Physicochemical binding properties of the proteoglycan receptor for serum lipoproteins.

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic polysugar side chains are stretched out into the blood substitute solution representing a co-receptor for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that oxLDL had a deleterious effect on heparan sulfate proteoglycan binding and conformation. Ca2+ binding to and storage on the proteoheparan sulfate/LDL compound formed a 'heterotrimeric' HS-PG/LDL/Ca2+ complex of high stability, aggregability and deposit coating. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan/lipoprotein complex.     

Unraveling principles of lead discovery: from unfrustrated energy landscapes to novel molecular anchors.

The search for novel leads is a critical step in the drug discovery process. Computational approaches to identify new lead molecules have focused on discovering complete ligands by evaluating the binding affinity of a large number of candidates, a task of considerable complexity. A new computational method is introduced in this work based on the premise that the primary molecular recognition event in the protein binding site may be accomplished by small core fragments that serve as molecular anchors, providing a structurally stable platform that can be subsequently tailored into complete ligands. To fulfill its role, we show that an effective molecular anchor must meet both the thermodynamic requirement of relative energetic stability of a single binding mode and its consistent kinetic accessibility, which may be measured by the structural consensus of multiple docking simulations. From a large number of candidates, this technique is able to identify known core fragments responsible for primary recognition by the FK506 binding protein (FKBP-12), along with a diverse repertoire of novel molecular cores. By contrast, absolute energetic criteria for selecting molecular anchors are found to be promiscuous. A relationship between a minimum frustration principle of binding energy landscapes and receptor-specific molecular anchors in their role as "recognition nuclei" is established, thereby unraveling a mechanism of lead discovery and providing a practical route to receptor-biased computational combinatorial chemistry.     

Crystal structure of the major histocompatibility complex class I H-2Kb molecule containing a single viral peptide: implications for peptide binding and T-cell receptor recognition.

To study the structure of a homogenous major histocompatibility complex (MHC) class I molecule containing a single bound peptide, a complex of recombinant mouse H-2Kb, beta 2-microglobulin (beta 2m), and a fragment of the vesicular stomatitis virus (VSV) nuclear capsid protein, VSV-(N52-59) octapeptide (Arg-Gly-Tyr-Val-Tyr-Gln-Gly-Leu), was prepared by exploiting a high-yield bacterial expression system and in vitro cocomplex formation. The structure of mouse H-2Kb revealed its similarity to three human class I HLA molecules, consistent with the high primary sequence homology and common function of these peptide-presenting molecules. Electron density was located in the peptide-binding groove, to which a single peptide in a unique conformation was unambiguously fit. The peptide extends the length of the groove, parallel to the alpha-helices, and assumes an extended, mostly beta-strand conformation. The peptide is constrained within the groove by hydrogen bonding of its main-chain atoms and by contacts of its side chains with the H-2Kb molecule. The amino-terminal nitrogen atom of the peptide forms a hydrogen bond with the hydroxyl group of Tyr-171 of H-2Kb at one end of the groove, while the carboxyl-terminal oxygen forms a hydrogen bond with the hydroxyl group of Tyr-84 at the other end. Since the amino acids at both ends are conserved among human and mouse MHC molecules, this anchoring of each end of the peptide appears to be a general feature of peptide-MHC class I molecule binding and imposes restrictions on its length. The side chains of residues Tyr-3, Tyr-5, and Leu-8 of the VSV octapeptide fit into the interior of the H-2Kb molecule with no appreciable surface exposure, a finding in support of previous biological studies that showed the importance of these residues for binding. Thus, the basis for binding of specific peptide sequences to the MHC class I molecule is the steric restriction imposed on the peptide side chains by the architecture of the floor and sides of the groove. The side chains of Arg-1, Val-4, and Gln-6 and the main-chain of Gly-7 of the octapeptide are exposed on the surface of the complex, thus confirming their availability for T-cell receptor contact, as previously demonstrated by T-cell recognition experiments.     

The lack of consensus for I-A(g7)-peptide binding motifs: is there a requirement for anchor amino acid side chains?

We discuss here the problems in identifying sequence motifs of peptides that bind to I-A(g7), the class II histocompatibility molecule of NOD diabetic mice. We present studies that indicate a minor contribution of amino acid side chains for binding. A peptide from the Ealpha chain binds to I-A(g7) molecules and is recognized by CD4 T cells. By producing single-residue mutations we identified four residues that were considered to contact the T cell receptor. No residue was found to be essential for binding to I-A(g7): a peptide that contained the T cell contact residues, on a backbone of alanines, bound to I-A(g7) and stimulated the T cells. We conclude that peptides can bind to I-A(g7) without the requirement for residues with prominent side chains to anchor them.     

A peptide motif that recognizes A.T tracts in DNA.

The DAT1 gene of Saccharomyces cerevisiae encodes a DNA binding protein that specifically interacts with nonalternating oligo(A).oligo(T) tracts (A.T tracts). Deletion analysis of DAT1 coding information showed that the amino-terminal 36 residues are sufficient for specific DNA binding activity. Furthermore, a 35-residue synthetic peptide corresponding to amino acids 2-36 bound to A.T tracts with an equilibrium dissociation constant of 4 x 10(-10) M. Within this region the pentad Gly-Arg-Lys-Pro-Gly is repeated three times. Mutational analysis revealed that the Arg side chains are required for high-affinity binding, whereas the other pentad side chains are dispensable. Chemical interference experiments showed that the DAT1 protein interacts with the minor groove of the double helix. The data suggest that the pentad arginines interact in a cooperative manner with a repeated minor groove feature of A.T tract DNA to achieve high-affinity recognition. Amino acid similarities with other DNA binding proteins suggest that the DAT1 protein pentad represents a specialized example of a widespread motif used by proteins to recognize A.T base pairs.     

Mutations in lambda repressor''s amino-terminal domain: implications for protein stability and DNA binding.

The DNA binding properties of 52 different single-amino acid substitutions in lambda repressor's amino-terminal domain have been characterized. Seven proteins bearing mutations that change solvent-exposed side chains have been purified. The amino-terminal domains of these mutant repressors are folded and are comparable to the wild-type amino-terminal domain in thermal stability. In contrast, a purified mutant repressor bearing a substitution in a buried side chain contains an amino-terminal domain with decreased thermal stability. We argue that mutations that alter solvent-exposed wild-type side chains define residues that form the operator DNA binding surface of lambda repressor whereas completely or partially buried mutations exert their effect by decreasing protein stability.     

Specificity of the juvenile hormone binding protein: The geometrical isomers of juvenile hormone I

The binding of the geometrical isomers (≥99% pure) of juvenile hormone I to the hemolymph juvenile hormone binding protein of Manduca sexta (Lepidoptera, Sphingidae) was analyzed. A technique is described for isomer separation by micropreparative high-resolution liquid chromatography. Analysis of competition was performed by using a “batch adsorption” hydroxylapatite binding assay. Competition studies indicate that the naturally occurring isomer, 2E,6E,10cis, is bound with the highest affinity. Optimal binding appears to depend most heavily upon the configuration of the 2,3 double bond. Juvenile hormone binding protein shows a higher affinity for the 2E than for the 2Z configuration. The 6,7 double bond is of less importance in determining binding activity, and isomerism about the epoxide appears least important in conferring binding activity. The binding site may be a groove along the surface of the binding protein interacting with the side chains of juvenile hormone, including the ester methyl group. The grouping of the side chains and the ester methyl group thus constitutes a distinct hydrophobic face, and the hydrophobic interactions are essential in maintenance of the bound ligand.     

Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition.

Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor alpha chain (hIL-5R alpha). A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor alpha chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5R alpha were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues Glu-110 and Trp-111, and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important alpha chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5R alpha.     

Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: structural basis for recognition of B-cell receptors and superantigen activity

Staphylococcus aureus produces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-A resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (V(H)) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human V(H)3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig V(H) regions and the T-cell receptor V(beta) regions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor V(beta) backbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.     

High-affinity binding of short peptides to major histocompatibility complex class II molecules by anchor combinations.

We have previously identified four anchor positions in HLA-DRB1*0101-binding peptides, and three anchors involved in peptide binding to DRB1*0401 and DRB1*1101 molecules, by screening of an M13 peptide display library (approximately 20 million independent nonapeptides) for DR-binding activity. In this study, high stringency screening of the M13 library for DRB1*0401 binding has resulted in identification of three further anchor positions. Taken together, a peptide-binding motif has been obtained, in which six of seven positions show enrichment of certain residues. We have demonstrated an additive effect of anchors in two different ways: (i) the addition of more anchors is shown to compensate for progressive truncation of designer peptides; (ii) the incorporation of an increasing number of anchors into 6- or 7-residue-long designer peptides is shown to result in a gradual increase of binding affinity to the level of 13-residue-long high-affinity epitopes. The anchor at relative position 1 seems to be obligatory, in that its substitution abrogates binding completely, whereas the elimination of other anchors results only in partial loss of binding affinity. The spacing between anchors is critical, since their effect is lost by shifting them one position toward the N or C terminus. The information born out of this study has been successfully used to identify DR-binding sequences from natural proteins.     

Shared fine specificity between T-cell receptors and an antibody recognizing a peptide/major histocompatibility class I complex.

Cytotoxic T cells recognize mosaic structures consisting of target peptides embedded within self-major histocompatibility complex (MHC) class I molecules. This structure has been described in great detail for several peptide-MHC complexes. In contrast, how T-cell receptors recognize peptide-MHC complexes have been less well characterized. We have used a complete set of singly substituted analogs of a mouse MHC class I, Kk-restricted peptide, influenza hemagglutinin (Ha)255-262, to address the binding specificity of this MHC molecule. Using the same peptide-MHC complexes we determined the fine specificity of two Ha255-262-specific, Kk-restricted T cells, and of a unique antibody, pSAN, specific for the same peptide-MHC complex. Independently, a model of the Ha255-262-Kk complex was generated through homology modeling and molecular mechanics refinement. The functional data and the model corroborated each other showing that peptide residues 1, 3, 4, 6, and 7 were exposed on the MHC surface and recognized by the T cells. Thus, the majority, and perhaps all, of the side chains of the non-primary anchor residues may be available for T-cell recognition, and contribute to the stringent specificity of T cells. A striking similarity between the specificity of the T cells and that of the pSAN antibody was found and most of the peptide residues, which could be recognized by the T cells, could also be recognized by the antibody.     

Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid

Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles.     

RNA-binding domain of the A protein component of the U1 small nuclear ribonucleoprotein analyzed by NMR spectroscopy is structurally similar to ribosomal proteins.

An RNA recognition motif (RRM) of approximately 80 amino acids constitutes the core of RNA-binding domains found in a large family of proteins involved in RNA processing. The U1 RNA-binding domain of the A protein component of the human U1 small nuclear ribonucleoprotein (RNP), which encompasses the RRM sequence, was analyzed by using NMR spectroscopy. The domain of the A protein is a highly stable monomer in solution consisting of four antiparallel beta-strands and two alpha-helices. The highly conserved RNP1 and RNP2 consensus sequences, containing residues previously suggested to be involved in nucleic acid binding, are juxtaposed in adjacent beta-strands. Conserved aromatic side chains that are critical for RNA binding are clustered on the surface of the molecule adjacent to a variable loop that influences recognition of specific RNA sequences. The secondary structure and topology of the RRM are similar to those of ribosomal proteins L12 and L30, suggesting a distant evolutionary relationship between these two types of RNA-associated proteins.     

A primary lesion model for arteriosclerotic microplaque formation

Proteoheparan sulfate can be adsorbed to a methylated silica surface in a monomolecular layer via its transmembrane hydrophobic protein core domain. Due to electrostatic repulsion, its anionic glycosaminoglycan side chains are stretched out into the blood substitute solution representing a receptor site for specific lipoprotein binding through basic amino acid-rich residues within their apolipoproteins. The binding process was studied by ellipsometric techniques showing that HDL has a high binding affinity to the receptor and a protective effect on interfacial heparan sulfate proteoglycan layers with respect to LDL and Ca²⁺ complexation. LDL was found to deposit strongly at the proteoheparan sulfate, particularly in the presence of Ca²⁺ thus creating the ternary complex formation proteoglycan-LDL-calcium. This heterotrimeric complex build-up may be interpreted as arteriosclerotic microplaque formation on the molecular level responsible for the primary lesion. On the other hand, HDL bound to heparan sulfate proteoglycan protected against LDL docking and completely suppressed calcification of the proteoglycan-lipoprotein complex. In addition, HDL and garlic extract were able to reduce the ternary complex deposition.Presented in part at the 41st Annual Congress, International College of Angiology, Sapporo, Japan, July 1999.     

Molecular dynamics analysis of a buckyball-antibody complex

This is a multinanosecond molecular dynamics study of a bio-nano complex formed by a carbon nanoparticle, a buckyball C(60), and a biological molecule, an antibody, with high binding affinity and specificity. In the simulation, the ball is completely desolvated by the binding site of the antibody by means of a nearly perfect shape complementarity and extensive side-chain interactions, with the exception that about 17% of the surface is persistently exposed to solvent and could be used for functional derivatization. The interactions are predominantly hydrophobic, but significant polar interactions occur as well. There exists a rich body of various pi-stacking interactions. Aromatic side chains are involved in both double and triple stackings with the ball. Some ionic side chains, such as the guanidinium group of arginine, also form pi-stackings with the ball. The results suggest that pi-stackings are very efficient and common modes of biological recognition of pi-electron-rich carbon nanoparticles. Most importantly, the results demonstrate that, in general, an ordinary protein binding site, such as that of an antibody, can readily bind to a carbon nanoparticle with high affinity and specificity through recognition modes that are common in protein-ligand recognition.     

Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.     

Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

The natively disordered N-terminal 83-aa translocation (T) domain of E group nuclease colicins recruits OmpF to a colicin-receptor complex in the outer membrane (OM) as well as TolB in the periplasm of Escherichia coli, the latter triggering translocation of the toxin across the OM. We have identified the 16-residue TolB binding epitope in the natively disordered T-domain of the nuclease colicin E9 (ColE9) and solved the crystal structure of the complex. ColE9 folds into a distorted hairpin within a canyon of the six-bladed beta-propeller of TolB, using two tryptophans to bolt the toxin to the canyon floor and numerous intramolecular hydrogen bonds to stabilize the bound conformation. This mode of binding enables colicin side chains to hydrogen-bond TolB residues in and around the channel that runs through the beta-propeller and that constitutes the binding site of peptidoglycan-associated lipoprotein (Pal). Pal is a globular binding partner of TolB, and their association is known to be important for OM integrity. The structure is therefore consistent with translocation models wherein the colicin disrupts the TolB-Pal complex causing local instability of the OM as a prelude to toxin import. Intriguingly, Ca(2+) ions, which bind within the beta-propeller channel and switch the surface electrostatics from negative to positive, are needed for the negatively charged T-domain to bind TolB with an affinity equivalent to that of Pal and competitively displace it. Our study demonstrates that natively disordered proteins can compete with globular proteins for binding to folded scaffolds but that this can require cofactors such as metal ions to offset unfavorable interactions.     

The PD-1/PD-L1 complex resembles the antigen-binding Fv domains of antibodies and T cell receptors

Signaling through the programmed death 1 (PD-1) inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays an important role in the maintenance of peripheral immune tolerance. Release from PD-1 inhibitory signaling revives "exhausted" virus-specific T cells in chronic viral infections. Here we present the crystal structure of murine PD-1 in complex with human PD-L1. PD-1 and PD-L1 interact through the conserved front and side of their Ig variable (IgV) domains, as do the IgV domains of antibodies and T cell receptors. This places the loops at the ends of the IgV domains on the same side of the PD-1/PD-L1 complex, forming a surface that is similar to the antigen-binding surface of antibodies and T cell receptors. Mapping conserved residues allowed the identification of residues that are important in forming the PD-1/PD-L1 interface. Based on the structure, we show that some reported loss-of-binding mutations involve the PD-1/PD-L1 interaction but that others compromise protein folding. The PD-1/PD-L1 interaction described here may be blocked by antibodies or by designed small-molecule drugs to lower inhibitory signaling that results in a stronger immune response. The immune receptor-like loops offer a new surface for further study and potentially the design of molecules that would affect PD-1/PD-L1 complex formation and thereby modulate the immune response.     

Antigenic peptides containing large PEG loops designed to extend out of the HLA-A2 binding site form stable complexes with class I major histocompatibility complex molecules.

Recognition of peptides bound to class I major histocompatibility complex (MHC) molecules by specific receptors on T cells regulates the development and activity of the cellular immune system. We have designed and synthesized de novo cyclic peptides that incorporate PEG in the ring structure for binding to class I MHC molecules. The large PEG loops are positioned to extend out of the peptide binding site, thus creating steric effects aimed at preventing the recognition of class I MHC complexes by T-cell receptors. Peptides were synthesized and cyclized on polymer support using high molecular weight symmetrical PEG dicarboxylic acids to link the side chains of lysine residues substituted at positions 4 and 8 in the sequence of the HLA-A2-restricted human T-lymphotrophic virus type I Tax peptide. Cyclic peptides promoted the in vitro folding and assembly of HLA-A2 complexes. Thermal denaturation studies using circular dichroism spectroscopy showed that these complexes are as stable as complexes formed with antigenic peptides.     

A seven-helix coiled coil

Coiled-coil proteins contain a characteristic seven-residue sequence repeat whose positions are designated a to g. The interacting surface between alpha-helices in a classical coiled coil is formed by interspersing nonpolar side chains at the a and d positions with hydrophilic residues at the flanking e and g positions. To explore how the chemical nature of these core amino acids dictates the overall coiled-coil architecture, we replaced all eight e and g residues in the GCN4 leucine zipper with nonpolar alanine side chains. Surprisingly, the alanine-containing mutant forms a stable alpha-helical heptamer in aqueous solution. The 1.25-A resolution crystal structure of the heptamer reveals a parallel seven-stranded coiled coil enclosing a large tubular channel with an unusual heptad register shift between adjacent staggered helices. The overall geometry comprises two interleaved hydrophobic helical screws of interacting cross-sectional a and d layers that have not been seen before. Moreover, asparagines at the a positions play an essential role in heptamer formation by participating in a set of buried interhelix hydrogen bonds. These results demonstrate that heptad repeats containing four hydrophobic positions can direct assembly of complex, higher-order coiled-coil structures with rich diversity for close packing of alpha-helices.