Solution Structure of the IIAChitobiose-HPr Complex of the N,N′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System*

Background: The bacterial phosphoryl transfer system (PTS) couples phosphoryl transfer to sugar transport. Results: The structure of the IIAchitobiose-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the PTS. Conclusion: Phosphoryl transfer occurs without any significant backbone conformational changes. Significance: Recognition of multiple, structurally diverse partners is facilitated by complementary interaction surfaces and side chain conformational plasticity. The solution structure of the complex of enzyme IIA of the N,N′-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIAChb-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIAChb was mutated to Glu to mimic the phosphorylated state. IIAChb(H89E) and HPr form a weak complex with a KD of ∼0.7 mm. The interacting binding surfaces, concave for IIAChb and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIAChb formed at the interface of two adjacent subunits of the IIAChb trimer, thereby coming into close proximity with the active site residue, H89E, of IIAChb. A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIAChb-HPr complex with the IIAChb-IIBChb complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.

extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.
The phosphoenolpyruvate:sugar phosphotransferase system (PTS) 4 is a central bacterial signal transduction pathway in which phosphoryl transfer, via a series of bimolecular proteinprotein complexes, is coupled to both sugar transport across the membrane and the regulation of many cellular processes, including catabolite repression (1)(2)(3)(4)(5)(6). The first component of the PTS, enzyme I, is autophoshorylated by phosphoenolpyruvate and subsequently transfers the phosphoryl group to the histidine phosphocarrier protein (HPr). HPr then transfers the phosphoryl group to the A domain of the sugar-specific enzymes II, which are divided into four structurally distinct families corresponding to the glucose, mannose, mannitol, and lactose/chitobiose branches of the PTS. All enzymes II have similar organizations comprising A and B cytoplasmic domains, and a membrane bound sugar transporter comprising the C domain, and sometimes a D domain as well. In some instances the domains are expressed as a contiguous protein, in others as separate proteins. From IIA, the phosphoryl group is transferred to IIB, and finally onto the incoming sugar molecule bound to the transmembrane IIC domain. Despite the similar domain organization of the enzymes II, the A and B cytoplasmic domains from the different branches of the PTS bear no sequence similarity to one another, and with the exception of IIB Mtl (7,8) and IIB Chb (9 -11), no similarity in either ternary or quaternary structures either.
Structures of the individual cytoplasmic components of the PTS have been solved by either NMR (7, 8, 10 -20) or crystal-lography (9,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). Structures of the cytoplasmic protein-protein complexes of the PTS, however, have been intractable to crystallography, presumably due to their weak affinity making successful co-crystallization difficult. Weak binding, however, is not an impediment to NMR, and we have solved the solution structures of all the cytoplasmic binary protein complexes of the PTS (15, 16, 18, 36 -43) with the exception of the IIA Chb -HPr complex. These complexes provide a wealth of information for understanding the unifying mechanism whereby a common interface, coupled with side chain conformational plasticity, can be used to recognize multiple, structurally dissimilar partners, and in addition, have yielded the first direct experimental evidence for the existence of highly transient, sparsely populated encounter complexes (44 -46).
In this paper we present the solution structure of the IIA Chb -HPr complex, the remaining outstanding cytoplasmic complex of the PTS, thereby completing our long term goal of solving all the cytoplasmic complexes of the PTS.

EXPERIMENTAL PROCEDURES
Protein Expression and Mutagenesis-Genes encoding IIA Chb * (corresponding to a N⌬13/D92L mutant of wild-type IIA Chb ) (20) and HPr (39,47) were cloned into the pET-11 vector. H89E and H15D mutations of the active site histidines of IIA Chb * and HPr, respectively, were introduced using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). (Residues of HPr are denoted in italics throughout.) Both mutations were designed to mimic the charge effect of phosphorylation of the active site histidines.
Structure Calculations-NOE-derived interproton distance restraints were classified into loose approximate distance ranges of 1.8 -2.7, 1.8 -3.5, 1.8 -5.0, and 1.8 -6.0 Å corresponding to strong, medium, weak, and very weak NOE cross-peak intensities, respectively (55). An empirical correction of 0.5 Å was added to the upper distance bounds of distance restraints involving methyl groups to account for the higher apparent intensity of methyl resonances (56), and NOEs involving nonstereospecifically assigned methyl, methylene, and aromatic protons were represented by a (Αr Ϫ6 ) Ϫ1/6 sum (57). Backbone torsion angle restraints for the active site region (residues 13-17) of HPr were derived from backbone 1 H/ 15 N/ 13 C chemical shifts using the program TALOSϩ (58) and used in the calculations of the phosphoryl transition state. The current experiments yielded interproton distance restraints and interfacial side chain torsion angle restraints.
Structures were calculated using conjoined rigid body/torsion angle-simulated annealing (59,60) with the program Xplor-NIH (61). The target function that is minimized comprises NOE-derived interproton distance restraints, torsion angle restraints, residual dipolar coupling restraints, 13 C␣/ 13 C␤ chemical shift restraints, a quartic van der Waals repulsion term for the nonbonded contacts, a multidimensional torsion angle data base potential of mean force (62), and a gyration volume potential to ensure optimal packing (63). Structure figures were generated using the programs VMD-XPLOR (64) and GRASP (65). Reweighted atomic probability density maps were calculated as described previously (66). The atomic coordinates and NMR experimental restraints (accession codes 2lrk and 2lrl for the unphosphorylated and phosphoryl transition state complexes, respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ.

RESULTS AND DISCUSSION
Equilibrium Binding of IIA Chb *(H89E) and HPr-At concentrations used in NMR experiments, wild type IIA Chb is highly prone to nonspecific aggregation promoted by the presence of a disordered 13-residue N-terminal tail and divalent cations required to neutralize and coordinate three symmetry-related, buried aspartate side chains (Asp-92) located at the center of the trimer interface (20). As in previous work (20,43), we therefore made use of the IIA Chb * construct throughout the current study. IIA Chb * forms a stable monodisperse trimer, and comprises a deletion of the disordered 13-residue N-terminal tail and mutation of the buried Asp-92 to Leu (20). Leu and Asp have similarly branched side chains, and the methyl groups of the three Leu-92 side chains, one from each subunit, substitute well packed hydrophobic methyl-methyl interactions at the trimer interface in place of the role fulfilled by the metal cation. These mutations do not affect phosphoryl transfer activity.
The interaction between IIA Chb * and HPr was assessed by monitoring 1 H N / 15 N chemical shift perturbations of 15 N-labeled HPr upon addition of unlabeled IIA Chb * (Fig. 1). Studies were carried out with HPr, IIA Chb *, IIA Chb *(H89E), and HPr(H15D). (Note that throughout the text, residues of HPr are printed in italics to distinguish them from residues of IIA Chb *.) The latter two mutations are designed to mimic the charge effects of phosphorylation of the active site histidines at the N⑀2 (H89E) and N␦1 (H15D) positions. At pH 7.4 and 20°C we could not detect any significant chemical shift perturbations upon addition of IIA Chb * to either HPr or HPr(H15D) at the concentrations employed (up to ϳ1.2 mM in subunits of IIA Chb * with ϳ0.4 mM HPr). However, measurable chemical shift perturbations were obtained upon addition of IIA Chb *(H89E) to HPr yielding a K D of 0.7 Ϯ 0.1 mM (Fig. 1). Therefore all structural studies were carried out with the IIA Chb *(H89E) phosphomimetic mutant. Given that three molecules of HPr can bind to the IIA Chb * symmetric trimer and binding is weak, all NMR experiments were carried on samples comprising 1 mM IIA Chb * (in trimer) and 1 mM HPr. Under these conditions, there is 24% free HPr and 42, 29, and 5%, singly, doubly and triply bound HPr; and 42% free IIA Chb *, and 42, 14, and 2% IIA Chb * with one, two, and three HPr molecules bound. Given molecular masses of ϳ34 and 9.5 kDa for free IIA Chb * and HPr, respectively, the population weighted average masses of IIA Chb * and HPr, which determine the line widths in the NMR experiments, are ϳ40 kDa each. Note that the existence of multiple bound states, as well as the presence of a significant fraction of free proteins, precludes the use of residual dipolar couplings for determining the relative orientation of the two proteins in the complex, because the apparent alignment tensor can no longer be deconvoluted into individual alignment tensors for each component in the system (43).
The structure of the IIA Chb *-HPr complex was largely based on intermolecular NOE data derived from three-dimensional 12 C-filtered/ 13 C-separated three-dimensional NOE experiments in which NOEs are exclusively observed between protons attached to 12 C and protons attached to 13 C. An array of different combinations of isotopically labeled samples, com-

Solution Structure of the IIA Chb -HPr Complex
prising both uniform and residue-specific labeling (Table 1), was employed to remove any ambiguities in assignment of intermolecular NOEs. An example of the quality of the intermolecular data is provided in Fig. 2. The calculation strategy used to determine the structure of the complex made use of conjoined rigid body/torsion angle dynamics simulated annealing (60). In this instance, the backbone and noninterfacial side chains of the 2.0-Å resolution x-ray coordinates of free HPr (30) were treated as a rigid body with rotational and translational degrees of freedom, whereas interfacial side chains were given torsional degrees of freedom. The only coordinates of free IIA Chb * available are NMR coordinates (20), which are inherently less accurate than x-ray coordinates (especially in terms of translation and packing). Thus for IIA Chb * full torsional, rotational, and translational degrees of freedom were allowed with the coordinates restrained by the experimental NMR restraints (NOEs, torsion angles, dipolar couplings) obtained for free IIA Chb * (20). This approach, rather than using the restrained regularized mean coordinates of free IIA Chb * (20) as a rigid body, was employed for the following reasons: the interface of both partners is largely helical and structurally rigid; the active site residue (H89E) is located within a deep cleft at the interface of adjacent subunits; and therefore small errors in the backbone coordinates of the free NMR structure of IIA Chb * can readily propagate and distort the docking of HPr onto IIA Chb *. The backbone coordinate shifts relative to the free IIA Chb * coordinates, however, are small (Ͻ1 Å) and well within the uncertainties of the NMR coordinates. In the case of the IIA Chb *-IIB Chb complex, on the other hand, the IIB Chb interaction site comprises a loop so that uncertainties in the IIA Chb * coordinates could be assimilated by simply giving the backbone of the active site loop of IIB Chb torsional degrees of freedom, while treating the remaining backbone of IIB Chb as well as the backbone of IIA Chb * (excluding the disordered loop from residues 75-84) as rigid bodies (43).
As in the case of the weak IIA Chb *-IIB Chb complex, a heuristic approach was employed for interfacial side chains since the samples comprised a mixture of free and bound states (43). Thus, the interfacial side chains were given torsion angle degrees of freedom within the 1 and where appropriate 2 rotamers of the free structures, unless contradicted by the intermolecular NOE data. A summary of the structural statistics is provided in Table 2, a best fit superposition of the final ensemble of 100 simulated annealing structures of the complex is displayed in Fig. 3A, and a reweighted atomic probability density map for some interfacial side chains is shown in Fig. 3C.
The Overall Structure of the IIA Chb *-HPr Complex-A ribbon diagram of the overall complex showing two and three molecules of HPr bound per trimer is displayed in Fig. 3B. Each HPr molecule interacts with two adjacent subunits of IIA Chb *: specifically subunits A and C, C and B, and B and A, where the first subunit in each pair contributes the active site residue at position 89. For the purposes of describing intermolecular contacts between HPr and IIA Chb *, we will restrict ourselves to the interaction surface formed at the interface of the A and C subunits of IIA Chb *.
Each subunit of IIA Chb * comprises 3 helices in an up, down, up topology comprising residues 17-43 (helix 1), 47-74 (helix 2), and 85-114 (helix 3) (20). HPr has three helices formed by residues 16 -28 (helix 1), 47-52 (helix 2), and 70 -83 (helix 3), as well as a four-stranded antiparallel ␤-sheet (30). The active site histidine at position 89, as well as His-93, of the A subunit of IIA Chb * are located deep within a cleft formed at the interface of subunits A and C (Figs. 3B and 4A), whereas the active site His-15 of HPr is exposed at the tip of a convex protrusion on the  13
The total accessible surface area buried upon complex formation is ϳ1580 Å 2 , comprising ϳ350 Å 2 and ϳ450 Å 2 for subunits A and C, respectively, of IIA Chb *, and ϳ780 Å 2 for HPr (subdivided into ϳ350 and ϳ430 Å 2 for contacts with the A and C subunits of IIA Chb *, respectively). The binding site on IIA Chb * for both subunits A and C comprises ϳ45% hydrophobic residues, with the remainder equally divided between polar and charged residues (Fig. 4A); for HPr, the portion of the binding surface that interacts with the A subunit of IIA Chb * is ϳ40% hydrophobic, with the remainder equally divided between polar and charged residues (Fig. 4B, left  half), while the portion of the HPr binding surface that interacts with the C subunit of IIA Chb * is composed of ϳ55% hydrophobic and ϳ45% uncharged polar residues (Fig. 4B,  right half). As in the other complexes of the PTS (15, 16, , and C subunits of the IIA Chb *(H89E) symmetric trimer in blue, gold, and green, respectively, and HPr in red. HPr is shown interacting with the A and C subunits of IIB Chb *. The purple meshes represent the atomic density probability maps (66) for the two active site residues, H89E of subunit A of IIA Chb *(H89E) and His-15 of HPr. (The probability maps are drawn at a value of 20% maximum.) Note that because IIA Chb *(H89E) is a symmetric trimer there are three identical binding sites formed at the interfaces between the A and C subunits, the C and B subunits, and the B and A subunits. B, ribbon diagrams of the complex showing two HPr molecules bound to the IIA Chb *(H89E) trimer (left panel) and an orthogonal view depicting three molecules of HPr bound to the IIA Chb *(H89E) trimer (right panel). The color coding is the same as in A. C, stereoview showing a reweighted atomic probability density map (drawn at a value of 20% maximum and calculated from the final 100 simulated annealing structures) for some of the interfacial side chains displayed as blue and green meshes for the A and C subunits for IIA Chb *(H89E) and as a red mesh for HPr. The backbones are shown as tubes color coded as in A; the side chains of the restrained regularized mean structure are color coded according to atom type (carbon, gray; oxygen, red; nitrogen, blue; sulfur, yellow). Residues of HPr are labeled in italics. are largely, but not exclusively, hydrophobic, interspersed by uncharged polar residues, whereas the outer edges are predominantly charged or polar (Fig. 4).
Detailed views of the side chain interactions between HPr and the A and C subunits of IIA Chb * are shown in Fig. 5, A and B, respectively, together with a schematic summary of the intermolecular contacts in Fig. 5C. In contrast, to EIN, IIA Glc , IIA Mtl , and IIA Man , where the charged residues in the binding site for HPr are largely negative, with very few positively charged residues (36 -39), the binding surface on IIA Chb * (Fig. 4A) contains an equal number of positively and negatively charged residues (three of each), of which two negative (Glu-19 and Glu-73) and two positive (Arg-58 and Lys-62) residues participate in intermolecular electrostatic interactions (Fig. 5). Indeed there are a quite number of potential hydrogen bonding, salt bridge, and longer range electrostatic interactions that serve to orient HPr and IIA Chb * correctly.
Thus, at the interface between HPr and the A subunit of IIA Chb *, the hydroxyl group of Thr-16 forms a hydrogen bond with the N⑀2 atom of His-93 A ; the guanidino group of Arg-17 forms potential salt bridges with the hydroxyl group of Ser-33 A and the side chain carbonyl of Gln-30 A , with the orientation of the side chain of Arg-17 further stabilized by an intramolecular interaction between its guanidino group and the side chain carbonyl of Gln-21; and Lys-24 and Lys-27 form potential salt bridge and longer range electrostatic interactions with the carboxylate of Glu-19 A (Fig. 5A). In addition, the carboxylate of H89E A is sufficiently close (Ͻ5 Å) to the hydroxyl group of Thr-16 to allow for an electrostatic interaction that may explain why the IIA Chb *(H89E)-HPr complex is of higher affinity than either the IIA Chb *-HPr or IIA Chb *-HPr(H15D) complexes.
At the interface of HPr and the C subunit of IIA Chb *, the side chain carbonyl of Gln-57 has electrostatic interactions with the guanidino group of Arg-58 C and the side chain amide group of Asn-62 C ; the side chain amide group of Asn-12 forms a potential hydrogen bond with the S␦ atom of Met-98 C ; the backbone amide of Leu-53 donates a potential hydrogen bond to the carboxylate of Glu-73 C ; and the carboxyamide group of Gln-51 forms potential hydrogen bonds with the carboxylate of Glu-73 C and the side chain amino group of Lys-82 C (Fig. 5B). Given that the interaction surfaces of HPr and IIA Chb * are complementary both in terms of shape and distribution of residue type, it is likely that many of the above intermolecular electrostatic interactions are rather weak and transient, thereby accounting for the high equilibrium dissociation constant (K D ϳ 0.7 mM; cf. Fig. 1) for the complex.
The Phosphoryl Transition State-It is known from isotope labeling experiments that the phosphoryl transition state in complexes of the PTS comprises a pentacoordinate phosphoryl group in a trigonal bipyramidal geometry, with donor and acceptor atoms at apical positions and the oxygen atoms of the phosphoryl group lying in an equatorial plane (67,68). The His-89 A (N⑀2)-P and His-15(N␦1)-P distances can lie anywhere between 1.8 and 3.5 Å corresponding to pure associative and pure dissociative transition states, respectively, and the phosphorus atom lies in the plane of the imidazole group of both active site histidines.
To model the transition state, we therefore carried out conjoined rigid body/torsion angle-simulated annealing calculations using exactly the same protocol and experimental restraints as those used for the unphosphorylated complex but with the addition of covalent geometry restraints for the pentacoordinate phosphoryl group and the introduction of backbone torsional degrees of freedom for residues 13-17 of HPr encompassing the active site. The overall backbone r.m.s. shift between the restrained regularized mean structures of the transition state and unphosphorylated complexes is 0.5 Å overall, and 0.3 Å for the interface (Fig. 6B), which is well within the errors of the NMR coordinates. In addition, there are only minor perturbations in side chain positions (Fig. 6B). Thus, one can conclude that the transition state can be readily accommodated without any significant perturbation in backbone conformation. Furthermore, agreement with the experimental restraints and indicators of structural quality are unaffected by the introduction of the phosphoryl transition state ( Table 1).
The phosphoryl group in the transition state is hydrogen bonded to the hydroxyl group of Thr-16 of HPr, the H⑀2 atom of His-93 A of subunit A of IIA Chb *, and the carboxyamide group of Gln-91 C of subunit C of IIA Chb * (Fig. 6A). As in other PTS complexes, the phosphoryl group is surrounded by a cluster of hydrophobic groups: Leu-47 and Phe-48 of HPr; Val-21 A , Ile-25 A , Val-86 A , and Leu-92 A of the A subunit of IIA Chb *; and Ile-72 C , Val-83 C , and Met-95 C , as well as the aliphatic portion of the side chain of Lys-82 C , of the C subunit of IIA Chb *.
Comparison with the IIA Chb *-IIB Chb Complex-HPr (30) and IIB Chb (9 -11) share no similarity in either overall structure or local structure surrounding the active site residue, His-15 in the case of HPr, and Cys-11 for IIB Chb . Yet both proteins bind to highly overlapping binding sites on IIA Chb * (this paper and Ref. 43). The interaction surfaces share 10 residues in common for subunit A and 9 for subunit C. The residues that are not shared by the two interaction surfaces are located at the peripheries of the binding sites. In the view shown in Fig. 4, the binding surface for HPr extends slightly upwards to include Ser-33 A of subunit A and Arg-58 C , Asn-62 C , and Met-98 C of subunit C, whereas the binding surface for IIB Chb extends slightly downwards to include Glu-15 A of subunit A, and Gly-74 C and Gly-77 C of subunit C (43). These small differences can be readily appreciated by the superposition of the two complexes shown in Fig. 7, and probably reflect two factors: first, the slightly larger size of the binding site on IIB Chb , which comprises 29 residues versus 19 for HPr; and second the slightly more peripheral location of His-15 relative to the interaction surface compared with Cys-11 of IIB Chb .
Although small, the above differences nicely illustrate the concept of redundancy in a system in which one partner, IIA Chb *, recognizes multiple partners, while making use of the same active site residue (His-89 A ) to effect phosphoryl transfer. Thus, the four additional residues at the top edge of the IIA Chb * binding surface for HPr that are not used in the interaction with IIB Chb , namely Ser-33 A , Arg-58 C , Asn-62 C , and Met-98 C (Fig.  4A), are all involved in potential hydrogen bonding and electrostatic interactions with HPr (Fig. 5, A and B) that contribute to correctly orienting HPr relative to IIA Chb *. The same is true of Glu-15 A , located at the bottom edge of the IIA Chb * binding surface for IIB Chb but absent from the interaction with HPr, which forms a salt bridge with Lys-86 of IIB Chb (43).
At the same time, side chain conformational plasticity allows side chains to participate in similar interactions (cf.  very similar to that with Tyr-84 of IIB Chb , except that the hydrophobic contacts between these two pairs of residues is supplemented by a potential hydrogen bond between the S␦ atom of Met-22 A and the hydroxyl group of Tyr-84. Concluding Remarks-The structure of the IIA Chb *-HPr complex in the present paper completes the structure elucida-tion of representative soluble complexes for all four sugar branches of the PTS (15, 16, 18, 36 -40, 43). This collection of structures provides a paradigm of protein recognition in signal transduction pathways that allows for multiple recognition partners, transient interactions, and specificity.
Although the structures of the IIA components of the four sugar branches bear no sequence or structural similarity to one another, their recognition surfaces for HPr are remarkably similar in shape and residue composition. Moreover, each enzyme IIA makes use of highly overlapping surfaces to recognize both its upstream partner HPr and its downstream partner, enzyme IIB (this paper and Refs. 15, 16, 37-40, and 43).
The ability to recognize multiple different partners relies on a number of design features. First, similar surfaces are constructed from completely different underlying structural elements. Thus, the shape of the binding surfaces on HPr and the four classes of enzymes IIB are convex in shape and similar in size. Likewise, all four classes of enzymes IIA have a concave binding surface of similar size. Second, all the surfaces generally share similar features comprising predominantly hydrophobic residues, interspersed by uncharged polar residues, at the center of the interface surrounded by polar and charged residues at the periphery. Third, the interactions surfaces are all large (600 -1000 Å 2 ), thereby allowing considerable redundancy in the intermolecular interactions that have to be formed to achieve appropriate docking and orientation of the phosphoryl transfer complexes. A corollary to large surfaces and redundancy of specific intermolecular interactions is that all the complexes are transient and weak ranging from K D values of ϳ10 M to the millimolar range (this paper and Refs. 15, 16, 36 -40, 42, and 43)). Fourth, conformational plasticity of amino acids with long side chains (such as Arg, Lys, and Glu) permit similar types of intermolecular interactions to occur across complexes involving one shared partner. Finally, although HPr uses the same binding surface to recognize enzyme I and all four classes of enzyme IIA, and the binding surfaces on enzymes IIA used to interact with HPr and the corresponding enzymes IIB are highly overlapping, the absence of any detectable interaction between enzyme I and any of the enzymes IIB arises through electrostatic selection. The binding surface on HPr contains no negative charges, and the charged residues on the binding surface of enzyme I are predominantly negative. In contrast, the binding surfaces on enzymes IIA and IIB comprise a mixture of positively and negatively charged residues that largely complement one another. Thus these charged residues are either involved in intermolecular salt bridges, hydrogen-bonding interactions, or participate in van der Waals contacts. Intermolecular electrostatic repulsion, however, between like-charged residues is avoided. The positively charged residues located in the binding surface of the enzymes IIA are accommodated by the binding surface of HPr, either by making use of their long side chains in hydrophobic contacts, or by electrostatic interactions with polar groups (e.g. in the case of the IIA Chb *-HPr complex, Arg-58 C and Lys-82 C of subunit C of IIA Chb * interact with the side chain carbonyls of Gln-57 and Gln-51 of HPr, respectively). A, environment surrounding the His-89 A -P-His-15 pentacoordinate phosphoryl transition state. The backbone is displayed as transparent tubes with HPr in red, and the A and C subunits of IIA Chb * in blue and green, respectively. B, identical view to A showing a superposition of the structure of the IIA Chb *(H89E)-HPr complex (transparent tubes and bonds) with the structure of the IIA Chb *-P-HPr transition state (opaque tubes and bonds). Exactly the same experimental restraints are used to calculate the two structures, but, in addition, the calculations for the transition state include geometric restraints specifying the geometry of the phosphoryl transition state and backbone torsion angle degrees of freedom for residues 13-17 of HPr encompassing the active side His-15. Color coding: red, HPr; blue, A subunit of IIA Chb *; green, C subunit of IIA Chb *. Side chains are displayed as stick diagrams with the atoms color coded according to type (carbon, gray; nitrogen, blue; oxygen, red; phosphorus, gold; sulfur, yellow). Residues of HPr are labeled in italics. Dashed black lines indicate hydrogen bonds to the phosphoryl group in the transition state, and the dashed gray line indicates a potential intermolecular hydrogen bond between the carboxyamide group of Asn-12 of HPr and the Met-98(S␦) atom of the C subunit of IIA Chb *. A, overall stereoview with IIA Chb * from the two complexes best-fitted to one another, and, B, close up of the His-P-His and His-P-Cys phosphoryl transition states for the IIA Chb *-HPr and IIA Chb *-IIB Chb complexes, respectively. The backbone is displayed as a ribbon diagram and the His-P-His and His-P-Cys transition states as stick diagrams with the atoms color coded according to type (carbon, gray; nitrogen, blue; oxygen, red; phosphorus, gold; sulfur, yellow). For the IIA Chb *-HPr complex, IIA Chb * and HPr are shown in red and blue, respectively; for the IIA Chb *-IIB Chb complex, IIA Chb * and IIB Chb are shown in gray and purple, respectively. The coordinates of the IIA Chb *-IIB Chb complex are taken from Ref. 43 (PDB code 2WWV). The small differences in the IIA Chb * coordinates from the two structures is within coordinate error. Also note that the region that displays the largest apparent differences is the loop from residues 77 to 84 of IIA Chb *, which is disordered in solution.