Biomedical subjects

Rongjin Guan

Publications and source records attributed to Rongjin Guan.

12 recordsLinked to original sources

PubMed2006

Crystal structure of staphylococcal enterotoxin I (SEI) in complex with a human major histocompatibility complex class II molecule.

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 beta-chain. However, the basis for promiscuous MHC recognition by zinc-dependent superantigens is not obvious, because the beta-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 2.0 A 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 beta-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 beta-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.

Amino Acid Sequence↗

PubMed2006

Crystal structure of the murine cytomegalovirus MHC-I homolog m144.

Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy. The m144 glycoprotein, expressed by murine cytomegalovirus, is thought to be an MHC-I homolog whose expression prolongs viral survival in vivo by preventing natural killer cell activation. To explore the structural basis of this m144 function, we have determined the three-dimensional structure of an m144/beta2-microglobulin (beta2m) complex at 1.9A resolution. This structure reveals the canonical features of MHC-I molecules including readily identifiable alpha1, alpha2, and alpha3 domains. A unique disulfide bond links the alpha1 helix to the beta-sheet floor, explaining the known thermal stability of m144. Close juxtaposition of the alpha1 and alpha2 helices and the lack of critical residues that normally contribute to anchoring the peptide N and C termini eliminates peptide binding. A region of 13 amino acid residues, corresponding to the amino-terminal portion of the alpha2 helix, is missing in the electron density map, suggesting an area of structural flexibility that may be involved in ligand binding.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Dual strategies for peptidoglycan discrimination by peptidoglycan recognition proteins (PGRPs).

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 peptidoglycan 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 PGRP-binding site revealed by these mutants suggests an intrinsic capacity of the innate immune system to rapidly evolve specificities to meet new microbial challenges.

Animals↗

PubMed2006

Crystal structure of human peptidoglycan recognition protein I alpha bound to a muramyl pentapeptide from Gram-positive bacteria.

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 A resolution, of the C-terminal PGN-binding domain of human PGRP-I alpha 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-I alpha 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.

Carrier Proteins↗

PubMed2005

Selective recognition of synthetic lysine and meso-diaminopimelic acid-type peptidoglycan fragments by human peptidoglycan recognition proteins I{alpha} and S.

The interactions of a range of synthetic peptidoglycan derivatives with PGRP-Ialpha and PGRP-S have been studied in real-time using surface plasmon resonance. A dissociation constant of K(D) = 62 mum was obtained for the interaction of peptidoglycan recognition protein (PGRP)-Ialpha with the lysine-containing muramyl pentapeptide (compound 6). The normalized data for the lysine-containing muramyl tetra- (compound 5) and pentapeptide (compound 6) showed that these compounds have similar affinities, whereas a much lower affinity for muramyl tripeptide (compound 3) was measured. Similar affinities were obtained when the lysine moiety of the muramyl peptides was replaced by meso-diaminopimelic acid (DAP). Furthermore, the compounds that contained only a stem peptide (pentapeptide, compound 1) and (DAP-PP, compound 2) as well as muramyldipeptide (compound 3) exhibited no binding indicating that the muramyltripeptide (compound 4) is the smallest peptidoglycan fragment that can be recognized by PGRP-Ialpha. Surprisingly, PGRP-S derived significantly higher affinities for the DAP-containing fragments to similar lysine-containing derivatives, and the following dissociation constants were measured: muramylpentapeptide-DAP, K(D) = 104 nm; muramyltetrapeptide-DAP, 92.4 nm; and muramyltripeptide-DAP, 326 nm. The binding profiles were rationalized by using a recently reported x-ray crystal structure of PGRP-Ialpha with the lysine-containing muramyltripeptide (4).

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2005

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.

Amino Acid Sequence↗

PubMed2005

Structural basis of affinity maturation and intramolecular cooperativity in a protein-protein interaction.

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.

Amino Acid Substitution↗

PubMed2005

Crystal structure of a peptidoglycan recognition protein (PGRP) in complex with a muramyl tripeptide from Gram-positive bacteria.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyse, bacterial peptidoglycans (PGNs). We determined the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptide representing the conserved core of lysine-type PGNs. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end could accommodate N-acetylglucosamine. Both peptide and glycan moieties are essential for binding by PGRPs. Conservation of key PGN-contacting residues indicates that all PGRPs employ this basic PGN-binding mode. The structure identifies variable residues that likely mediate discrimination between lysine- and diaminopimelic acid-type PGNs. In addition, we propose a mechanism for PGN hydrolysis by Zn2+-containing catalytic PGRPs.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2004

Structural basis for peptidoglycan binding by peptidoglycan recognition proteins.

Peptidoglycan (PGN) recognition proteins (PGRPs) are pattern-recognition receptors of the innate immune system that bind and, in some cases, hydrolyze bacterial PGNs. We determined the crystal structure, at 2.30-A resolution, of the C-terminal PGN-binding domain of human PGRP-Ialpha in complex with a muramyl tripeptide representing the core of lysine-type PGNs from Gram-positive bacteria. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end can accommodate a linked N-acetylglucosamine. Although most interactions are with the peptide, the glycan moiety also seems to be essential for specific recognition by PGRPs. Conservation of key PGN-contacting residues shows that all PGRPs employ this basic PGN-binding mode. The structure pinpoints variable residues that likely mediate discrimination between lysine- and diaminopimelic acid-type PGNs. We also propose a mechanism for PGN hydrolysis by Zn(2+)-containing PGRPs.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed2004

Crystal structure of the C-terminal peptidoglycan-binding domain of human peptidoglycan recognition protein Ialpha.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind, and in some cases hydrolyze, peptidoglycans (PGNs) on bacterial cell walls. These molecules, which are highly conserved from insects to mammals, participate in host defense against both Gram-positive and Gram-negative bacteria. We report the crystal structure of the C-terminal PGN-binding domain of human PGRP-Ialpha in two oligomeric states, monomer and dimer, to resolutions of 2.80 and 1.65 A, respectively. In contrast to PGRPs with PGN-lytic amidase activity, no zinc ion is present in the PGN-binding site of human PGRP-Ialpha. The structure reveals that PGRPs exhibit extensive topological variability in a large hydrophobic groove, located opposite the PGN-binding site, which may recognize host effector proteins or microbial ligands other than PGN. We also show that full-length PGRP-Ialpha comprises two tandem PGN-binding domains. These domains differ at most potential PGN-contacting positions, implying different fine specificities. Dimerization of PGRP-Ialpha, which occurs through three-dimensional domain swapping, is mediated by specific binding of sodium ions to a flexible hinge loop, stabilizing the conformation found in the dimer. We further demonstrate sodium-dependent dimerization of PGRP-Ialpha in solution, suggesting a possible mechanism for modulating PGRP activity through the formation of multivalent adducts.

Amino Acid Sequence↗

PubMed2003

Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b).

The Ly49 family of natural killer (NK) receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I. Ly49 receptors show complex patterns of MHC class I cross-reactivity and, in certain cases, peptide selectivity. To investigate whether specificity differences result from topological differences in MHC class I engagement, we determined the structure of the peptide-selective receptor Ly49C in complex with H-2K(b). The Ly49C homodimer binds two MHC class I molecules in symmetrical way, a mode distinct from that of Ly49A, which binds MHC class I asymmetrically. Ly49C does not directly contact the MHC-bound peptide. In addition, MHC crosslinking by Ly49C was demonstrated in solution. We propose a dynamic model for Ly49-MHC class I interactions involving conformational changes in the receptor, whereby variations in Ly49 dimerization mediate different MHC-binding modes.

Animals↗

PubMed2003

Dissecting cooperative and additive binding energetics in the affinity maturation pathway of a protein-protein interface.

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 Vbeta domain that exhibit an overall affinity range of approximately 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 Vbeta domain, exhibit negative cooperativity.

Algorithms↗