PapG is the adhesin at the tip of the P pilus that mediates attachment of uropathogenic Escherichia coli to the uroepithelium of the human kidney. The human specific allele of PapG binds to globoside (GbO4), which consists of the tetrasaccharide GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc linked to ceramide. Here, we present the crystal structures of a binary complex of the PapG receptor binding domain bound to GbO4 as well as the unbound form of the adhesin. The biological importance of each of the residues involved in binding was investigated by site-directed mutagenesis. These studies provide a molecular snapshot of a host-pathogen interaction that determines the tropism of uropathogenic E. coli for the human kidney and is critical to the pathogenesis of pyelonephritis.

In addition to its procoagulant and anticoagulant roles in the blood coagulation cascade, thrombin works as a signaling molecule when it interacts with the G-protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are almost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90-fold) specificity of PAR1 relative to PAR3 and PAR4. The most critical residues for the interaction with the PARs are located around the active site where mutations affect recognition in the order PAR4 > PAR3 > PAR1. Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of effects, the mutation W215A selectively compromises PAR4 cleavage, whereas the mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 and PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies.

Residue 225 in serine proteases of the chymotrypsin family is Pro or Tyr in more than 95% of nearly 300 available sequences. Proteases with Y225 (like some blood coagulation and complement factors) are almost exclusively found in vertebrates, whereas proteases with P225 (like degradative enzymes) are present from bacteria to human. Saturation mutagenesis of Y225 in thrombin shows that residue 225 affects ligand recognition up to 60,000-fold. With the exception of Tyr and Phe, all residues are associated with comparable or greatly reduced catalytic activity relative to Pro. The crystal structures of three mutants that differ widely in catalytic activity (Y225F, Y225P, and Y225I) show that although residue 225 makes no contact with substrate, it drastically inf luences the shape of the water channel around the primary specificity site. The activity profiles obtained for thrombin also suggest that the conversion of Pro to Tyr or Phe documented in the vertebrates occurred through Ser and was driven by a significant gain (up to 50-fold) in catalytic activity. In fact, Ser and Phe are documented in 4% of serine proteases, which together with Pro and Tyr account for almost the entire distribution of residues at position 225. The unexpected crucial role of residue 225 in serine proteases explains the evolutionary selection of residues at this position and shows that the structural determinants of protease activity and specificity are more complex than currently believed. These findings have broad implications in the rational design of enzymes with enhanced catalytic properties.

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Inwardly rectifying K؉ currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteineinteracting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K ATP channels. The ability of Cd 2؉ ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd 2؉ accessibility is consistent with an ␣-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. Potassium channels are tetrameric assemblies of subunits, each of which contains a conserved extracellular hydrophobic P-loop, flanked by at least two transmembrane domains. The P-loop and the following transmembrane domain (S6 or M2) determine pore properties (1-12). In the crystal structure of the core region of KcsA (a two-transmembrane domain bacterial K ϩ channel), the P-loop forms the outer ion selectivity filter, and ␣-helical M2 lines the long inner vestibule between the selectivity filter and the cytoplasm (13). No direct structural information is available for mammalian K ϩ channels. However, pore structure has been inferred by use of the substituted cysteine accessibility method using silver (14), cadmium (15, 16), or organic sulfhydryl reagents (17, 18) as cysteine-reactive probes. The study of Liu et al. (18) indicates no obvious structure of S6 in voltage-gated K ϩ (Kv) channels, and recent systematic mutageneses of M2 in Kir2.1 (IRK1) (19 -21) question the general applicability of the KcsA crystal structure to the structure of mammalian K ϩ channels. K ATP channels are tetramers of Kir6.2 subunits, and hallmark nucleotide inhibition results from an interaction of ATP with cytoplasmic regions of Kir6.2 (22-25). Each Kir6.2 subunit is normally associated with a sulfonylurea receptor (SUR1 or SUR2) (22, 26 -28), which provides additional nucleotide and drug sensitivities (29 -32). The similarity of Kir6.2 to other inwardly rectifying K ϩ (Kir) channels, the ability to control gating by nucleotides, and a relatively large single channel conductance (ϳ80 picosiemens) make this an attractive model system in which to study the pore structure and dynamics of Kir channels. We introduced cysteines throughout M2 of Kir6.2 and examined their accessibility to Cd 2ϩ ions. Our results are consistent with M2 being ␣-helical and structurally and dynamically similar to M2 of the bacterial KcsA channel (13, 33). EXPERIMENTAL PROCEDURES Expression of K ATP Channels in COSm6Cells-COSm6 cells were transfected with pCMV6b-Kir6.2 (with mutations as described), pECE-SUR1, and pGreenLantern (Life Technologies, Inc.) as described previously (24).Generation of a Non-reactive Background and Introduction of Cysteines-Point mutations were prepared by overlap extension at the junctions of relevant residues by sequential polymerase chain react...

Isothermal titration calorimetry and X-ray crystallography have been used to determine the structural and thermodynamic consequences associated with constraining the pTyr residue of the pYEEI ligand for the Src Homology 2 domain of the Src kinase (Src SH2 domain). The conformationally constrained peptide mimics that were used are cyclopropane-derived isosteres whereby a cyclopropane ring substitutes to the N-Calpha-Cbeta atoms of the phosphotyrosine. Comparison of the thermodynamic data for the binding of the conformationally constrained peptide mimics relative to their equivalent flexible analogues as well as a native tetrapeptide revealed an entropic advantage of 5-9 cal mol(-1) K(-1) for the binding of the conformationally constrained ligands. However, an unexpected drop in enthalpy for the binding of the conformationally constrained ligands relative to their flexible analogues was also observed. To evaluate whether these differences reflected conformational variations in peptide binding modes, we have determined the crystal structure of a complex of the Src SH2 domain bound to one of the conformationally constrained peptide mimics. Comparison of this new structure with that of the Src SH2 domain bound to a natural 11-mer peptide (Waksman et al. Cell 1993, 72, 779-790) revealed only very small differences. Hence, cyclopropane-derived peptides are excellent mimics of the bound state of their flexible analogues. However, a rigorous analysis of the structures and of the surface areas at the binding interface, and subsequent computational derivation of the energetic binding parameters, failed to predict the observed differences between the binding thermodynamics of the rigidified and flexible ligands, suggesting that the drop in enthalpy observed with the conformationally constrained peptide mimic arises from sources other than changes in buried surface areas, though the exact origin of the differences remains unclear.

The adenylate cyclase (CyaA) secreted by Bordetella pertussis is a toxin that is able to enter eukaryotic cells and cause a dramatic increase in cAMP level. In addition, the toxin also exhibits an intrinsic hemolytic activity that is independent from the ATP cycling catalytic activity of the toxin. Both the cytotoxic and hemolytic activities are calcium-dependent. In this work, we have analyzed the calcium interacting properties of CyaA. We have shown that CyaA exposed to CaCl 2 could retain membrane binding capability and hemolytic activity when it was further assayed in the presence of an excess of EGTA. Determination of the calcium content of CyaA exposed first to calcium and subsequently to EGTA indicated that some (3-5) calcium ions remained bound to the protein, suggesting the existence of Ca

Nanofabrication by molecular self-assembly involves the design of molecules and self-assembly strategies so that shape and chemical complementarities drive the units to organize spontaneously into the desired structures. The power of self-assembly makes it the ubiquitous strategy of living organized matter and provides a powerful tool to chemists. However, a challenging issue in the self-assembly of complex supramolecular structures is to understand how kinetically efficient pathways emerge from the multitude of possible transition states and routes. Unfortunately, very few systems provide an intelligible structure and formation mechanism on which new models can be developed. Here, we elucidate the molecular and supramolecular self-assembly mechanism of synthetic octapeptide into nanotubes in equilibrium conditions. Their complex hierarchical self-assembly has recently been described at the mesoscopic level, and we show now that this system uniquely exhibits three assembly stages and three intermediates: (i) a peptide dimer is evidenced by both analytical centrifugation and NMR translational diffusion experiments; (ii) an open ribbon and (iii) an unstable helical ribbon are both visualized by transmission electron microscopy and characterized by small angle X-ray scattering. Interestingly, the structural features of two stable intermediates are related to the final nanotube organization as they set, respectively, the nanotube wall thickness and the final wall curvature radius. We propose that a specific self-assembly pathway is selected by the existence of such preorganized and stable intermediates so that a unique final molecular organization is kinetically favored. Our findings suggests that the rational design of oligopeptides can encode both molecular- and macro-scale morphological characteristics of their higher-order assemblies, thus opening the way to ultrahigh resolution peptide scaffold engineering.