegans
37, the region of strong
electron density near the catalytic α1 Nudix helix (Fig. 4A) was modeled with a single phosphate anion bound to
a network of magnesium ions and waters (Fig.
4B,D). Bailey et al. proposed that this phosphate
molecule occupied the P4-phosphate position of Ap4A,
primarily due to its proximity to the Nudix helix and distance from the
P1-phosphate 37. Thus, all further discussions of this site will refer to it as
the P4-phosphate.</p><p>Within the CT771/AMP-PO4 structure the
P4-phosphate is maintained in a tightly bound position through
numerous solvent-ligand contacts and a single direct protein interaction
(His45). Solvent-ligand contacts are primarily mediated by three hexacoordinated
Mg2+ ions, which themselves form tight metal coordination bonds
(average bond length, 2.13 Å) with three Glutamate residues (Glu59, Glu63
and Glu111), 6 water molecules and the aforementioned phosphate (Fig. 4D). A fourth Glutamate residue (Glu62)
forms hydrogen bonds with two of the active site waters involved in magnesium
hexacoordination. Arg58 appears to stabilize the position of Glu59, which
coordinates with two of the three Mg2+ ions. As these residues are
found within the Nudix motif, it is unsurprising that they are all highly
conserved amongst Ap4A hydrolases (red circles,
Fig. 2B). Intriguingly, only three of
the oxygens within the P4-phosphate participate in extensive
contacts, suggesting the fourth oxygen would likely be linked to the second
adenosyl moiety of Ap4A. Thus, as the CT771/AMP-PO4
structure was crystallized in the presence of a high concentration of magnesium
(200 mM MgCl2), the active site metals appear to properly maintain
the P4-phosphate in a highly specific orientation, priming the
substrate for subsequent nucleophilic attack and resulting catalysis at the
P3-P4-phosphate bond. While substrates other than
Ap4A were not tested for enzymatic activity in the current study,
it has previously been reported that this family of enzymes can hydrolyze
adenosine polyphosphates with four, five or six phosphates, but not three
3, indicating
positioning of the P4-phosphate is critical to substrate
catalysis.</p><p>Despite efforts to co-crystallize Ap4A with CT771, it appears
that the substrate was hydrolyzed prior to data collection, with the product AMP
and a magnesium-bound phosphate complex being retained in the final structure.
Despite the absence of interpretable electron density for P2- and
P3-phosphates in the ligand-bound CT771 structure, comparison
with the sulfate anion bound in the apo-CT771 structure (described in Supporting Information)
suggests that both Lys43 and Lys92 could hydrogen bond with multiple phosphate
oxygen atoms of Ap4A (Fig. 5).
In agreement with the ATP-bound A. aeolicus Ap4A
hydrolase structure (Fig.
S3A), the P2-phosphate of Ap4A would likely not
make contacts with CT771. Finally, the second adenosyl binding site, if there is
one, remains unoccupied, as seen in all previously reported Ap4A
hydrolase binary complexes 7, 8, 37. Analysis of both structures presented herein reveals
that a minimal binding module of Np4 is required for CT771 and this
family of enzymes 40.</p><!><p>Structural comparison of the apo- and ligand-bound forms of CT771
presents insight into substrate binding and conformational changes required for
ligand catalysis. Structural superposition of these two CT771 structures results
in the alignment of 140/147 Cα atoms with an RMSD of 1.41 Å (Fig. 6). Notably, the catalytic α1
Nudix helix and surrounding loops are almost identical (highlighted
purple in Fig. 6) with
only the side chain of Glu63 reorienting to coordinate a magnesium ion in the
ligand-bound structure (Fig. 7). While the
aforementioned structures are highly similar from a secondary structure
perspective, several key loop regions have constricted inward towards the active
site. Glu111 directly coordinates two magnesium ions in the ligand-bound
structure (shifting ~2.9 Å from its apo-position, (Fig. 7)), as the loop connecting β6
and β7 shifts ~3.0 Å in order to accommodate this
interaction (region two in Fig. 6).
Additionally within this region, the sidechain of His38 moves ~6.7
Å as it rotates nearly 120° (Fig.
7), where it hydrogen bonds with the P1-phosphate of the
bound AMP molecule. Minor conformational changes within this region of CT771
also disrupt an intrachain disulfide bond between Cys32 and Cy113 upon ligand
binding (Fig. S5). An
extra turn of α3 is formed (region 3 in Fig. 6, residues Pro130 – Arg133) upon ligand binding, which
places Phe129 in the appropriate position (shifting ~12.2 Å from
its apo-position) to participate in π-π stacking interactions with
the adenine ring of AMP (Fig. 4C). The
extra turn of α3 facilitates an ~9.3 Å shift of Glu131 away
from the active site cleft (Fig. 7), where
it hydrogen bonds with the N6 atom of AMP. The loop connecting β5 and
β6 shifts ~4.0 Å (region 4 in Fig. 6), however, it does not contact any of the bound ligands.
Rather, this shift appears to facilitate bonding interactions involving Tyr80
(shifting ~4.9 Å from its apo-position) on β5 and Lys92 on
β6, as these β-sheets subtly bow towards the bound AMP molecule
(Fig. 7). Finally, the loop region
connecting β1 and β2 has been significantly reordered in the
ligand-bound structure (region 1 in Fig.
6); however, this likely arises from protein-protein contacts in crystal
packing and does not appear to affect the CT771 active site.</p><!><p>CT771 is conserved across all sequenced members of
Chlamydiaceae (sequence identities ranging from 71-99%).
However, BLAST analysis of Chlamydiales, members of which
include environmental Chlamydiae isolates, reveals the presence
of genes orthologous to CT771 (sequence identities ranging from 35-42%) that
were not previously clustered with other Chlamydiaceae members
(aligned with CT771 in Fig. 2B). It thus
appears that the presence of Ap4A hydrolase is required in the basic
biology of Chlamydiae. As such we recommend that the annotation
of CT771, along with its current orthologs and the ones identified herein, be
updated to asymmetric diadenosine tetraphosphatase (nudH).</p><!><p>BLAST analysis with the Nudix motif identifies a single target (CT771)
within the obligate intracellular human pathogen Chlamydia
trachomatis. This singular protein is conserved throughout both
pathogenic and environmental Chlamydiales (Fig. 2B), suggesting it might play an important role in
chlamydial biology. In order to better understand the function of this protein,
structural studies were undertaken on CT771 from C. trachomatis, in
both apo- and ligand-bound forms. Together, the structural and enzymatic
characterization of CT771 suggests that this protein functions as an asymmetric
diadenosine tetraphosphate (Ap4A) hydrolase, with the likely ability to
cleave diadenosine polyphosphates (n≥4). Insights into substrate binding and
catalysis have been obtained by efforts to co-crystallize CT771 with
Ap4A, with very similar active site electron density occupancy to the
previously determined C. elegans Ap4A binary complex
37. Intriguingly, the
structure of CT771, only the second Ap4A hydrolase determined from
eubacteria, most closely resembles both bacterial and mammalian Ap4A
hydrolases, further supporting that these two classes are more closely linked than
previously thought.</p><p>Both the apo- and ligand-bound CT771 crystal structures contained
sulfate/phosphate anions bound near the Nudix motif within the putative active site
(Fig. 4, S2). Numerous highly
conserved side chains interact with these anions either directly or through
coordinated metal ions. Based upon similarities to binary complexes from C.
elegans
37 and A.
aeolicus
7 Ap4A hydrolases, these
anions have been designated P1-, P3- and
P4-phosphates; to our knowledge, this is the first description of all 3
contacts within the same protein. While a bound AMP molecule, which represents the
first/primary nucleotide binding pocket, was also found within the putative active
site, the absence of any appreciable binding contacts for the second adenosine group
is consistent with the ability of this subclass of Nudix hydrolases to cleave
diadenosine polyphosphates that are linked by four to six phosphates. Given the use
of different substrates (AppCH2ppA and Ap4A, respectively),
the highly similar ligands bound within the active sites of the C.
elegans
37 and CT771 binary complexes are
surprising. Despite the fact that Bailey et al. used a
theoretically non-hydrolysable Ap4A analog, it appears both compounds
were cleaved by the crystallized hydrolases. Additionally, the previously discussed
A. aeolicus binary complex 7 also utilized Ap4A, which was also apparently
hydrolyzed. However, a bound ATP molecule was modeled by Jeyakanthan et
al. in the equivalent position of the AMP molecule within both the
C. elegans
37 and CT771 binary complexes. We
speculate that this difference in bound ligand arises from the common use (and
absence in the A. aeolicus binary complex) of high concentrations
of MgCl2 in the crystallization conditions of both the CT771 and
C. elegans binary structures that facilitated the presence of a
tightly bound Mg2+-phosphate complex.</p><p>Comparative analyses of the three phosphate binding sites (and the absence
of protein mediated contacts at the putative P2-phosphate site) from both
the apo- and ligand-bound CT771 structures presented herein are in agreement with
previously determined binary complexes; this supports the role of the Nudix motif in
metal binding and proper substrate positioning, prior to catalysis of the
P3-P4 phosphate bond. Previous studies have described the
asymmetric Ap4A hydrolase reaction mechanism as an
"in-line" nucleophilic attack, which is mediated by a conserved
glutamate functioning as a catalytic base, coordinating a water molecule and up to 3
magnesium ions 8. Structural and
mutagenic studies have suggested that Glu63 (CT771 numbering) is the most likely
candidate for the catalytic base; however the absence of structural evidence with an
intact substrate has precluded definitive assignment of this residue. Within the
CT771/AMP-PO4 structure, three glutamate residues (59, 63 and 111)
each coordinate with two of the three magnesium ions (Fig. 4D). Furthermore, Glu63, Glu111, and the Lys43 carbonyl coordinate
with a common magnesium ion (identified as 1 in Fig.
4D) that is bound to a water molecule in an appropriate position to
function as a nucleophile within the phosphoryl-binding groove of CT771 (denoted by
a dashed arrow in Fig. 4D).</p><p>Enzymatic characterization of CT771 was facilitated by monitoring hydrolyzed
Ap4A via released ATP and cognate luciferase activity. Unfortunately,
this method precluded the analysis of substrates other than Ap4A due to
an absence of released ATP. However, both in silico docking and
crystallographic studies on CT771 support a preference for Ap4A as a true
substrate. Furthermore, enzymatic analyses did confirm Ap4A hydrolase
activity for CT771 and reveal an intriguing inverse relationship between
concentration and the preferred divalent cation. While the majority of
Ap4A hydrolases preferentially utilize Mg2+
41, instances of maximal activity
in the presence of Mn2+ have been previously documented 42. Furthermore, in the presence of
Mn2+ the Km value for Ap4A was
roughly two orders of magnitude tighter than in the presence of Mg2+ for
CT771, yet increasing concentrations of Mn2+ resulted in a decreased
catalytic rate and efficiency (Table 4). The
results presented by Szurmak et al. are in agreement with those
reported here, particularly with respect to increasing catalytic rate in the
presence of decreasing Mn2+ and increasing Mg2+ cations. It is
difficult to speculate what physiological role this difference in metal ion affinity
would serve within Chlamydia, but it could be a reflection of the
intracellular environment where the chlamydial developmental cycle occurs.</p><p>Chlamydia species are obligate intracellular pathogens that
typically rely on the infected host cell for the majority of their energetic needs.
Thus, intracellular energy storage pools must be closely monitored in order to
prevent the accumulation of toxic byproducts, or alarmones, which could potentially
shutdown protein synthesis 43. One
such alarmone is Ap4A, which is typically generated when aminoacyl-tRNA
synthetases charge their cognate tRNA molecule 10. The chlamydial inclusion is not passively permeable to
small molecules ≥520 Da 44,
indicating that Ap4A (~800 Da) may not be freely diffusible and
would need to be processed within the inclusion, providing a clear role for CT771
throughout the developmental cycle. Substrate multispecificity has been documented
within a variety of Nudix hydrolase family members 9 and of particular note with respect to CT771,
bacterial Ap4A hydrolases have been demonstrated to efficiently remove
the 5′ termini of mRNA, suggesting a role in translation regulation
45. Clearly, further
studies exploring alternative substrate preferences and specificities are needed,
but at this time it appears that CT771 functions as a housekeeping gene, preventing
Ap4A levels from rising during periods of high metabolic activity. In
support of this role, quantifiable levels of CT771 protein are detected in EBs
46, indicating the
organism is primed to handle a rapid burst of nascent transcription (~1-2
hpi) 47 and a potential build-up
of Ap4A through tRNA charging during translation, upon infecting and
entering the host cell.</p>