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Solution NMR Structures of Productive and Non-productive Complexes between the A and B Domains of the Cytoplasmic Subunit of the Mannose Transporter of the Escherichia coli Phosphotransferase System*

Solution structures of complexes between the isolated A (IIAMan) and B (IIBMan) domains of the cytoplasmic component of the mannose transporter of Escherichia coli have been solved by NMR. The complex of wild-type IIAMan and IIBMan is a mixture of two species comprising a productive, phosphoryl transfer competent complex and a non-productive complex with the two active site histidines, His-10 of IIAMan and His-175 of IIBMan, separated by ∼25Å. Mutation of the active site histidine, His-10, of IIAMan to a glutamate, to mimic phosphorylation, results in the formation of a single productive complex. The apparent equilibrium dissociation constants for the binding of both wild-type and H10E IIAMan to IIBMan are approximately the same (KD ∼ 0.5 mm). The productive complex can readily accommodate a transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the Nϵ2 atom of His-10 of IIAMan and the Nδ1 atom of His-175 of IIBMan with negligible (<0.2Å) local backbone conformational changes in the immediate vicinity of the active site. The non-productive complex is related to the productive one by a ∼90° rotation and ∼37Å translation of IIBMan relative to IIAMan, leaving the active site His-175 of IIBMan fully exposed to solvent in the non-productive complex. The interaction surface on IIAMan for the non-productive complex comprises a subset of residues used in the productive complex and in both cases involves both subunits of IIAMan. The selection of the productive complex by IIAMan(H10E) can be attributed to neutralization of the positively charged Arg-172 of IIBMan at the center of the interface. The non-productive IIAMan-IIBMan complex may possibly be relevant to subsequent phosphoryl transfer from His-175 of IIBMan to the incoming sugar located on the transmembrane IICMan-IIDMan complex.

The two transmembrane components, IIC Man and IIB Man , form a tight complex (13). The cytoplasmic component, IIAB Man , is an obligate dimer with all dimerization contacts mediated by the A domain (14 -16). The A (residues 1-134) and B (residues 160 -323) domains of IIAB Man are covalently attached by a flexible 25-residue alanine/proline-rich linker (residues 135-159) and fold independently of one another (15). Phosphoryl transfer occurs between His-10 of IIA Man and His-175 of IIB Man (14,17). In the homologous sorbose (II Sor ) permease of Klebsiella pneumoniae (18) and fructose (II Lev ) permease of Bacillus subtilis (19), the A and B domains are expressed as separate polypeptide chains. To simplify the NMR spectroscopy and facilitate the identification of intermolecular contacts through isotope-filtered/separated nuclear Overhauser enhancement (NOE) experiments we therefore chose to carry out structural work on complexes of isolated IIA Man and IIB Man .
A high (1.7 Å) resolution crystal structure of E. coli IIA Man had previously been solved, but no structure was available for IIB Man . We therefore first solved the solution structure of E. coli IIB Man on the basis of NOE and dipolar coupling data in two alignment media, and then used this structure together with the x-ray structure of IIA Man to solve the structure of the IIA Man -IIB Man complex by conjoined rigid body/torsion angle dynamics on the basis of intermolecular NOE data. The intermolecular data recorded on the wild-type IIA Man -IIB Man complex were not compatible with the existence of a single species. Subsequent mutation of the active site His-10 of IIA Man to Glu to mimic phosphorylation of His-10 resulted in the formation of a single complex that was fully consistent with the stereochemical and geometric requirements for phosphoryl transfer between His-10 of IIA Man and His-175 of IIB Man . Selection of a single complex by the H10E mutation is due to neutralization of the positively charged Arg-172 of IIB Man at the center of the protein-protein interface of the productive complex. With the structure of the productive complex in hand, we were then able to determine the structure of the non-productive complex by accounting for the intermolecular NOE data on the basis of a mixture of productive and non-productive complexes. In the non-productive complex the active site histidines of IIA Man and IIB Man are ϳ25 Å apart, and the active site His-175 and associated active site loop of IIB Man are exposed to solvent. We suggest that the non-productive complex may therefore be relevant to subsequent phosphoryl transfer to the incoming sugar located on the cytoplasmic side of the transmembrane IIC Man -IID Man complex. This study illustrates how, for weak proteinprotein complexes, a relatively subtle change in a single interfacial residue can have a dramatic impact on the configuration of the resulting complex that, in this instance, may play an important role in both modulating and directing the phosphorylation cascade within the mannose transporter.

EXPERIMENTAL PROCEDURES
Cloning, Expression, and Purification of IIA Man and IIB Man -The A domain of wild-type IIAB Man (residues 1-136, IIA Man ) of E. coli was expressed and purified as described previously (9).
The DNA corresponding to the B domain of IIAB Man (residues 157-323, IIB Man ) was amplified by PCR using a DNA template derived from E. coli chromosomal DNA provided by A.
Peterkofsky. The PCR product contains an NcoI restriction site at the 5Ј site and a tandem pair of in-frame termination codons and a BamHI site at the 3Ј site introduced during the PCR reaction. The NcoI-and BamHI-cut PCR product was purified and subcloned into the corresponding expression sites of the modified pET32a vector (4) to form a thioredoxin fusion protein with a His 6 tag. The selected clone was verified by DNA sequencing.
The following mutations, H10E in IIA Man , and R172Q and H175E in IIB Man were introduced using the QuikChange mutagenesis kit (Stratagene), and the sequences confirmed by DNA sequencing. Expression and purification was carried out using the same protocols as that for the corresponding wildtype proteins.
The plasmids for IIA Man and IIB Man were introduced into E. coli BL21(DE3) (Novagen) cells for protein expression and induced at an A 600 ϳ 0.8 with 1 mM isopropyl-␤-D-thiogalactopyranoside at 37°C and 30°C, respectively. Cells were grown in either Luria-Bertani medium or minimal media (in either H 2 O or D 2 O, with 15  After induction (3 and 10 h for growths in H 2 O and D 2 O, respectively), cells expressing IIB Man protein were harvested, pelleted by centrifugation and resuspended in 50 ml (per liter of culture) of 30 mM Tris, pH 8.0, 10 mM imidazole and 200 mM NaCl. The cell suspension was lysed by three passages through a microfluidizer and centrifuged at 10,000 ϫ g for 20 min. The supernatant was loaded onto a 5-ml nickel-Sepharose column (HisTrap HP, Amersham Biosciences), and the fusion protein was eluted with a 50-ml gradient of imidazole (10 -500 mM). The eluted protein was collected and digested with 200 NIH units of thrombin after overnight dialysis against a 4-liter buffer of 25 mM Tris, pH 8.0, and 200 mM NaCl. Thrombin was removed by passage over a benzamidine-Sepharose column (1 ml, Amersham Biosciences), followed by the addition of 1 mM phenylmethylsulfonyl fluoride. The IIB Man protein after cleavage was collected after loading the digested mixture onto a 5-ml nickel-Sepharose column and further purified by gel filtration.
All NMR samples were prepared in a buffer of 20 mM sodium phosphate, pH 6.5, 0.01% sodium azide, and either 90% H 2 O/ 10% D 2 O or 99.996% D 2 O. IIA Man is a symmetric dimer with two non-overlapping but equivalent binding sites for IIB Man . To achieve optimal line widths we chose to record the majority of spectra on samples comprising a 1:1 mixture of IIA Man dimer to IIB Man monomer (see "Results" and "Discussion").
NMR Spectroscopy-NMR spectra were recorded at 30°C on Bruker DMX500, DMX600, DRX600, and DRX800 spectrometers equipped with either x-, y-, and z-shielded gradient triple resonance probes or z-shielded gradient triple resonance cryoprobes. Spectra were processed with the NMRPipe package (23) and analyzed using the programs PIPP, CAPP, and STAPP (24).
Assignments of free wild-type IIA Man were taken from previously published work by Williams et al. (9). Assignments for the H10E mutant of IIA Man were derived from three-dimensional double and triple resonance experiments with reference to the free wild-type IIA Man assignments.
Assignments of 13 C, 15 N, and 1 H chemical shifts in the IIA Man -IIB Man complex were based on the assignments of the free proteins in conjunction with data from titration experiments using constant time 1 H-13 C HMQC and TROSY 1 H-15 N spectra, as well as TROSY-based triple resonance through-bond correlation experiments (31).
Intermolecular NOEs were observed on the IIA Man -IIB Man complex in D 2 O buffer using three-dimensional 12 C-filtered(F 1 )/ 13 C-separated(F 2 ) or 13 C-separated(F 2 )/ 12 C-filtered(F 3 ) NOE experiments and in H 2 O buffer using two-dimensional 15 N separated/ 13 C-edited and two-dimensional 13  Structure Calculations-Interproton distance restraints were derived from the NOE spectra and classified into generous approximate distance ranges, 1.8 -2.7, 1.8 -3.5, 1.8 -5.0, and 1.8 -6.0 Å (with an additional 0.5 Å added to the upper limits for NOEs involving methyl groups), corresponding to strong, medium, weak, and very weak NOE cross-peak intensities, respectively (25,33). Non-stereospecifically assigned methyl, methylene, and aromatic protons and ambiguous intermolecular NOEs were represented by a (⌺r Ϫ6 ) Ϫ1/6 sum (26,34). / torsion angle restraints for free IIB Man were derived from backbone (N, CЈ, C␣, C␤, and H␣) chemical shifts using the program TALOS (35). Side-chain torsion angle restraints were derived from 3 J heteronuclear couplings and short mixing time NOE and ROE experiments using standard procedures (26). The minimum range for the torsion angle restraints was Ϯ20°.
All structure calculations were carried out using Xplor-NIH (36) and the IVM (21) module for torsion angle and rigid body dynamics. The structure of the free IIB Man was calculated by simulated annealing in torsion angle space (21). The structure deter- The torsion angle restraints comprise 160 , 157 , 108 1 , 44 2 , and 9 3 angles. c The RDC R-factor, which scales between 0 and 100%, is defined as the ratio of the r.m.s. deviation between observed and calculated values to the expected r.m.s. deviation if the vectors were randomly distributed, given by ͓2D a 2 (4 ϩ 3 2 )/5͔ 1/2 , where D a is the magnitude of the principal component of the alignment tensor, and is the rhombicity (61). The values of D a NH and , derived from the distribution of normalized RDCs, are Ϫ11.7 Hz and 0.30, respectively, for the data recorded in pf1 phage, and Ϫ10.0 Hz and 0.28, respectively, for the data in PEG/ hexanol (62). d Calculated with the program PROCHECK (48). The dihedral angle PROCHECK G factors for /, 1 / 2 , 1 , and 3 / 4 are 0.06 Ϯ 0.02, 0.57 Ϯ 0.06, 0.14 Ϯ 0.08, and 0.22 Ϯ 0.13, respectively. The WHATIF first generation packing score is 0.13; a value greater than Ϫ0.5 is considered to represent a high quality structure (63). e The precision of the coordinates is defined as the average atomic r.m.s. difference between the individual 130 simulated annealing structures and the corresponding mean coordinates best-fitted to the backbone atoms of residues 160 -321. (Residues 156 -159 and 322-323 at the N and C termini, respectively, are disordered.) mination of the IIA Man -IIB Man complex was carried out using conjoined rigid body/torsion angle dynamics (21,37). The target function for simulated annealing comprises the following: square well potentials for interproton distance and torsion angle restraints (38); harmonic potentials for 13 C␣/ 13 C␤ chemical shift restraints (39), RDC restraints (40), and covalent geometry; and a quartic van der Waals repulsion potential (41), a multidimensional torsion angle data base potential of mean force (42), a backbone hydrogen bonding data base potential of mean force with automatic hydrogen-bond selection (43), and a gyration volume term (44) to represent the non-bonded contacts. The gyration volume term represents a general, weak overall packing potential for any ellipsoidal-shaped molecule based on the observation that proteins pack to a constant density (45). Structures were displayed using VMD-XPLOR (46) and GRASP (47).

RESULTS AND DISCUSSION
Structure of IIB Man -The solution structure of IIB Man was solved on the basis of 3091 experimental NMR restraints, including 1467 NOE-derived interproton distance restraints and 717 backbone RDCs ( 1 D NH , 1 D NCЈ , and 2 D HNCЈ ) in two different alignment media (pf1 phage and polyethylene glycol/hexanol). RDCs provide orientational restraints relative to an external alignment tensor (28), and the two alignment media provide complementary information, because their alignment tensors are significantly different from one another with a normalized scalar product of 0.57. A summary of the structural statistics is provided in Table 1, and a stereoview of a best-fit superposition of the backbone atoms of the 130 final simulated annealing structures is shown in Fig. 1A. Residues 157-159 at the N terminus and 322-323 at the C terminus are disordered. The rest of the structure (residues 160 -321) is well defined with a backbone (N, C␣, CЈ, and O) precision of 0.26 Ϯ 0.04 Å. 93% of the residues occupy the most favorable region of Ramachandran space (48) and the hydrogen bonding data base potential (43) automatically identified 93 backbone hydrogen bonds (of which only 55 were explicitly identified based on the pattern of NOEs).
Binding of IIA Man to IIB Man -Three-dimensional 12 C-filtered/ 13 C-separated NOE experiments carried out on complexes of wild-type IIA Man with IIB Man revealed a pattern of intermolecular NOEs that was not consistent with a single spe- cies and suggested the presence of two co-existing complexes (Fig. 2). This is best illustrated by the top panel in Fig NOEs are observed to the methyl groups of Leu-207 and Val-211, which form one cluster on the surface of IIB Man , and to the methyl groups of Thr-180 and Thr-183, as well as the ␦-methylene group of Lys-184, which form a second cluster. Because the methyl groups of Val-211/Leu-207 are ϳ11/18, ϳ13/19, and ϳ16/22 Å away from the methyl groups of Thr-180 and Thr-183 and the ␦-methylene group of Lys-184, respectively, it is evident that the methyl group of Met-103 of IIA Man cannot be close to both clusters of residues simultaneously (Fig. 2C).
Further qualitative interpretation of the intermolecular NOE data suggested that a small number of NOEs were consistent with a productive complex, that is one in which the two active site histidines, His-10 of IIA Man and His-175 of IIB Man , are in close proximity and therefore capable of phosphoryl transfer, whereas the majority of NOEs arose from a non-productive complex. However, in the absence of prior detailed knowledge of one or the other structure, resolving the structures of two complexes simultaneously from the data was not feasible.
We reasoned that a possible explanation for the existence of two complexes could involve the conserved, solvent-exposed Arg-172 in close proximity to the active site His-175 of IIB Man . In the productive complex, Arg-172 would be buried at the interface and had been previously postulated to interact with the negatively charged phosphoryl group on His-10 of IIA Man (50). In the absence of histidine phosphorylation burial of the positively charged guanidinium group of Arg-172 at a protein-protein interface is likely to disfavor the formation of the productive complex. To test this hypothesis we mutated His-10 of IIA Man to Glu to mimic the effect of phosphorylation of His-10 (at its N⑀2 position). Analysis of the intermolecular NOE data for the resulting complex of the IIA Man (H10E) mutant with IIB Man was fully consistent with the formation of a single complex corresponding to the productive phosphoryl transfer complex. The change in the pattern of observed intermolecular NOEs can be seen by comparison of Figs. 2A and B.
Both wild-type and H10E IIA Man bind weakly to IIB Man , and the complexes are in fast exchange on the chemical shift time scale. The change in pattern of intermolecular NOEs between the complex of wild-type and H10E IIA Man with IIB Man is accompanied by differences in the chemical shift perturbation of methyl groups of IIB Man observed upon titration (Fig. 3A). Interestingly, the apparent affinity of the complex of IIB Man with both wild-type IIA Man and the H10E mutant are very comparable with an equilibrium dissociation constant (K D ) of ϳ0.5 mM (Fig.  3B). Although binding is weak, in the context of intact IIAB Man , where the A and B domains are connected by a flexible 25-residue linker, one can calculate (11,51), based upon the expected average end-to-end distance of ϳ50 Å for the linker (52), that there would be an ϳ85% probability of the two domains interacting with one another at any given time.
Structure Determination of the Productive and Non-productive IIA Man -IIB Man Complexes-The NMR samples used for structure determination comprised a mixture of 1 equivalent IIA Man dimer to 1 equivalent IIB Man monomer. IIA Man is a symmetric dimer, and the final stoichiometry of both the productive and non-productive complexes is 1 equivalent of IIA Man dimer to 2 equivalents of IIB Man . The use of a 1:1 mixture was chosen to optimize the line widths of both components, IIA Man and IIB Man , simultaneously. Because binding is weak, the samples will always comprise a mixture of free, 1:1 and 1:2 complexes in fast exchange with one another at the concentrations employed in the NMR experiments (0.5-1 mM). The line widths are proportional to the population average molecular weights. Given a K D of ϳ0.5 mM, a sample containing 1 mM IIA Man dimer and 1 mM IIB Man (with free molecular masses of 35 and 19 kDa, respectively), will comprise ϳ59 and ϳ72% complexed IIA Man and IIB Man , respectively, with an apparent molecular mass of ϳ49 kDa for both IIA Man and IIB Man . In contrast, a mixture of 1 mM IIA Man and 2 mM IIB Man will yield an effective molecular mass of 58 kDa for IIA Man and 48 kDa for IIB Man .
We first solved the structure of the productive complex using data exclusively from the IIA Man (H10E)-IIB Man samples. The changes in 1 H/ 15 N shifts upon complexation are very small indicating no significant structural perturbation in the backbone coordinates of either IIA Man or IIB Man occurs upon binding (at the level of detection of the NMR data). We therefore solved the structure of the productive complex using a hybrid approach that employs conjoined rigid body/torsion angle dynamics (20,21) on the basis of intermolecular NOE data with the coordinates of free IIA Man (x-ray, PDB code 1POD) (16) and IIB Man (the complete ensemble of NMR simulated annealing structures; this report) treated as rigid bodies and the interfacial side chains given torsional degrees of freedom. In addition, the backbone and side chains of residues 130 -134 of IIA Man were also given torsional degrees of freedom, because intermolecular NOEs were observed involving residues 129, 133, and 134, although residues 131-133 were not visible in the electron density map of the free crystal structure (16). 37 intermolecular NOEs were identified of which 33 are between unique proton pairs, and the remaining 4 are ambiguous involving potentially alternate partners and were therefore treated as (⌺r Ϫ6 ) Ϫ1/6 sums. The active site His-10 is located right at the interface of the two identical subunits of IIA Man . Because the C␣ atom positions of the two symmetrically related active site histidines, His-10 and His-10Ј, are separated by 20 Å, there is no issue attributing the intermolecular NOEs to one or other subunit of IIA Man . It should be noted that the use of RDCs to provide orientational information for the structure determination of the complex was precluded owing to uncertainties in the exact proportions of each component in the sample (i.e. free proteins, complex with one IIB Man bound and complex with two IIB Man molecules bound, all of which will have different alignment tensors), and the unfeasibility of deconvoluting the alignment tensors of the 1:1 and 1:2 complexes (because complete occupancy of the 1:2 complex cannot be achieved at concentrations compatible with the alignment media used for RDC measurements). A summary of the structural statistics is given in Table 2, and a best-fit superposition of the final 120 simulated annealing structures is shown in Fig. 4A. The relative orientation of IIB Man relative to the IIA Man dimer is well determined by the intermolecular NOE data with an overall backbone precision for the complex of 0.5 Å.
As noted above, samples of wild-type IIA Man and IIB Man comprise a mixture of productive and non-productive complexes. Of the 41 intermolecular NOEs identified, only 5 satisfied the structure of the productive complex with violations Ͻ 0.5 Å, and the remainder are violated by 4 -16 Å.
To obtain the structure of the non-productive complex, we therefore proceeded as follows. The structure of the productive complex (restrained regularized mean coordinates) was held fixed and a second molecule of IIB Man (restrained regularized mean structure of free IIB Man ) was introduced in random starting orientations and its position determined by conjoined rigid body/torsion angle dynamics (21)  lecular NOEs represented as ambiguous restraints, that is (⌺r Ϫ6 ) Ϫ1/6 sums (34), arising from both the productive and non-productive complexes. (Note that because the intermolecular NOEs are interpreted in terms of loose, conservative distance ranges, and because long distances do not contribute to the (⌺r Ϫ6 ) Ϫ1/6 sum, it is not necessary to know the proportion of productive and non-productive complexes present in the sample). A table of structural statistics is provided in Table 2, and a best-fit superposition of the ensemble of 120 simulated annealing structures of the non-productive complex is displayed in Fig. 4B. Although the relative orientation of IIB Man relative to IIA Man is not quite as well defined in the non-productive complex relative to the productive one, the backbone precision for the non-productive complex is still rather high (ϳ0.8 Å). It is worth noting that the same ensemble of structures was obtained for the non-productive complex by simply using the 36 intermolecular NOEs that were violated in the productive complex and not including the structure of the productive complex in the calculations. Structure of the Productive IIA Man -IIB Man Complex-A ribbon diagram of the productive complex derived from the IIA Man (H10E)-IIB Man data is shown in Fig. 5A. 1750 Å 2 of solvent-accessible surface area is buried at the interface, 870 Å 2 originating from IIA Man and 880 Å 2 from IIB Man . The dimensions of the interface are ϳ40 Å long and 30 Å wide. The gap volume index (ratio of gap volume to buried accessible surface area) is 3.5, which falls in the outer range observed for both hetero (2.4 Ϯ 1.0) and optional (2.7 Ϯ 0.9) protein-protein complexes (53), as expected given the weak binding.
In describing the intermolecular contacts involving IIA Man , the residues of the B-chain (red subunit in Figs. 5-8) are denoted by a prime symbol. (Note that the definition of A and B chains of IIA Man is purely arbitrary, and the relationship of the two IIA Man chains to IIB Man at one site is reversed in the symmetry related site.) The interface on IIA Man is made up of residues of both subunits and comprises helix ␣1, helix ␣4, and the C-terminal five residues of the A-chain: the active site His-10Ј, the loop between ␤2Ј and ␣2Ј, the N-terminal end of helix ␣2Ј, and helix ␣3Ј of the B-chain. The interface on IIB Man comprises the active site (residues 172-176, including His-175), helices ␣1 and ␣3, and the loops between strands ␤5 and ␤6, ␤6 and ␤7, and ␤8 and ␤9 (Fig. 5, A and C). A summary of the contacts is provided in Fig. 5C, and a stereoview showing the side-chain interactions is shown in Fig. 6A. The A chain of IIA Man primarily interacts with helices ␣1 and ␣3, and the loop between strands ␤8 and ␤9 of IIB Man , whereas the B-chain primarily contacts the active site loop, helix ␣3, and the loops between strands ␤5 and ␤6 and strands ␤6 and ␤7 of IIB Man .
The interface is made up of 57% non-polar atoms and 43% polar ones. The active site histidines, His-10Ј and His-175, are located at the center of the interface, as is Arg-172. In the IIA Man (H10E)-IIB Man complex, the positively charged guanidino group of Arg-172 is neutralized by the negative charge on the carboxylate of H10EЈ, mimicking phosphorylated His-10Ј. In the absence of neutralization of the guanidino group of Arg-172, the productive complex is destabilized allowing an alternative, non-productive complex to be formed. The majority of intermolecular interactions are hydrophobic in nature, and there are only three additional electrostatic interactions, between Asp-106 and Arg-180, and Glu-100 and Lys-184, which anchor helix ␣1 of IIB Man , and between Glu-43Ј and Arg-

Structural statistics for productive and non-productive IIA Man -IIB Man complexes
The notation is the same as that in Table 1. The final number of simulated annealing structures is 120. The number of experimental restraints for the various terms is given in parentheses, with the first number referring to the data for the productive complex derived from the IIA Man (H10E)-IIB Man complex, and the second to the data from the wild type IIA Man (H10)-IIB Man complex. The data obtained for the wild-type IIA Man (H10E)-IIB Man complex arise from a mixture of productive and unproductive complexes. The NOE data were therefore represented as ambiguous (⌺r Ϫ6 ) Ϫ1/6 sums. The productive complex was held fixed at the conformation determined from the IIA Man (H10E)-IIB Man data. Of the 41 intermolecular NOEs, only 5 (attributable to the productive complex) are not satisfied (i.e. violations Ͼ 0.5 Å) by the non-productive complex alone. c For the productive complex, the side-chain torsion angles comprise 11 1 and 5 2 for IIA Man , and 14 1 and 7 2 for IIB Man ; in addition, there are 4 and 3 backbone torsion angle restraints for residues 130 -134 of IIA Man , which were also given torsional degrees of freedom. For the non-productive complex, the side-chain torsion angles comprise 11 1 and 5 2 for IIA Man , and 5 1 and 1 2 for IIB Man . d The intermolecular repulsion energy is given by the value of the quartic van der Waals repulsion term calculated with a force constant of 4 kcal.mol Ϫ1 .Å Ϫ4 and a van der Waals radius scale factor of 0.78. The intermolecular Lennard-Jones van der Waals interaction energy is calculated using the CHARMM19/20 parameters and is not included in the target function used to calculate the structures. The percentage of residues present in the most favorable region of the Ramachandran map (48) for the x-ray structure of free IIA Man (PDB code 1PDO (16) (54,55). The transition state can readily be modeled on the basis of the structure of the IIA Man (H10E)-IIB Man complex using the procedures described previously (6 -9) in which the only portion of the complex allowed to move comprises the backbone and side chains of the active site histidines (His-10Ј and His-175), the immediately adjacent residues (residues 8 -12Ј of IIA Man and 173-177 of IIB Man ) and the phosphoryl group (Figs. 4A and 6B). For an N-P distance of 2.5 Å, which corresponds to a mechanism with substantial dissociative character consistent with many phosphoryl transfer reactions (56), the transition state can be accommodated with negligible (Ͻ0.2 Å) changes in backbone conformation for residues 9Ј-11Ј and 174 -176. Only minor additional backbone changes (ϳ0.2 Å) for these residues are required for an S N 2 mechanism (50% associative) with an N-P distance of 2 Å.
The His-10Ј-P-His-175 transition state is buried within a largely hydrophobic cavity comprising Leu-24, Phe-36Ј, Pro-38Ј, and Pro-73Ј of IIA Man , and Val-178 and the aliphatic portions of the long side chains of Arg-172 and Lys-305 of IIB Man . The phosphoryl group is within hydrogen bonding distance of the hydroxyl group of Ser-72Ј and the guanidino group of Arg-172, thereby stabilizing the transition state. In addition, there may be water-bridged interactions to the phosphoryl group from the hydroxyl group of Thr-68Ј, the carboxyamide of Gln-177, and the NH 3 group of Lys-305 (Fig. 6, B and C).
The C␣-C␣ distances between the N terminus of IIB Man (residue 209) and the C termini (residues 134 and 134Ј) of the A and B chains of IIA Man are 38 Å and 64 Å, respectively. The expected average end-to-end distance for a random-coil 25-residue linker is ϳ50 Å (52). This suggests that, in the intact IIAB Man dimer, phosphoryl transfer occurs predominantly in trans, that is between the IIA Man domain of one chain, and the IIB Man domain of the other chain.
The structure of the productive IIA Man -IIB Man complex displays both similarities and differences to the structure of the upstream IIA Man -HPr complex (9). Both IIB Man and HPr have an active site loop followed by an ␣-helix. The C␣ atomic r.m.s. difference between the element of structure Stereoviews showing best-fit superpositions of backbone (N, C␣, and C) atoms for the final 120 simulated annealing structures, with IIB Man in green, and the A and B subunits of IIA Man in blue and red, respectively; the active site side chains of the restrained regularized mean structure are shown in purple. A, structure corresponding to the productive, phosphoryl transfer competent complex, derived from the NOE data obtained with the phosphomimetic H10E mutant of IIA Man . The modeled phosphoryl transition state with a pentacoordinate phosphoryl group and Glu-10 replaced by His is shown in transparent orange. Only negligible changes (Ͻ0.2 Å) in local backbone conformation in the immediate vicinity of the active sites (residues 8Ј-12Ј and 173-177) are required to model the phosphoryl transition state. B, structure corresponding to the non-productive, phosphoryl transfer incompetent, complex derived from the NOE data obtained with wild-type IIA Man . The C␣-C␣ distance between the active site residues (residue 10Ј of the B chain of IIA Man and residue 175 of IIB Man ) is 11 Å for the productive complex versus 25 Å for the non-productive one.   Fig. 7. The similar disposition of the active site loop and helix ␣1 of IIB Man and HPr relative to IIA Man is evident. However, the interface is more extensive in the IIA Man -IIB Man complex than in the IIA Man -HPr complex (1750 Å 2 buried versus 1450 Å 2 ). Moreover, HPr has a large contact surface with the A chain of IIA Man , whereas IIB Man has more extensive contacts with the B chain. Despite the reduced contact area, the affinity of HPr for IIA Man (K D ϳ 30 M 9) is 15-to 20-fold higher than that of IIB Man (K D ϳ 0.5 mM; this report). This may be due to a higher degree of surface complementary, as exemplified, for example, by a 2-fold greater number of electrostatic/hydrogen-bonding interactions between HPr and IIA Man (9).
Structure of the Non-productive IIA Man -IIB Man Complex-A ribbon diagram of the non-productive complex is shown in Fig. 8A. The interaction involves a highly hydrophobic ridge-like protrusion on the surface of IIB Man formed exclusively by helix ␣3, interacting with a subset of residues on IIA Man that comprise the central portion of the interface involved in the productive complex (Fig. 8C). This subset comprises the predominantly hydrophobic surface formed by helices ␣1 (Met-23, Leu-24, and Leu-25) and ␣4 (Pro-96, Val-99, and Met-103) of the A-chain of IIA Man , and the active site His-10Ј, the loop following strand ␤2Ј (Phe-36Ј), the loop between strand ␤3Ј and helix ␣3Ј (Thr-68Ј, Trp-69Ј, and Gly-70Ј), and helix ␣3Ј (Ser-72Ј and Asn-75Ј) of the B chain of IIA Man (Fig. 8, B and C). The buried accessible surface area at the interface is 790 Å 2 , of which 380 Å 2 originates from IIA Man and 410 Å 2 from IIB Man , approximately half that of the productive complex. The C␣-C␣ distance between the two active site histidines, His-10Ј and His-175, is 25 Å and therefore incompatible with phosphoryl transfer between IIA Man and IIB Man . The C␣-C␣ distances between the N terminus of IIB Man and the C termini of the A and B chains of IIA Man are 60 and 30 Å, respectively (Fig. 8A). Thus, just as in the case of the productive complex, the interaction between the IIB Man domain and IIA Man domain is likely to occur in trans in the intact IIAB Man dimer.
The orientation of IIB Man relative to IIA Man in the non-productive complex is related by a ϳ90-Å rotation and ϳ37-Å translation relative to the productive one. This is readily appreciated from a comparison of the location of helix ␣3 of IIB Man on the surface of IIA Man in the two complexes provided by Figs. 5B and 8C (left-hand panels).
The active site of IIB Man , including His-175 and Arg-172, is fully exposed to solvent in the non-productive complex, and thus His-175 is potentially available to transfer a phosphoryl group onto the incoming sugar located on the transmembrane IIC Man -IID Man complex. It is interesting to note that His-175 lies close to the upper edge of a deep V-shaped hydrophobic cleft at the bottom of which lies helix ␣3 of IIB Man , with an outer rim of negatively charged residues (Asp-106, Asp-107, and Asp-108) provided by the A chain of IIA Man and positively charged residues (Arg-172, Arg-181, Arg-204, and Lys-305) by IIB Man . The walls of the cleft are formed by helices ␣1 and ␣4 of the A chain of IIA Man , and the active site loop, helix ␣1, and the loops between strands ␤5 and ␤6 and between ␤6 and ␤7 of IIB Man . It is tempting to speculate that this cleft comprises part of the binding site for the membrane-bound IIC Man -IID Man complex. In this regard, it is worth noting that IIAB Man has been reported to form a stable complex with the transmembrane IIC Man -IID Man component of the mannose transporter with an apparent K D of 5-10 nM (57), and a IIAB Man -IIC Man -IID Man complex can be copurified (58).
Probing the Role of Arg-172 in Complex Formation-The successful elimination of the non-productive complex as a consequence of the introduction of the phosphomimetic H10E mutation providing charge neutralization of Arg-172, strongly suggests a major role for Arg-172 in conjunction with phosphorylation of His-10 in modulating whether a productive or nonproductive complex is formed. To probe the role of Arg-172 further, intermolecular NOEs involving the leucine (Leu-24, Leu-25, and Leu129), valine (Val-99 and Val-134), and methionine (Met-23 and Met-103) methyl groups of IIA Man were analyzed for the following samples: IIA Man -IIB Man (H175E), IIA Man -IIB Man (R172Q), and IIA Man (H10E)-IIB Man (R172Q). A qualitative assessment of the proportion of productive to non-productive complexes in these samples relative to the wild-type IIA Man -IIB Man sample can be obtained by examining (a) the fraction of observed intermolecular NOEs attributable to the productive complex and (b) the ratio of the intermolecular NOE cross-peak intensities involving the methyl group of Met-103 of IIA Man attributable to the productive and non-productive complexes (cf. Fig.  2, top panels). The latter has a value of ϳ0.8 for the wild-type sample. (Note this value cannot be converted to populations of the two species in the wild-type sample, because the NOE intensities are related not only to population but also to specific intermolecular interproton distances in the two complexes.) The pattern of intermolecular NOEs observed for the IIA Man -IIB Man (H175E) sample is very similar to that of the wild-type IIA Man -IIB Man sample, with only ϳ15% of the intermolecular NOEs attributable to the productive complex compared with ϳ10% for the wild-type sample. The ratio of the cross-peak intensities for the productive to non-productive complexes is increased by ϳ1.2 relative to wild-type. These data indicate that the proportion of non-productive complex is only slightly decreased relative to wild type and, therefore, suggest that the H175E mutation does not provide adequate intramolecular charge neutralization of Arg-172. This finding may be relevant to the postulated role of the nonproductive complex in transferring a phosphoryl group on to the incoming sugar on the transmembrane IIC Man -IID Man complex. In particular, this result may suggest that, once the phosphoryl group is transferred from His-10Ј to His-175, the equilibrium between productive and non-productive complex may be shifted toward the non-productive complex if the intramolecular charge neutralization of Arg-172 by phosphorylated His-175 is less effective than the intermolecular charge neutralization by phosphorylated His-10Ј. This hypothesis may be supported by the observation that the side chain of Arg-172 is disordered in the crystal structure of B. subtilis IIB Lev (49).
For the IIA Man -IIB Man (R172Q) sample, intermolecular NOEs corresponding to both productive and non-productive complexes were observed, but the fraction attributable to the productive complex was increased to ϳ30%, and the ratio of the cross-peak intensities of productive to non-productive complexes was increased by ϳ5-fold relative to wild type. Thus, the population of productive complex in the IIA Man -IIB Man (R172Q) sample was increased substantially relative to the wild-type IIA Man -IIB Man sample. The R172Q mutation removes the positive charge of Arg-172 but still leaves a potentially unfavorable polar residue in the middle of the interface of the productive complex, thereby accounting for the continued presence of non-productive complex.
Finally, ϳ90% of intermolecular NOEs observed for the IIA Man (H10E)-IIB Man (R172Q) sample arise from the productive complex, and the few from the non-productive complex were extremely weak relative to their intensities in the wild-type sample. The ratio of NOE cross-peak intensities of productive to non-productive complexes for Met-103 was increased ϳ9-fold relative to wild type. Thus, the productive complex constitutes the major species. This result is consistent with the observation that IIB Man (R172Q) can still be phosphorylated by IIA Man , albeit somewhat less efficiently than wild-type IIB Man (59).
Concluding Remarks-This report completes the structures of cytoplasmic complexes of the mannose branch of the PTS. The intriguing finding is that the nature of the IIA Man -IIB Man complex can be modulated by the presence or absence of charge neutralization between the active sites, and in particular of the guanidino group of Arg-172. With wild-type IIA Man , two forms of complex with IIB Man were observed: the predominant one arises from a non-productive complex in which the active site histidines (His-10Ј and His-175) are separated by ϳ25 Å and therefore incompatible with phosphoryl transfer between IIA Man and IIB Man , whereas the minor one arises from a productive complex in which the active site histidines are in close proximity. Mutation of His-10Ј of IIA Man to a Glu to mimic histidine phosphorylation results in the exclusive formation (at the level of detection) of a productive complex that is fully consistent with the formation of a pentacoordinate phosphoryl transition state and in which the positive charge on the guanidinium group of Arg-172 is neutralized by interaction with the negative carboxylate group of H10EЈ. In the non-productive complex, Arg-172 and the active site His-175 of IIB Man are fully exposed to solvent and potentially available to transfer a phosphoryl group to the sugar located on the cytoplasmic side of the IIC Man -IID Man transmembrane complex.  ) is as in A, and the side-chain bonds are colored coded according to atom type (see Fig.  6 legend). C, interaction surfaces for the non-productive IIA Man -IIB Man . The left and right panels display the interaction surfaces on IIA Man and IIB Man , respectively, with the color coding as in Fig. 6B. D, diagrammatic representation of the intermolecular contacts with the active site His-10Ј colored in purple, and residues involved in potential side chain-side chain intermolecular electrostatic interactions colored in red (acceptor) and blue (donor).
The structural transition between the productive and nonproductive states is dramatic and involves a 90°rotation and concomitant 37-Å translation of IIB Man relative to IIA Man . The interaction surface on IIA Man in the non-productive complex comprises a subset of residues located in the central region of the interaction surface employed in the productive complex, including the active site His-10Ј. Thus, the non-productive complex does not allow for the formation of a ternary HPr-IIA Man -IIB Man complex, because the interaction surface on IIA Man used by IIB Man is also a subset of the interaction surface on IIA Man used by HPr. The interaction surface on IIB Man in the productive and non-productive complexes also partially overlap insofar that the interaction surface in the non-productive complex comprises exclusively helix ␣3, which is used in a completely different set of interactions with IIA Man in the productive complex.
The existence of the non-productive IIA Man -IIB Man complex may be fortuitous owing to the presence of a highly hydrophobic protrusion on the surface of IIB Man formed by helix ␣3 that can readily fit in a groove between the two subunits of IIA Man . Nevertheless, it seems likely that weak binding complexes with K D values in the 0.1-2 mM range may be particularly susceptible to multiple alternative configurations arising from rather small changes at the interface. Indeed, two distinct quaternary structures resulting from a relatively small number of changes at an interface have been observed in the much tighter homodimeric complexes of the chemokine family where the CXC and CC chemokines have high sequence identity, the same monomer folds, but entirely different dimeric structures employing completely different interfaces (60).