Peptidoglycan recognition proteins (PGRPs) are highly conserved pattern-recognition molecules of the innate immune system that bind bacterial peptidoglycans (PGNs), which are polymers of alternating ...N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) cross-linked by short peptide stems. Human PRGPs are bactericidal against pathogenic and nonpathogenic Gram-positive bacteria, but not normal flora bacteria. Like certain glycopeptide antibiotics (e.g., vancomycin), PGRPs kill bacteria by directly interacting with their cell wall PGN, thereby interfering with PGN maturation. To better understand the bactericidal mechanism of PGRPs, we determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-Iβ in complex with NAG-NAM-L-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala, a synthetic glycopeptide comprising a complete PGN repeat. This structure, in conjunction with the previously reported NMR structure of a dimeric PGN fragment, permitted identification of major conformational differences between free and PGRP-bound PGN with respect to the relative orientation of saccharide and peptide moieties. These differences provided structural insights into the bactericidal mechanism of human PGRPs. On the basis of molecular modeling, we propose that these proteins disrupt cell wall maturation not only by sterically encumbering access of biosynthetic enzymes to the nascent PGN chains, but also by locking PGN into a conformation that prevents formation of cross-links between peptide stems in the growing cell wall.
The innate immune system constitutes the first line of defense against microorganisms in both vertebrates and invertebrates. Although much progress has been made toward identifying key receptors and ...understanding their role in host defense, far less is known about how these receptors recognize microbial ligands. Such studies have been severely hampered by the need to purify ligands from microbial sources and a reliance on biological assays, rather than direct binding, to monitor recognition. We used synthetic peptidoglycan (PGN) derivatives, combined with microcalorimetry, to define the binding specificities of human and insect peptidogycan recognition proteins (PGRPs). We demonstrate that these innate immune receptors use dual strategies to distinguish between PGNs from different bacteria: one based on the composition of the PGN peptide stem and another that senses the peptide bridge crosslinking the stems. To pinpoint the site of PGRPs that mediates discrimination, we engineered structure-based variants having altered PGN-binding properties. The plasticity of the PGRPbinding site revealed by these mutants suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges.
Superantigens are bacterial or viral proteins that elicit massive T cell activation through simultaneous binding to major histocompatibility complex (MHC) class II and T cell receptors. This ...activation results in uncontrolled release of inflammatory cytokines, causing toxic shock. A remarkable property of superantigens, which distinguishes them from T cell receptors, is their ability to interact with multiple MHC class II alleles independently of MHC-bound peptide. Previous crystallographic studies have shown that staphylococcal and streptococcal superantigens belonging to the zinc family bind to a high affinity site on the class II β-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the β-chain is polymorphic and the MHC-bound peptide forms part of the binding interface. To understand how zinc-dependent superantigens recognize MHC, we determined the crystal structure, at 2.0Å resolution, of staphylococcal enterotoxin I bound to the human class II molecule HLA-DR1 bearing a peptide from influenza hemagglutinin. Interactions between the superantigen and DR1 β-chain are mediated by a zinc ion, and 22% of the buried surface of peptide·MHC is contributed by the peptide. Comparison of the staphylococcal enterotoxin I·peptide·DR1 structure with ones determined previously revealed that zinc-dependent superantigens achieve promiscuous binding to MHC by targeting conservatively substituted residues of the polymorphic β-chain. Additionally, these superantigens circumvent peptide specificity by engaging MHC-bound peptides at their conformationally conserved N-terminal regions while minimizing sequence-specific interactions with peptide residues to enhance cross-reactivity.
We report here, the first solution state evidence for the role of water molecules in the specific interaction of carbohydrates with a legume lectin, concanavalin A. Concanavalin A from Canavalia ...ensiformis is a protein containing 237 amino acid residues with each monomer possessing one sugar binding site as well as sites for transition-metal ions, Mn2+ and Ca2+. The lectin binds specifically to α-anomers of monosaccharides, d-glucopyranoside and d-mannopyranoside, and recognizes the trimannosidic core of N-linked glycoproteins, 3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside with high specificity, which constitutes the minimum carbohydrate epitope that completely fills the sugar binding site. Sensitive isothermal titration microcalorimetry coupled with osmotic stress strategy on concanavalin A was used to dissect out the differential involvement of water molecules in the recognition of the branched trimannoside (3,6-di-O-(α-d-mannopyranosyl)-α-d-mannopyranoside), the individual dimannosidic arms (3-O-(α-d-mannopyranosyl)-α-d-mannopyranoside and 6-O-(α-d-mannopyranosyl)-α-d-mannopyranoside) as well as the monomer unit, d-mannopyranoside. The specific binding of concanavalin A to different sugars, is accompanied by differential uptake of water molecules during the binding process. These results not only complement the X-ray crystallographic studies of legume lectin−sugar complexes displaying structurally conserved water molecules mediating the specific ligation of the sugars with the corresponding sites in the binding pocket but also provide a rationale for the observed compensatory behavior of enthalpies with entropies in lectin−sugar interactions.
Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind bacterial peptidoglycans (PGNs). We determined the crystal structure, to 2.1 Å ...resolution, of the C‐terminal PGN‐binding domain of human PGRP‐Iα in complex with a muramyl pentapeptide (MPP) from Gram‐positive bacteria containing a complete peptide stem (L‐Ala‐D‐isoGln‐L‐Lys‐D‐Ala‐D‐Ala). The structure reveals important features not observed previously in the complex between PGRP‐Iα and a muramyl tripeptide lacking D‐Ala at stem positions 4 and 5. Most notable are ligand‐induced structural rearrangements in the PGN‐binding site that are essential for entry of the C‐terminal portion of the peptide stem and for locking MPP in the binding groove. We propose that similar structural rearrangements to accommodate the PGN stem likely characterize many PGRPs, both mammalian and insect.
When two proteins associate they form a molecular interface that is a structural and energetic mosaic. Within such interfaces, individual amino acid residues contribute distinct binding energies to ...the complex. In combination, these energies are not necessarily additive, and significant positive or negative cooperative effects often exist. The basis of reliable algorithms to predict the specificities and energies of protein-protein interactions depends critically on a quantitative understanding of this cooperativity. We have used a model protein-protein system defined by an affinity maturation pathway, comprising variants of a T cell receptor Vβ domain that exhibit an overall affinity range of ∼1500-fold for binding to the superantigen staphylococcal enterotoxin C3, in order to dissect the cooperative and additive energetic contributions of residues within an interface. This molecular interaction has been well characterized previously both structurally, by x-ray crystallographic analysis, and energetically, by scanning alanine mutagenesis. Through analysis of group and individual maturation and reversion mutations using surface plasmon resonance spectroscopy, we have identified energetically important interfacial residues, determined their cooperative and additive energetic properties, and elucidated the kinetic and thermodynamic bases for molecular evolution in this system. The summation of the binding free energy changes associated with the individual mutations that define this affinity maturation pathway is greater than that of the fully matured variant, even though the affinity gap between the end point variants is relatively large. Two mutations in particular, both located in the complementarity determining region 2 loop of the Vβ domain, exhibit negative cooperativity.
Although protein-protein interactions are involved in nearly all cellular processes, general rules for describing affinity and selectivity in protein-protein complexes are lacking, primarily because ...correlations between changes in protein structure and binding energetics have not been well determined. Here, we establish the structural basis of affinity maturation for a protein-protein interaction system that we had previously characterized energetically. This model system exhibits a 1500-fold affinity increase. Also, its affinity maturation is restricted by negative intramolecular cooperativity. With three complex and six unliganded variant X-ray crystal structures, we provide molecular snapshots of protein interface remodeling events that span the breadth of the affinity maturation process and present a comprehensive structural view of affinity maturation. Correlating crystallographically observed structural changes with measured energetic changes reveals molecular bases for affinity maturation, intramolecular cooperativity, and context-dependent binding.
The transmembrane protein, linker for activation of T cells (LAT), is essential for T‐cell activation and development. Phosphorylation of LAT at multiple tyrosines creates binding sites for the ...adaptors Gads and Grb2, leading to nucleation of multiprotein signaling complexes. Human LAT contains five potential binding sites for Gads, of which only those at Tyr171 and Tyr191 appear necessary for T‐cell function. We asked whether Gads binds preferentially to these sites, as differential recognition could assist in assembling defined LAT‐based complexes. Measured calorimetrically, Gads‐SH2 binds LAT tyrosine phosphorylation sites 171 and 191 with higher affinities than the other sites, with the differences ranging from only several fold weaker binding to no detectable interaction. Crystal structures of Gads‐SH2 complexed with phosphopeptides representing sites 171, 191 and 226 were determined to 1.8−1.9 Å resolutions. The structures reveal the basis for preferential recognition of specific LAT sites by Gads, as well as for the relatively greater promiscuity of the related adaptor Grb2, whose binding also requires asparagine at position +2 C‐terminal to the phosphorylated tyrosine.
Energetically competent binary recognition of the cofactorS-adenosyl-l-methionine (AdoMet) and the product S-adenosyl-l-homocysteine (AdoHcy) by the DNA (cytosine C-5) methyltransferase (M.HhaI) is ...demonstrated herein. Titration calorimetry reveals a dual mode, involving a primary dominant exothermic reaction followed by a weaker endothermic one, for the recognition of AdoMet and AdoHcy by M.HhaI. Conservation of the bimodal recognition in W41I and W41Y mutants of M.HhaI excludes the cation−π interaction between the methylsulfonium group of AdoMet and the π face of the Trp41 indole ring from a role in its origin. Small magnitude of temperature-independent heat capacity changes upon AdoMet or AdoHcy binding by M.HhaI preclude appreciable conformational alterations in the reacting species. Coupled osmotic-calorimetric analyses of AdoMet and AdoHcy binding by M.HhaI indicate that a net uptake of nearly eight and 10 water molecules, respectively, assists their primary recognition. A change in water activity at constant temperature and pH is sufficient to engender and conserve enthalpy−entropy compensation, consistent with a true osmotic effect. The results implicate solvent reorganization in providing the major contribution to the origin of this isoequilibrium phenomenon in AdoMet and AdoHcy recognition by M.HhaI. The observations provide unequivocal evidence for the binding of AdoMet as well as AdoHcy to M.HhaI in solution state. Isotope partitioning analysis and preincubation studies favor a random mechanism for M.HhaI-catalyzed reaction. Taken together, the results clearly resolve the issue of cofactor recognition by free M.HhaI, specifically in the absence of DNA, leading to the formation of an energetically and catalytically competent binary complex.
The thermodynamics of a monoclonal antibody (mAb)-peptide interaction have been characterized by isothermal titration microcalorimetry. GCC:B10 mAb, generated against human guanylyl cyclase C, a ...membrane-associated receptor and a potential marker for metastatic colon cancer, recognizes the cognate peptide epitope HIPPENIFPLE and its two contiguous mimotopes, HIPPEN and ENIFPLE, specifically and reversibly. The exothermic binding reactions between 6.4 and 42 °C are driven by dominant favorable enthalpic contributions between 20 and 42 °C, with a large negative heat capacity (ΔCp) of −421 ± 27 cal mol−1 K−1. The unfavorable negative value of entropy (ΔSb0) at 25 °C, an unusual feature among protein-protein interactions, becomes a positive one below an inversion temperature of 20.5 °C. Enthalpy-entropy compensation due to solvent reorganization accounts for an essentially unchanged free energy of interaction (ΔΔGb0 ≅ 0). The role of water molecules in the recognition process was tested by coupling an osmotic stress technique with isothermal titration microcalorimetry. The results provide direct and compelling evidence that GCC :B10 mAb recognizes the peptides HIPPENIFPLE, HIPPEN, and ENIFPLE differentially, with a concomitant release of variable and nonadditive numbers of water molecules (15, 7, and 3, respectively) from the vicinity of the binding site.
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