Interaction of the bacterial division regulator MinE with lipid bicelles studied by NMR spectroscopy.

The bacterial MinE and MinD division regulatory proteins form a standing wave enabling MinC, which binds MinD, to inhibit FtsZ polymerization everywhere except at the midcell, thereby assuring correct positioning of the cytokinetic septum and even distribution of contents to daughter cells. The MinE dimer undergoes major structural rearrangements between a resting six-stranded state present in the cytoplasm, a membrane-bound state, and a four-stranded active state bound to MinD on the membrane, but it is unclear which MinE motifs interact with the membrane in these different states. Using NMR, we probe the structure and global dynamics of MinE bound to disc-shaped lipid bicelles. In the bicelle-bound state, helix α1 no longer sits on top of the six-stranded ß-sheet, losing any contact with the protein core, but interacts directly with the bicelle surface; the structure of the protein core remains unperturbed and also interacts with the bicelle surface via helix α2. Binding may involve a previously identified excited state of free MinE in which helix α1 is disordered, thereby allowing it to target the membrane surface. Helix α1 and the protein core undergo nanosecond rigid body motions of differing amplitudes in the plane of the bicelle surface. Global dynamics on the sub-millisecond time scale between a ground state and a sparsely populated excited state are also observed and may represent a very early intermediate on the transition path between the resting six-stranded and active four-stranded conformations. In summary, our results provide insights into MinE structural rearrangements important during bacterial cell division.

A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks.

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val 1H/13C methyl protonated proteins from 100 ml cultures in M9 ++ /D2O medium induced at high (OD600 ~ 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of 2H/15N/13C and 1H/13C labeled proteins, yields comparable quantities of protein from 100 ml cell culture to those obtained using a conventional 1 L culture with M9/D2O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-13C4; 3,4',4',4'-d4) and α-ketobutyric (13C4; 3,3-d2) acid precursors.

XIPP: multi-dimensional NMR analysis software.

Here we present the XIPP (eXtensible Interactive Peak Picker) NMR software for analyzing multidimensional NMR data of proteins, DNA, RNA and protein-nucleic acid complexes. XIPP organizes experiments into pre-defined studies and replaces our original PIPP software suite which is no longer supported. Default study types exist for backbone assignment, sidechain assignment, NOE assignment and several relaxation series experiments, used in solution NMR studies. XIPP is written in Java and Jython. The default study types are defined in Jython which can be modified and extended to create new types of studies.

A simple protocol for expression of isotope-labeled proteins in Escherichia coli grown in shaker flasks at high cell density.

Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria-Bertani (LB) medium at 37 °C. We recently showed that the E. coli linear growth phase can be extended to higher cell density when cells are cultured under less than optimal conditions such as in minimal medium and/or at lower temperatures. Maximizing the yield of protein per unit volume of culture is important for reducing the costs, especially when isotopically labeling is required. Here, we present a modified minimal medium and a simple protocol that can increase the protein yield up to fourfold in a pH-stabilized LB medium and up to sevenfold in a modified M9+ medium (M9++). When M9++ medium coupled with the high density (OD600 ~ 6) cell growth protocol are used to express uniformly 15N- or 15N/13C-labeled proteins, the amount of 15NH4Cl and 13C6-glucose for a given cell mass is reduced by 50% and ~ 65%, respectively, relative to the traditional low density (OD600 ~ 1) cell growth protocol with M9 medium; the inclusion of 0.1% LB in the minimal medium permits a reduction in the concentration of both the trace element solution and MgCl2, which can cause precipitation. Mass data indicate that inclusion of 0.1% LB does not significantly affect the isotope enrichment level.

Probing transient excited states of the bacterial cell division regulator MinE by relaxation dispersion NMR spectroscopy.

Bacterial MinD and MinE form a standing oscillatory wave which positions the cell division inhibitor MinC, that binds MinD, everywhere on the membrane except at the midpoint of the cell, ensuring midcell positioning of the cytokinetic septum. During this process MinE undergoes fold switching as it interacts with different partners. We explore the exchange dynamics between major and excited states of the MinE dimer in 3 forms using 15N relaxation dispersion NMR: the full-length protein (6-stranded ß-sheet sandwiched between 4 helices) representing the resting state; a 10-residue N-terminal deletion (Δ10) mimicking the membrane-binding competent state where the N-terminal helix is detached to interact with membrane; and N-terminal deletions of either 30 (Δ30) or 10 residues with an I24N mutation (Δ10/I24N), in which the ß1-strands at the dimer interface are extruded and available to bind MinD, leaving behind a 4-stranded ß-sheet. Full-length MinE samples 2 "excited" states: The first is similar to a full-length/Δ10 heterodimer; the second, also sampled by Δ10, is either similar to or well along the pathway toward the 4-stranded ß-sheet form. Both Δ30 and Δ10/I24N sample 2 excited species: The first may involve destabilization of the ß3- and ß3'-strands at the dimer interface; changes in the second are more extensive, involving further disruption of secondary structure, possibly representing an ensemble of states on the pathway toward restoration of the resting state. The quantitative information on MinE conformational dynamics involving these excited states is crucial for understanding the oscillation pattern self-organization by MinD-MinE interaction dynamics on the membrane.

A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks.

We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600 of up to 10. Inducing the cells with isopropyl ß-D-1-thiogalactopyranoside at an OD600 of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD600 between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H2O-based to D2O-based medium is also key for ensuring high levels of protein expression in D2O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H2O to LB in D2O to modified-M9 medium in D2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.

A Novel Chemotherapeutic Agent to Treat Tumors with DNA Mismatch Repair Deficiencies.

Impairing the division of cancer cells with genotoxic small molecules has been a primary goal to develop chemotherapeutic agents. However, DNA mismatch repair (MMR)-deficient cancer cells are resistant to most conventional chemotherapeutic agents. Here we have identified baicalein as a small molecule that selectively kills MutSα-deficient cancer cells. Baicalein binds preferentially to mismatched DNA and induces a DNA damage response in a MMR-dependent manner. In MutSα-proficient cells, baicalein binds to MutSα to dissociate CHK2 from MutSα leading to S-phase arrest and cell survival. In contrast, continued replication in the presence of baicalein in MutSα-deficient cells results in a high number of DNA double-strand breaks and ultimately leads to apoptosis. Consistently, baicalein specifically shrinks MutSα-deficient xenograft tumors and inhibits the growth of AOM-DSS-induced colon tumors in colon-specific MSH2 knockout mice. Collectively, baicalein offers the potential of an improved treatment option for patients with tumors with a DNA MMR deficiency. Cancer Res; 76(14); 4183-91. ©2016 AACR.

SGTA recognizes a noncanonical ubiquitin-like domain in the Bag6-Ubl4A-Trc35 complex to promote endoplasmic reticulum-associated degradation.

Elimination of aberrantly folded polypeptides from the endoplasmic reticulum (ER) by the ER-associated degradation (ERAD) system promotes cell survival under stress conditions. This quality control mechanism requires movement of misfolded proteins across the ER membrane for targeting to the cytosolic proteasome, a process facilitated by a "holdase" complex, consisting of Bag6 and the cofactors Ubl4A and Trc35. This multiprotein complex also participates in several other protein quality control processes. Here, we report SGTA as a component of the Bag6 system, which cooperates with Bag6 to channel dislocated ERAD substrates that are prone to aggregation. Using nuclear magnetic resonance spectroscopy and biochemical assays, we demonstrate that SGTA contains a noncanonical ubiquitin-like-binding domain that interacts specifically with an unconventional ubiquitin-like protein/domain in Ubl4A at least in part via electrostatics. This interaction helps recruit SGTA to Bag6, enhances substrate loading to Bag6, and thus prevents the formation of nondegradable protein aggregates in ERAD.

Solution structure of the IIAChitobiose-HPr complex of the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system.

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 IIA(Chb)-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 IIA(Chb) was mutated to Glu to mimic the phosphorylated state. IIA(Chb)(H89E) and HPr form a weak complex with a K(D) of ~0.7 mM. The interacting binding surfaces, concave for IIA(Chb) 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 IIA(Chb) formed at the interface of two adjacent subunits of the IIA(Chb) trimer, thereby coming into close proximity with the active site residue, H89E, of IIA(Chb). 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 IIA(Chb)-HPr complex with the IIA(Chb)-IIB(Chb) 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.

Structure of the Plasmodium 6-cysteine s48/45 domain.

The s48/45 domain was first noted in Plasmodium proteins more than 15 y ago. Previously believed to be unique to Plasmodium, the s48/45 domain is present in other aconoidasidans. In Plasmodium, members of the s48/45 family of proteins are localized on the surface of the parasite in different stages, mostly by glycosylphosphatydylinositol-anchoring. Members such as P52 and P36 seem to play a role in invasion of hepatocytes, and Pfs230 and Pfs48/45 are involved in fertilization in the sexual stages and have been consistently studied as targets of transmission-blocking vaccines for years. In this report, we present the molecular structure for the s48/45 domain corresponding to the C-terminal domain of the blood-stage protein Pf12 from Plasmodium falciparum, obtained by NMR. Our results indicate that this domain is a ß-sandwich formed by two sheets with a mixture of parallel and antiparallel strands. Of the six conserved cysteines, two pairs link the ß-sheets by two disulfide bonds, and the third pair forms a bond outside the core. The structure of the s48/45 domain conforms well to the previously defined surface antigen 1 (SAG1)-related-sequence (SRS) fold observed in the SAG family of surface antigens found in Toxoplasma gondii. Despite extreme sequence divergence, remarkable spatial conservation of one of the disulfide bonds is observed, supporting the hypothesis that the domains have evolved from a common ancestor. Furthermore, a homologous domain is present in ephrins, raising the possibility that the precursor of the s48/45 and SRS domains emerged from an ancient transfer to Apicomplexa from metazoan hosts.

No interaction of barrier-to-autointegration factor (BAF) with HIV-1 MA, cone-rod homeobox (Crx) or MAN1-C in absence of DNA.

Barrier-to-autointegration factor is a cellular protein that protects retroviral DNA from autointegration. Its cellular role is not well understood, but genetic studies show that it is essential and depletion or knockout results in lethal nuclear defects. In addition to binding DNA, BAF interacts with the LEM domain, a domain shared among a family of lamin-associated polypeptides. BAF has also been reported to interact with several other viral and cellular proteins suggesting that these interactions may be functionally relevant. We find that, contrary to previous reports, BAF does not interact with HIV-1 MA, cone-rod homeobox (Crx) or MAN1-C. The reported interactions can be explained by indirect association through DNA binding and are unlikely to be biologically relevant. A mutation that causes a premature aging syndrome lies on the previously reported MAN1-C binding surface of BAF. The absence of direct binding of BAF to MAN1-C eliminates disruption of this interaction as the cause of the premature aging phenotype.

Structural basis of the association of HIV-1 matrix protein with DNA.

HIV-1 matrix (MA) is a multifunctional protein that is synthesized as a polyprotein that is cleaved by protease during viral maturation. MA contains a cluster of basic residues whose role is controversial. Proposed functions include membrane anchoring, facilitating viral assembly, and directing nuclear import of the viral DNA. Since MA has been reported to be a component of the preintegration complex (PIC), we have used NMR to probe its interaction with other PIC components. We show that MA interacts with DNA and this is likely sufficient to account for its association with the PIC.

Solution structure of the IIAChitobiose-IIBChitobiose complex of the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system.

The solution structure of the IIA-IIB complex of the N,N'-diacetylchitobiose (Chb) transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state and the active site Cys-10 of IIB(Chb) was substituted by serine to prevent intermolecular disulfide bond formation. Binding is weak with a K(D) of approximately 1.3 mm. The two complementary interaction surfaces are largely hydrophobic, with the protruding active site loop (residues 9-16) of IIB(Chb) buried deep within the active site cleft formed at the interface of two adjacent subunits of the IIA(Chb) trimer. The central hydrophobic portion of the interface is surrounded by a ring of polar and charged residues that provide a relatively small number of electrostatic intermolecular interactions that serve to correctly align the two proteins. The conformation of the active site loop in unphosphorylated IIB(Chb) is inconsistent with the formation of a phosphoryl transition state intermediate because of steric hindrance, especially from the methyl group of Ala-12 of IIB(Chb). Phosphorylation of IIB(Chb) is accompanied by a conformational change within the active site loop such that its path from residues 11-13 follows a mirror-like image relative to that in the unphosphorylated state. This involves a transition of the phi/psi angles of Gly-13 from the right to left alpha-helical region, as well as smaller changes in the backbone torsion angles of Ala-12 and Met-14. The resulting active site conformation is fully compatible with the formation of the His-89-P-Cys-10 phosphoryl transition state without necessitating any change in relative translation or orientation of the two proteins within the complex.

Barrier-to-autointegration factor (BAF) condenses DNA by looping.

Barrier-to-autointegration factor (BAF) is a protein that has been proposed to compact retroviral DNA, making it inaccessible as a target for self-destructive integration into itself (autointegration). BAF also plays an important role in nuclear organization. We studied the mechanism of DNA condensation by BAF using total internal reflection fluorescence microscopy. We found that BAF compacts DNA by a looping mechanism. Dissociation of BAF from DNA occurs with multiphasic kinetics; an initial fast phase is followed by a much slower dissociation phase. The mechanistic basis of the broad timescale of dissociation is discussed. This behavior mimics the dissociation of BAF from retroviral DNA within preintegration complexes as monitored by functional assays. Thus the DNA binding properties of BAF may alone be sufficient to account for its association with the preintegration complex.

Unprecedented glycosidase activity at a lectin carbohydrate-binding site exemplified by the cyanobacterial lectin MVL.

Carbohydrate binding proteins, or lectins, are engendered with the ability to bind specific carbohydrate structures, thereby mediating cell-cell and cell-pathogen interactions. Lectins are distinct from carbohydrate modifying enzymes and antibodies, respectively, as they do not carry out glycosidase or glycosyl transferase reactions, and they are of nonimmune origin. Cyanobacterial and algal lectins have become prominent in recent years due to their unique biophysical traits, such as exhibiting novel protein folds and unusually high carbohydrate affinity, and ability to potently inhibit HIV-1 entry through high affinity carbohydrate-mediated interactions with the HIV envelope glycoprotein gp120. The antiviral cyanobacterial lectin Microcystis viridis lectin (MVL), which contains two high affinity oligomannose binding sites, is one such example. Here we used glycan microarray profiling, NMR spectroscopy, and mutagenesis to show that one of the two oligomannose binding sites of MVL can catalyze the cleavage of chitin fragments (such as chitotriose) to GlcNAc, to determine the mode of MVL binding to and cleavage of chitotriose, to identify Asp75 as the primary catalytic residue involved in this cleavage, and to solve the solution structure of an inactive mutant of MVL in complex with this unexpected substrate. These studies represent the first demonstration of dual catalytic activity and carbohydrate recognition for discrete oligosaccharides at the same carbohydrate-binding site in a lectin. Sequence comparisons between the N- and C-domains of MVL, together with the sequences of new MVL homologues identified through bioinformatics, provide insight into the evolving roles of carbohydrate recognition.

Impact of phosphorylation on structure and thermodynamics of the interaction between the N-terminal domain of enzyme I and the histidine phosphocarrier protein of the bacterial phosphotransferase system.

The structural and thermodynamic impact of phosphorylation on the interaction of the N-terminal domain of enzyme I (EIN) and the histidine phosphocarrier protein (HPr), the two common components of all branches of the bacterial phosphotransferase system, have been examined using NMR spectroscopy and isothermal titration calorimetry. His-189 is located at the interface of the alpha and alphabeta domains of EIN, resulting in rather widespread chemical shift perturbation upon phosphorylation, in contrast to the highly localized perturbations seen for HPr, where His-15 is fully exposed to solvent. Residual dipolar coupling measurements, however, demonstrate unambiguously that no significant changes in backbone conformation of either protein occur upon phosphorylation: for EIN, the relative orientation of the alpha and alphabeta domains remains unchanged; for HPr, the backbone /Psi torsion angles of the active site residues are unperturbed within experimental error. His --> Glu/Asp mutations of the active site histidines designed to mimic the phosphorylated states reveal binding equilibria that favor phosphoryl transfer from EIN to HPr. Although binding of phospho-EIN to phospho-HPr is reduced by a factor of approximately 21 relative to the unphosphorylated complex, residual dipolar coupling measurements reveal that the structures of the unphosphorylated and biphosphorylated complexes are the same. Hence, the phosphorylation states of EIN and HPr shift the binding equilibria predominantly by modulating intermolecular electrostatic interactions without altering either the backbone scaffold or binding interface. This facilitates highly efficient phosphoryl transfer between EIN and HPr, which is estimated to occur at a rate of approximately 850 s(-1) from exchange spectroscopy.

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 (IIA(Man)) and B (IIB(Man)) domains of the cytoplasmic component of the mannose transporter of Escherichia coli have been solved by NMR. The complex of wild-type IIA(Man) and IIB(Man) 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 IIA(Man) and His-175 of IIB(Man), separated by approximately 25A. Mutation of the active site histidine, His-10, of IIA(Man) 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 IIA(Man) to IIB(Man) are approximately the same (K(D) approximately 0.5 mM). The productive complex can readily accommodate a transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the Nepsilon2 atom of His-10 of IIA(Man) and the Ndelta1 atom of His-175 of IIB(Man) with negligible (<0.2A) local backbone conformational changes in the immediate vicinity of the active site. The non-productive complex is related to the productive one by a approximately 90 degrees rotation and approximately 37A translation of IIB(Man) relative to IIA(Man), leaving the active site His-175 of IIB(Man) fully exposed to solvent in the non-productive complex. The interaction surface on IIA(Man) for the non-productive complex comprises a subset of residues used in the productive complex and in both cases involves both subunits of IIA(Man). The selection of the productive complex by IIA(Man)(H10E) can be attributed to neutralization of the positively charged Arg-172 of IIB(Man) at the center of the interface. The non-productive IIA(Man)-IIB(Man) complex may possibly be relevant to subsequent phosphoryl transfer from His-175 of IIB(Man) to the incoming sugar located on the transmembrane IIC(Man)-IID(Man) complex.

From bacterial genomes to novel antibacterial agents: discovery, characterization, and antibacterial activity of compounds that bind to HI0065 (YjeE) from Haemophilus influenzae.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.

Solution NMR structure of the barrier-to-autointegration factor-Emerin complex.

The barrier-to-autointegration factor BAF binds to the LEM domain (Em(LEM)) of the nuclear envelope protein emerin and plays an essential role in the nuclear architecture of metazoan cells. In addition, the BAF(2) dimer bridges and compacts double-stranded DNA nonspecifically via two symmetry-related DNA binding sites. In this article we present biophysical and structural studies on a complex of BAF(2) and Em(LEM). Light scattering, analytical ultracentrifugation, and NMR indicate a stoichiometry of one molecule of Em(LEM) bound per BAF(2) dimer. The equilibrium dissociation constant (K(d)) for the interaction of the BAF(2) dimer and Em(LEM), determined by isothermal titration calorimetry, is 0.59 +/- 0.03 microm. Z-exchange spectroscopy between corresponding cross-peaks of the magnetically non-equivalent subunits of the BAF(2) dimer in the complex yields a dissociation rate constant of 78 +/- 2s(-1). The solution NMR structure of the BAF(2)-Em(LEM) complex reveals that the LEM and DNA binding sites on BAF(2) are non-overlapping and that both subunits of the BAF(2) dimer contribute approximately equally to the Em(LEM) binding site. The relevance of the implications of the structural and biophysical data on the complex in the context of the interaction between the BAF(2) dimer and Em(LEM) at the nuclear envelope is discussed.

Solution structure of a post-transition state analog of the phosphotransfer reaction between the A and B cytoplasmic domains of the mannitol transporter IIMannitol of the Escherichia coli phosphotransferase system.

The solution structure of the post-transition state complex between the isolated cytoplasmic A (IIAMtl) and phosphorylated B (phospho-IIBMtl) domains of the mannitol transporter of the Escherichia coli phosphotransferase system has been solved by NMR. The active site His-554 of IIAMtl was mutated to glutamine to block phosphoryl transfer activity, and the active site Cys-384 of IIBMtl (residues of IIBMtl are denoted in italic type) was substituted by serine to permit the formation of a stable phosphorylated form of IIBMtl. The two complementary interaction surfaces are predominantly hydrophobic, and two methionines on IIBMtl, Met-388 and Met-393, serve as anchors by interacting with two deep pockets on the surface of IIAMtl. With the exception of a salt bridge between the conserved Arg-538 of IIAMtl and the phosphoryl group of phospho-IIBMtl, electrostatic interactions between the two proteins are limited to the outer edges of the interface, are few in number, and appear to be weak. This accounts for the low affinity of the complex (Kd approximately 3.7 mm), which is optimally tuned to the intact biological system in which the A and B domains are expressed as a single polypeptide connected by a flexible 21-residue linker. The phosphoryl transition state can readily be modeled with no change in protein-protein orientation and minimal perturbations in both the backbone immediately adjacent to His-554 and Cys-384 and the side chains in close proximity to the phosphoryl group. Comparison with the previously solved structure of the IIAMtl-HPr complex reveals how IIAMtl uses the same interaction surface to recognize two structurally unrelated proteins and explains the much higher affinity of IIAMtl for HPr than IIBMtl.