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J Tormo

Publications and source records attributed to J Tormo.

At least 19 recordsLinked to original sources
PubMed2000

Estimation of the hydrophobic effect in an antigen-antibody protein-protein interface.

Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V(L)W92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine, and phenylalanine) were determined to resolutions between 1.75 and 2.00 A. In the wild-type complex, V(L)W92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic "hot spot" for antigen binding. The losses in apolar buried surface area in the mutant complexes, relative to wild-type, range from 25 (V(L)F92) to 115 A(2) (V(L)A92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V(L)A92, where there is a peptide flip. The affinities of the mutant Fv fragments for HEL are 10-100-fold lower than that of the original antibody. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V(L)92 mutant series and changes in the enthalpy of formation. Conversely, there exist linear correlations between losses of apolar buried surface and decreases in binding free energy (R(2) = 0.937) as well as increases in the solvent portion of the entropy of binding (R(2) = 0.909). The correlation between binding free energy and apolar buried surface area corresponds to 21 cal mol(-1) A(-2) (1 cal = 4.185 J) for the effective hydrophobicity at the V(L)92 mutation site. Furthermore, the slope of the line defined by the correlation between changes in binding free energy and solvent entropy approaches unity, demonstrating that the exclusion of solvent from the binding interface is the predominant energetic factor in the formation of this protein complex. Our estimate of the hydrophobic contribution to binding at site V(L)92 in the D1.3-HEL interface is consistent with values for the hydrophobic effect derived from classical hydrocarbon solubility models. We also show how residue V(L)W92 can contribute significantly less to stabilization when buried in a more polar pocket, illustrating the dependence of the hydrophobic effect on local environment at different sites in a protein-protein interface.

Animals↗
PubMed2000

Mapping the ligand of the NK inhibitory receptor Ly49A on living cells.

We have used a recombinant, biotinylated form of the mouse NK cell inhibitory receptor, Ly49A, to visualize the expression of MHC class I (MHC-I) ligands on living lymphoid cells. A panel of murine strains, including MHC congenic lines, was examined. We detected binding of Ly49A to cells expressing H-2D(d), H-2D(k), and H-2D(p) but not to those expressing other MHC molecules. Cells of the MHC-recombinant strain B10.PL (H-2(u)) not only bound Ly49A but also inhibited cytolysis by Ly49A(+) effector cells, consistent with the correlation of in vitro binding and NK cell function. Binding of Ly49A to H-2D(d)-bearing cells of different lymphoid tissues was proportional to the level of H-2D(d) expression and was not related to the lineage of the cells examined. These binding results, interpreted in the context of amino acid sequence comparisons and the recently determined three-dimensional structure of the Ly49A/H-2D(d) complex, suggest a role for amino acid residues at the amino-terminal end of the alpha1 helix of the MHC-I molecule for Ly49A interaction. This view is supported by a marked decrease in affinity of an H-2D(d) mutant, I52 M, for Ly49A. Thus, allelic variation of MHC-I molecules controls measurable affinity for the NK inhibitory receptor Ly49A and explains differences in functional recognition in different mouse strains.

Amino Acid Sequence↗
PubMed2000

Crystal structure of the C-type lectin-like domain from the human hematopoietic cell receptor CD69.

CD69, one of the earliest specific antigens acquired during lymphoid activation, acts as a signal-transducing receptor involved in cellular activation events, including proliferation and induction of specific genes. CD69 belongs to a family of receptors that modulate the immune response and whose genes are clustered in the natural killer (NK) gene complex. The extracellular portion of these receptors represent a subfamily of C-type lectin-like domains (CTLDs), which are divergent from true C-type lectins and are referred to as NK-cell domains (NKDs). We have determined the three-dimensional structure of human CD69 NKD in two different crystal forms. CD69 NKD adopts the canonical CTLD fold but lacks the features involved in Ca(2+) and carbohydrate binding by C-type lectins. CD69 NKD dimerizes noncovalently, both in solution and in crystalline state. The dimer interface consists of a hydrophobic, loosely packed core, surrounded by polar interactions, including an interdomain beta sheet. The intersubunit core shows certain structural plasticity that may facilitate conformational rearrangements for binding to ligands. The surface equivalent to the binding site of other members of the CTLD superfamily reveals a hydrophobic patch surrounded by conserved charged residues that probably constitutes the CD69 ligand-binding site.

Amino Acid Sequence↗
PubMed2000

Structural and biochemical features distinguish the foot-and-mouth disease virus leader proteinase from other papain-like enzymes.

The structures of the two leader protease (Lpro) variants of foot-and-mouth disease virus known to date were solved using crystals in which molecules were organized as molecular fibers. Such crystals diffract to a resolution of only approximately 3 A. This singular, pseudo-polymeric organization is present in a new Lpro crystal form showing a cubic packing. As molecular fiber formation appeared unrelated to crystallization conditions, we mutated the reactive cysteine 133 residue, which makes a disulfide bridge between adjacent monomers in the fibers, to serine. None of the intermolecular contacts found in the molecular fibers was present in crystals of this variant. Analysis of this Lpro structure, refined at 1.9 A resolution, enables a detailed definition of the active center of the enzyme, including the solvent organization. Assay of Lpro activity on a fluorescent hexapeptide substrate showed that Lpro, in contrast to papain, was highly sensitive to increases in the cation concentration and was active only across a narrow pH range. Examination of the Lpro structure revealed that three aspartate residues near the active site, not present in papain-like enzymes, are probably responsible for these properties.

Amino Acid Substitution↗
PubMed1999

Crystal structure of a lectin-like natural killer cell receptor bound to its MHC class I ligand.

Natural killer (NK) cell function is regulated by NK receptors that interact with MHC class I (MHC-I) molecules on target cells. The murine NK receptor Ly49A inhibits NK cell activity by interacting with H-2D(d) through its C-type-lectin-like NK receptor domain. Here we report the crystal structure of the complex between the Ly49A NK receptor domain and unglycosylated H-2D(d). The Ly49A dimer interacts extensively with two H-2D(d) molecules at distinct sites. At one interface, a single Ly49A subunit contacts one side of the MHC-I peptide-binding platform, presenting an open cavity towards the conserved glycosylation site on the H-2D(d) alpha2 domain. At a second, larger interface, the Ly49A dimer binds in a region overlapping the CD8-binding site. The smaller interface probably represents the interaction between Ly49A on the NK cell and MHC-I on the target cell, whereas the larger one suggests an interaction between Ly49A and MHC-I on the NK cell itself. Both Ly49A binding sites on MHC-I are spatially distinct from that of the T-cell receptor.

Antigens, Ly↗
PubMed1999

Crystal structures of two H-2Db/glycopeptide complexes suggest a molecular basis for CTL cross-reactivity.

Two synthetic O-GlcNAc-bearing peptides that elicit H-2Db-restricted glycopeptide-specific cytotoxic T cells (CTL) have been shown to display nonreciprocal patterns of cross-reactivity. Here, we present the crystal structures of the H-2Db glycopeptide complexes to 2.85 A resolution or better. In both cases, the glycan is solvent exposed and available for direct recognition by the T cell receptor (TCR). We have modeled the complex formed between the MHC-glycopeptide complexes and their respective TCRs, showing that a single saccharide residue can be accommodated in the standard TCR-MHC geometry. The models also reveal a possible molecular basis for the observed cross-reactivity patterns of the CTL clones, which appear to be influenced by the length of the CDR3 loop and the nature of the immunizing ligand.

Acetylglucosamine↗
PubMed1998

Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition.

The leader protease of foot-and-mouth disease virus, as well as cleaving itself from the nascent viral polyprotein, disables host cell protein synthesis by specific proteolysis of a cellular protein: the eukaryotic initiation factor 4G (eIF4G). The crystal structure of the leader protease presented here comprises a globular catalytic domain reminiscent of that of cysteine proteases of the papain superfamily, and a flexible C-terminal extension found intruding into the substrate-binding site of an adjacent molecule. Nevertheless, the relative disposition of this extension and the globular domain to each other supports intramolecular self-processing. The different sequences of the two substrates cleaved during viral replication, the viral polyprotein (at LysLeuLys/GlyAlaGly) and eIF4G (at AsnLeuGly/ArgThrThr), appear to be recognized by distinct features in a narrow, negatively charged groove traversing the active centre. The structure illustrates how the prototype papain fold has been adapted to the requirements of an RNA virus. Thus, the protein scaffold has been reduced to a minimum core domain, with the active site being modified to increase specificity. Furthermore, surface features have been developed which enable C-terminal self-processing from the viral polyprotein.

Amino Acid Sequence↗
PubMed1998

Production, crystallization, and preliminary X-ray analysis of the human MHC class Ib molecule HLA-E.

HLA-E is the first human class Ib major histocompatibility complex molecule to be crystallized. HLA-E is highly conserved and almost nonpolymorphic, and has recently been shown to be the first specialized ligand for natural killer cell receptors. In functional studies, HLA-E is unlike the class Ia MHC molecules in having tightly restricted peptide binding specificity. HLA-E binds a limited set of almost identical leader sequence peptides derived from class Ia molecules and presents these at the cell surface for recognition by natural killer cell receptors. We now show that the extracellular region of HLA-E forms a stable complex with beta2 microglobulin and can be refolded around synthetic peptide. Crystals of this complex formed slowly over four to six months in the presence of ammonium sulphate. The crystals diffract to 2.85 A with space group P3(1)21 and unit cell dimensions a = 182.2 A, b = 182.2 A, c = 88.4 A.

Amino Acid Sequence↗
PubMed1998

Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E.

The crystal structure of the nonclassical human class lb MHC molecule HLA-E has been determined in complex with a prototypic ligand, the nonamer peptide (VMAPRTVLL), derived from the highly conserved residues 3-11 of the human MHC class la leader sequence. The mode of peptide binding retains some of the standard features observed in MHC class la complexes, but novel features imply that HLA-E has evolved to mediate specific binding to a tightly defined set of almost identical hydrophobic peptides from the highly conserved class l leader sequences. These molecular adaptations make HLA-E a rigorous checkpoint at the cell surface reporting on the integrity of the antigen processing pathway to CD94/NKG2 receptor-bearing natural killer cells.

Crystallography↗
PubMed1998

Recognition surfaces of MHC class I.

Recent crystallographic results have provided close to atomic resolution views of the recognition events mediated by MHC class I molecules. The specificity-conferring interaction of MHC class I/peptide with a T-cell antigen receptor (TCR) appears dependent on certain key interactions with the MHC scaffold. These interactions, in particular those of the TCR V alpha domain, define a standard orientation for TCR binding. Previous studies on biologically significant variations in the TCR recognition surface presented by a series of MHC/variant peptide complexes can be reassessed in the light of this TCR-binding mode. The interaction of CD8 with MHC class I resembles that between antibody and antigen in the use of loops from the CD8 structure. The interaction is of very low affinity and buries equivalent surface area to that between the TCR and MHC class I but while the TCR/MHC interface shows poor surface shape complementarity the match in the conservative interaction between MHC and CD8 is precise.

Animals↗
PubMed1997

Crystal structure of the complex between human CD8alpha(alpha) and HLA-A2.

The dimeric cell-surface glycoprotein CD8 is crucial to the positive selection of cytotoxic T cells in the thymus. The homodimer CD8alpha(alpha) or the heterodimer alpha beta stabilizes the interaction of the T-cell antigen receptor (TCR) with major histocompatibility complex (MHC) class I/peptide by binding to the class I molecule. Here we report the crystal structure at 2.7 A resolution of a complex between CD8alpha(alpha) and the human MHC molecule HLA-A2, which is associated with peptide. CD8alpha(alpha) binds one HLA-A2/peptide molecule, interfacing with the alpha2 and alpha3 domains of HLA-A2 and also contacting beta2-microglobulin. A flexible loop of the alpha3 domain (residues 223-229) is clamped between the complementarity-determining region (CDR)-like loops of the two CD8 subunits in the classic manner of an antibody-antigen interaction, precluding the binding of a second MHC molecule. The position of the alpha3 domain is different from that in uncomplexed HLA-A2, being most similar to that in the TCR/Tax/HLA-A2 complex, but no conformational change extends to the MHC/peptide surface presented for TCR recognition. Although these shifts in alpha3 may provide a synergistic modulation of affinity, the binding of CD8 to MHC is clearly consistent with an avidity-based contribution from CD8 to TCR-peptide-MHC interactions.

CD8 Antigens↗
PubMed1996

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.

Amino Acid Sequence↗
PubMed1995

Crystal structure of catalase HPII from Escherichia coli.

BACKGROUND: Catalase is a ubiquitous enzyme present in both the prokaryotic and eukaryotic cells of aerobic organisms. It serves, in part, to protect the cell from the toxic effects of small peroxides. Escherichia coli produces two catalases, HPI and HPII, that are quite distinct from other catalases in physical structure and catalytic properties. HPII, studied in this work, is encoded by the katE gene, and has been characterized as an oligomeric, monofunctional catalase containing one cis-heme d prosthetic group per subunit of 753 residues. RESULTS: The crystal structure of catalase HPII from E. coli has been determined to 2.8 A resolution. The asymmetric unit of the crystal contains a whole molecule, which is a tetramer with accurate 222 point group symmetry. In the model built, that includes residues 27-753 and one heme group per monomer, strict non-crystallographic symmetry has been maintained. The crystallographic agreement R-factor is 20.1% for 58,477 reflections in the resolution shell 8.0-2.8 A. CONCLUSIONS: Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known. In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase. This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown. In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases. HPII is the largest catalase for which the structure has been determined to almost atomic resolution.

Amino Acid Sequence↗
PubMed1995

Docking of a human rhinovirus neutralizing antibody onto the viral capsid.

The structure of the complex between the Fab fragment of a human rhinovirus serotype 2 (HRV2) neutralizing antibody (8F5) and a cross-reactive synthetic peptide derived from the viral capsid protein VP2 has been recently determined by crystallographic methods. The conformation adopted by the peptide was very similar to and could be superimposed onto the corresponding region of the viral protein VP2 of human rhinovirus 1A (HRV1A) whose three-dimensional structure is known. The structure of the Fab fragment determined in the complex was docked onto the viral capsid using the superimposition transformation found for the peptide. In the resulting model the Fab protrudes almost radially to about 60 A from the surface of the virion without any major steric problem. The Fab fragment was then placed on each one of the 60 equivalent epitopes using the T = 1 icosahedral symmetry of the virus. The closest pairs of Fab fragments are related by viral 2-fold axes and run almost parallel to each other without clashing. These axes of symmetry from the viral particle could thus be coincident with the dyad axes of the antibodies. Furthermore, comparison of the three-dimensional structure of the Fab/peptide complex with the structure of the Fab fragment alone indicates that the flexibility of the antibody's elbow would facilitate bivalent attachment to the same viral particle. In accordance with the docking results, experimental determination of the stoichiometry of binding yielded a ratio of 30 IgG molecules per virion also suggesting bivalent attachment of antibody 8F5 onto the viral particle. The neutralization of viral infectivity, being neither aggregation (this paper) nor inhibition of receptor binding, might be mainly achieved by reducing viral spread from cell to cell and/or inhibition of uncoating.

Amino Acid Sequence↗
PubMed1995

Differences in production of several extracellular virulence factors in clinical and food Aeromonas spp. strains.

Production of several extracellular virulence factors (lipase, protease and haemolysin) was compared in 15 Aeromonas spp. isolated from faeces of patients with Aeromonas-associated gastroenteritis and 81 strains isolated from food. Strains from food did not show differences in production of these factors when compared with strains isolated from faeces. However, if strains were considered in relation to autoagglutination (AA) character, the AA+ differed from AA- strains in lipase and protease production. Supernatant fluids of AA+ food and human strains showed 2.5-fold more protease production than that observed in AA- strains. These two characteristics of certain Aeromonas strains could be related with the more virulent capacity.

Aeromonas↗
PubMed1994

Crystallization and preliminary X-ray analysis of the major cellulose-binding domain of the cellulosome from Clostridium thermocellum.

The cellulose-binding domain from the scaffoldin subunit of the cellulosome from Clostridium thermocellum strain YS has been expressed in Escherichia coli, purified to homogeneity, and crystallized. Crystals were grown by vapor diffusion using polyethylene glycol as precipitant. They belong to the monoclinic space group C2 with unit cell dimensions of a = 64.68 A, b = 50.36 A, c = 96.27 A; beta = 99.43 degrees, and density packing considerations suggest that the asymmetric unit contains two molecules. The crystals diffract beyond 2.0 A resolution using a laboratory rotating anode source.

Cellulase↗
PubMed1994

Domains of Escherichia coli primase: functional activity of a 47-kDa N-terminal proteolytic fragment.

Endoproteinase Asp-N cleaves the 581-amino acid Escherichia coli primase (65,564 Da) into several major fragments. One of these, a 47-kDa fragment containing the complete N terminus and the first 422 amino acids of primase, is capable of primer RNA (pRNA) synthesis in the G4oric/single-stranded DNA binding protein/primase pRNA synthesis system. A cloned 398-amino acid N-terminal fragment of primase can also synthesize pRNA. The sizes of the pRNA synthesized by these N-terminal fragments, however, are smaller than those synthesized by intact primase, suggesting that the C-terminal region of primase plays a role in processivity or regulation of pRNA synthesis. Primase mutants with the last 10 and 40 C-terminal amino acids deleted synthesize pRNA as wild-type primase, indicating that any regulatory sequences must be internal to the C terminus of primase.

Amino Acid Sequence↗