Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide

We report the crystal structure of a soluble form of human urokinase-type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor-stromal microenvironment in many human cancers. The structure was solved at 2.7 A in association with a competitive peptide inhibitor of the urokinase-type plasminogen activator (uPA)-uPAR interaction. uPAR is composed of three consecutive three-finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA-uPAR complex. This model reveals that the receptor-binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA-focalized proteolysis on the cell surface and control cell adhesion and migration.     

Modeling of the spatial structure of an HIV-haiti immunodominant epitope

A model of the spatial structure of the immunodominant epitope of human immunodeficiency virus type 1 (HIV-Haiti virion) consistent with the NMR data was constructed using an earlier proposed method based on theoretical calculations. It was found that, in aqueous solution, the main immunogenic region of the virus protein gp120 took the prevalent conformation of a double β-turn IV–IV. It is shown that two structures of this fragment found in the crystal were present in the set of preferable conformations of the HIV-Haiti immunodominant epitope. On the basis of comparative analysis of the 3D structures of the immunodominant epitopes of type 1 human immunodeficiency virus (HIV-Haiti, HIV-Thailand, and HIV-MN virions), it was concluded that the studied gp120 fragment forms similar spatial backbone structures in these three virus subtypes; however, these structures are formed from different local minimums of the constituent amino acids. It was assumed that the detected distinctions between the local structures of this fragment determine the specificity of HIV-1 binding to virus-neutralizing antibodies.     

MHC class I bound to an immunodominant Theileria parva epitope demonstrates unconventional presentation to T cell receptors

T cell receptor (TCR) recognition of peptide-MHC class I (pMHC) complexes is a crucial event in the adaptive immune response to pathogens. Peptide epitopes often display a strong dominance hierarchy, resulting in focusing of the response on a limited number of the most dominant epitopes. Such T cell responses may be additionally restricted by particular MHC alleles in preference to others. We have studied this poorly understood phenomenon using Theileria parva, a protozoan parasite that causes an often fatal lymphoproliferative disease in cattle. Despite its antigenic complexity, CD8+ T cell responses induced by infection with the parasite show profound immunodominance, as exemplified by the Tp1(214-224) epitope presented by the common and functionally important MHC class I allele N*01301. We present a high-resolution crystal structure of this pMHC complex, demonstrating that the peptide is presented in a distinctive raised conformation. Functional studies using CD8+ T cell clones show that this impacts significantly on TCR recognition. The unconventional structure is generated by a hydrophobic ridge within the MHC peptide binding groove, found in a set of cattle MHC alleles. Extremely rare in all other species, this feature is seen in a small group of mouse MHC class I molecules. The data generated in this analysis contribute to our understanding of the structural basis for T cell-dependent immune responses, providing insight into what determines a highly immunogenic p-MHC complex, and hence can be of value in prediction of antigenic epitopes and vaccine design.     

Crystal structure of an RNA aptamer bound to thrombin

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.     

Amyloid-beta-anti-amyloid-beta complex structure reveals an extended conformation in the immunodominant B-cell epitope

Alzheimer's disease (AD) is the most common form of dementia. Amyloid-β (Aβ) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on Aβ, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-Aβ antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the Aβ peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the Aβ peptide. The structures reveal the molecular basis for WO2 recognition and binding of Aβ. The Aβ peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound Aβ peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of Aβ, such as WO2, hold promise for therapeutic development.     

Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence

The first X-ray structures of an intein-DNA complex, that of the two-domain homing endonuclease PI-SceI bound to its 36-base pair DNA substrate, have been determined in the presence and absence of Ca(2+). The DNA shows an asymmetric bending pattern, with a major 50 degree bend in the endonuclease domain and a minor 22 degree bend in the splicing domain region. Distortions of the DNA bound to the endonuclease domain cause the insertion of the two cleavage sites in the catalytic center. DNA binding induces changes in the protein conformation. The two overlapping non-identical active sites in the endonucleolytic center contain two Ca(+2) ions that coordinate to the catalytic Asp residues. Structure analysis indicates that the top strand may be cleaved first.     

Induced peptide conformations in different antibody complexes: molecular modeling of the three-dimensional structure of peptide-antibody complexes using NMR-derived distance restraints.

Intramolecular interactions in bound cholera toxin peptide (CTP3) in three antibody complexes were studied by two-dimensional transferred NOE spectroscopy. These measurements together with previously recorded spectra that show intermolecular interactions in these complexes were used to obtain restraints on interproton distances in two of these complexes (TE32 and TE33). The NMR-derived distance restraints were used to dock the peptide into calculated models for the three-dimensional structure of the antibody combining site. It was found that TE32 and TE33 recognize a loop comprising the sequence VPGSQHID and a beta-turn formed by the sequence VPGS. The third antibody, TE34, recognizes a different epitope within the same peptide and a beta-turn formed by the sequence IDSQ. Neither of these two turns was observed in the free peptide. The formation of a beta-turn in the bound peptide gives a compact conformation that maximizes the contact with the antibody and that has greater conformational freedom than alpha-helix or beta-sheet secondary structure. A total of 15 antibody residues are involved in peptide contacts in the TE33 complex, and 73% of the contact area in the antibody combining site consists of the side chains of aromatic amino acids. A comparison of the NMR-derived models for CTP3 interacting with TE32 and TE33 with the previously derived model for TE34 reveals a relationship between amino acid sequence and combining site structure and function. (a) The three aromatic residues that interact with the peptide in TE32 and TE33 complexes, Tyr 32L, Tyr 32H, and Trp 50H, are invariant in all light chains sharing at least 65% identity with TE33 and TE32 and in all heavy chains sharing at least 75% identity with TE33. Although TE34 differs from TE32 and TE33 in its fine specificity, these aromatic residues are conserved in TE34 and interact with its antigen. Therefore, we conclude that the role of these three aromatic residues is to participate in nonspecific hydrophobic interactions with the antigen. (b) Residues 31, 31c, and 31e of CDR1 of the light chain interact with the antigen in all three antibodies that we have studied. The amino acids in these positions in TE34 differ from those in TE32 and TE33, and they are involved in specific polar interactions with the antigen. (c) CDR3 of the heavy chain varies considerably both in length and in sequence between TE34 and the two other anti-CTP3 antibodies. These changes modify the shape of the combining site and the hydrophobic and polar interactions of CDR3 with the peptide antigen.     

Induced fit of an epitope peptide to a monoclonal antibody probed with a novel parallel surface plasmon resonance assay

Class II major histocompatibility complex proteins bind peptides for presentation to T-cells as part of the immune response process. Monoclonal antibody MEM-265 recognizes the peptide-free conformation of the major histocompatibility complex class II protein HLA-DR1 through specific binding to an epitope contained between residues 50-67 of the beta-chain. In previous work using alanine scanning (1), we identified residues Leu-53, Asp-57, Tyr-60, Trp-61, Ser-63, and Leu-67 as essential for specific recognition by MEM-265. The spacing of these residues approximates a 3.5-residue repeat, suggesting that MEM-265 may recognize the epitope in an alpha-helical conformation. In the folded, peptide-loaded DR1 structure, the beta-chain residues 50-67 contain a kinked alpha-helical segment spanning Glu-52-Ser-63 (2). However, the conformation of this segment in the peptide-free form is unknown. We have used a new surface plasmon resonance approach in a SpotMatrix format to compare the kinetic rates and affinities for 18 alanine scanning mutants comprising epitope residues 50-67. In addition to the six essential residues described previously, we found two additional residues, Glu-52 and Gln-64, that contribute by enhancing MEM-265 binding. By contrast, mutation of either Gly-54 or Pro-56 to an alanine actually improved binding to MEM-265. In essentially all cases peptide substitutions that either improve or reduce MEM-265 recognition could be traced to differences in the dissociation rate (k off). The kinetic details of the present study support the presence of a structural component in the antigenic epitope recognized by MEM-265 in the peptide-free form of major histocompatibility complex II DR1 beta-chain.     

Crystal structure of an antigen-binding fragment bound to single-stranded DNA.

Antibodies to DNA are characteristic of the autoimmune disease systemic lupus erythematosus (SLE) and they also serve as models for the study of protein-DNA recognition. Anti-DNA antibodies often play an important role in disease pathogenesis by mediating kidney damage via antibody-DNA immune complex formation. The structural underpinnings of anti-DNA antibody pathogenicity and antibody-DNA recognition, however, are not well understood, due in part to the lack of direct, experimental three-dimensional structural information on antibody-DNA complexes. To address these issues for anti-single-stranded DNA antibodies, we have determined the 2.1 A crystal structure of a recombinant Fab (DNA-1) in complex with dT5. DNA-1 was previously isolated from a bacteriophage Fab display library from the immunoglobulin repertoire of an SLE-prone mouse. The structure shows that DNA-1 binds oligo(dT) primarily by sandwiching thymine bases between Tyr side-chains, which allows the bases to make sequence-specific hydrogen bonds. The critical stacking Tyr residues are L32, L49, H100, and H100A, while His L91 and Asn L50 contribute hydrogen bonds. Comparison of the DNA-1 structure to other anti-nucleic acid Fab structures reveals a common ssDNA recognition module consisting of Tyr L32, a hydrogen bonding residue at position L91, and an aromatic side-chain from the tip of complementarity determining region H3. The structure also provides a framework for interpreting previously determined thermodynamics data, and this analysis suggests that hydrophobic desolvation might underlie the observed negative enthalpy of binding. Finally, Arg side-chains from complementarity determining region H3 appear to play a novel role in DNA-1. Rather than forming ion pairs with dT5, Arg contributes to oligo(dT) recognition by helping to maintain the structural integrity of the combining site. This result is significant because antibody pathogenicity is thought to be correlated to the Arg content of anti-DNA antibody hypervariable loops.     

Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence.

The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns.     

Crystal structure of an activated response regulator bound to its target

The chemotactic regulator CheY controls the direction of flagellar rotation in Escherichia coli. We have determined the crystal structure of BeF3--activated CheY from E. coli in complex with an N-terminal peptide derived from its target, FliM. The structure reveals that the first seven residues of the peptide pack against the beta4-H4 loop and helix H4 of CheY in an extended conformation, whereas residues 8-15 form two turns of helix and pack against the H4-beta5-H5 face. The peptide binds the only region of CheY that undergoes noticeable conformational change upon activation and would most likely be sandwiched between activated CheY and the remainder of FliM to reverse the direction of flagellar rotation.     

Crystal structure of thrombin bound to heparin

Thrombin is the final protease in the blood coagulation cascade and serves both pro- and anticoagulant functions through the cleavage of several targets. The ability of thrombin to specifically recognize a wide range of substrates derives from interactions that occur outside of the active site of thrombin. Thrombin possesses two anion binding exosites, which mediate many of its interactions with cofactors and substrates, and although many structures of thrombin have been solved, few such interactions have been described in molecular detail. Glycosaminoglycan binding to exosite II of thrombin plays a major role in switching off the procoagulant functions of thrombin by mediating its irreversible inhibition by circulating serpins and by its binding to the endothelial cell surface receptor thrombomodulin. Here we report the 1.85-A structure of human alpha-thrombin bound to a heparin fragment of eight monosaccharide units in length. The asymmetric unit is composed of two thrombin dimers, each sharing a single heparin octasaccharide chain. The observed interactions are fully consistent with previous mutagenesis studies and illustrate on a molecular level the cofactor interaction that is critical for the restriction of clotting to the site of blood vessel injury.     

Modeling of the 3D structure of the HIV-Haiti immunodominant epitope

The 3D structure for the HIV-Haiti immunodominant epitope was computed in terms of NMR spectroscopy data using the theoretical procedure including a probabilistic approach in conjunction with molecular mechanics algorithms and quantum chemical methods. The immunogenic crown of the virus protein gp120 was shown to form in solution a prevalent conformation whose geometric parameters match the double beta-turn IV-IV. Two structures observed in crystal were found in the ensemble of the best-energy conformations of the HIV-Haiti principal neutralizing epitope. From a comparison of simulated structures with those computed previously for the HIV-Thailand and HIV-MN isolates, it was concluded that the immunogenic tip of gp120 gives rise to similar spatial backbone forms in different HIV-1 strains but has some inherent conformational flexibility of its individual amino acid residues. The differences in local fragment structures revealed in three isolates of HIV-1 are supposed to be important for the specificity of its binding with neutralizing antibodies.     

Crystal structure of a superantigen bound to MHC class II displays zinc and peptide dependence.

The three-dimensional structure of a bacterial superantigen, Staphylococcus aureus enterotoxin H (SEH), bound to human major histocompatibility complex (MHC) class II (HLA-DR1) has been determined by X-ray crystallography to 2.6 A resolution (1HXY). The superantigen binds on top of HLA-DR1 in a completely different way from earlier co-crystallized superantigens from S.aureus. SEH interacts with high affinity through a zinc ion with the beta1 chain of HLA-DR1 and also with the peptide presented by HLA-DR1. The structure suggests that all superantigens interacting with MHC class II in a zinc-dependent manner present the superantigen in a common way. This suggests a new model for ternary complex formation with the T-cell receptor (TCR), in which a contact between the TCR and the MHC class II is unlikely.     

Crystal structure of a cross-reaction complex between an anti-HIV-1 protease antibody and an HIV-2 protease peptide

The monoclonal antibody 1696, elicited by HIV-1 protease, inhibits the activity of both HIV-1 and HIV-2 proteases with inhibition constants in the low nanomolar range. The antibody cross-reacts with peptides derived from the N-terminal region of both proteases. The crystal structure of the recombinant single-chain Fv fragment of 1696 complexed with an N-terminal peptide from the HIV-2 protease has been determined at 1.88A resolution. Interactions of the peptide with scFv1696 are compared with the previously reported structure of scFv1696 in complex with the corresponding peptide from HIV-1 protease. The origin of cross-reactivity of mAb1696 with HIV proteases is discussed.     

Crystal structure of aclacinomycin methylesterase with bound product analogues: implications for anthracycline recognition and mechanism

Aclacinomycin methylesterase (RdmC) is one of the tailoring enzymes that modify the aklavinone skeleton in the biosynthesis of anthracyclines in Streptomyces species. The crystal structures of this enzyme from Streptomyces purpurascens in complex with the product analogues 10-decarboxymethylaclacinomycin T and 10-decarboxymethylaclacinomycin A were determined to nominal resolutions of 1.45 and 1.95 A, respectively. RdmC is built up of two domains. The larger alpha/beta domain shows the common alpha/beta hydrolase fold, whereas the smaller domain is alpha-helical. The active site and substrate binding pocket are located at the interface between the two domains. Decarboxymethylaclacinomycin T and decarboxymethylaclacinomycin A bind close to the catalytic triad (Ser102-His276-Asp248) in a hydrophobic pocket, with the sugar moieties located at the surface of the enzyme. The binding of the ligands is dominated by hydrophobic interactions, and specificity appears to be controlled mainly by the shape of the binding pocket rather than through specific hydrogen bonds. Mechanistic key features consistent with the structure of complexes of RdmC with product analogues are Ser102 acting as nucleophile and transition state stabilization by an oxyanion hole formed by the backbone amides of residues Gly32 and Met103.     

B- and T-lymphocyte responses to an immunodominant epitope of human immunodeficiency virus.

Using synthetic peptides, we characterized the B-lymphocyte (antibody) and T-lymphocyte (proliferation) responses to an immunodominant epitope of human immunodeficiency virus type 1 (HIV-1) located near the amino-terminal end of the transmembrane glycoprotein (env amino acids 598 to 609). Both immunoglobulin M (IgM) and IgG antibodies against this epitope appeared early after primary infection with HIV-1. In an animal model, the IgG response to a synthetic peptide derived from this sequence was T-helper-cell dependent, whereas the IgM response was T-cell independent. In addition, antibody generated by immunization with this peptide had HIV-1-neutralizing activity. Greater than 99% (201 of 203) of patients infected with HIV-1 generated antibody to this peptide in vivo; however, only 24% (7 of 29) had T cells that proliferated in response to this peptide in vitro. These observations suggest that different HIV-1 gp41 epitopes elicit B-cell and T-cell immune responses.     

Editing of an immunodominant epitope of glutamate decarboxylase by HLA-DM

HLA-DM stabilizes peptide-receptive class II alphabeta dimers and facilitates the capture of high affinity peptides, thus influencing the peptide repertoire presented by class II molecules. Variations in DM levels may therefore have a profound effect on the antigenic focus of T cell-mediated immune responses. Specifically, DM expression may influence susceptibility and resistance to autoimmune diseases. In this study the role of DM in HLA-DR4-restricted presentation of an insulin-dependent diabetes mellitus autoantigen, glutamate decarboxylase (GAD), was tested. Presentation of immunodominant GAD epitope 273-285 was regulated by endogenous DM levels in human B lymphoblasts. T cell responses to exogenous GAD as well as an endogenous cytoplasmic form of this Ag were significantly diminished with increasing cellular expression of DM. Epitope editing by DM was observed only using Ag and not small synthetic peptides, suggesting that this process occurred within endosomes. Results with cytoplasmic GAD also indicated that peptides from this compartment intersect class II proteins in endocytic vesicles where DM editing was facilitated. Changes in DM levels within APC may therefore influence the presentation of autoantigens and the development of autoimmune disorders such as type I diabetes.     

Crystal structure of trioxacarcin A covalently bound to DNA

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule ‘caught in the act’ after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 Å resolution by MAD phasing employing brominated oligonucleotides. The DNA–drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3′-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.