Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes.

A detailed comparison of the structures of aspartate aminotransferase, alanine race-mase, the beta subunit of tryptophan synthase, D-amino acid aminotransferase and glycogen phosphorylase has revealed more extensive structural similarities among pyridoxal phosphate (PLP)-binding domains in these enzymes than was observed previously. These similarities consist of seven common structural segments of the polypeptide chain, which form an extensive common structural organization of the backbone chain responsible for the appropriate disposition of key residues, some from the aligned fragments and some from variable loops joined to these fragments, interacting with PLPs in these enzymes. This common structural organization contains an analogous hydrophobic minicore formed from four amino acid side chains present in the two most conserved structural elements. In addition, equivalent alpha-beta-alpha-beta supersecondary structures are formed by these seven fragments in three of the five structures: alanine racemase, tryptophan synthase and glycogen phosphorylase. Despite these similarities, it is generally accepted that these proteins do not share a common heritage, but have arisen on five separate occasions. The common and contiguous alpha-beta-alpha-beta structure accounts for only 28 residues and all five enzymes differ greatly in both the orientation of the PLP pyridoxal rings and their contacts with residues close to the common structural elements.

Analogous conformations of both binding and effector regions in cyclosporin A, FK506 and rapamycin.

Three immunosuppressant drugs, cyclosporin A, FK506 and rapamycin were compared in their three-dimensional structures by computer modelling. The pairwise comparisons of cyclosporin A, FK506 and rapamycin show two structurally common fragments. One fragment is Mle9-Bmt1 region in cyclosporin A, C22-O5 region in FK506 and C29-O5 region in rapamycin. Another fragment is Mle4-Mle6 region in cyclosporin A and C14-C21 regions in FK506 and rapamycin. The correspondence of the structurally analogous regions with the regions which are involved in the interactions with peptidyl-prolyl cis/trans isomerases and calcineurin or FKBP-rapamycin-associated protein is discussed.

Molecular models of two competitive inhibitors, IL-2delta2 and IL-2delta3, generated by alternative splicing of human interleukin-2.

Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.

Molecular model of an alternative splice variant of human IL-4, IL-4 delta 2, a naturally occurring inhibitor of IL-4-stimulated T cell proliferation.

The molecular model of IL-4 delta 2, a naturally occurring splice variant of human IL-4 with exons 1, 3, and 4 in an open reading frame, is described. The second exon codes the main part of the long loop AB connected the helices A and B in parallel superposition. Therefore the hydrophobic core and the native fold of the rest part of IL-4 delta 2 molecule could be preserved without any significant changes only in the case of revolution of the helix A relative to other helices. In the result, the dominated a left-handed four-helix bundle structure of IL-4 with an up-up-down-down structural pattern is transformed to the IL-4 delta 2 structure with a down-up-down-down structural pattern.

Influence of the conserved disulphide bond, exposed to the putative binding pocket, on the structure and function of the immunoglobulin-like molecular chaperone Caf1M of Yersinia pestis.

The Yersinia pestis protein Caf1M is a typical representative of a subfamily of periplasmic molecular chaperones with characteristic structural and functional features, one of which is the location of two conserved cysteine residues close to the putative binding pocket. We show that these residues form a disulphide bond, the reduction and alkylation of which significantly increases the dissociation constant of the Caf1M-Caf1 (where Caf 1 is a polypeptide subunit of the capsule) complex [from a Kd of (4.77+/-0.50)x10(-9) M for the intact protein to one of (3.68+/-0.68)x10(-8) M for the modified protein]. The importance of the disulphide bond for the formation of functional Caf1M in vivo was demonstrated using an Escherichia coli dsbA mutant carrying the Y. pestis f1 operon. In accordance with the CD and fluorescence measurements, the disulphide bond is not important for maintenance of the overall structure of the Caf1M molecule, but would appear to affect the fine structural properties of the subunit binding site. A three-dimensional model of the Caf1M-Caf1 complex was designed based on the published crystal structure of PapD (a chaperone required for Pap pili assembly) complexed with a peptide corresponding to the C-terminus of the papG subunit. In the model the disulphide bond is in close proximity to the invariant Caf1M Arg-23 and Lys-142 residues that are assumed to anchor the C-terminal group of the subunit. The importance of this characteristic disulphide bond for the orchestration of the binding site and subunit binding, as well as for the folding of the protein in vivo, is likely to be a common feature of this subfamily of Caf1M-like chaperones. A possible model for the role of the disulphide bond in Caf1 assembly is discussed.

Structure of cytokine hydrophobic cores.

Diagrams were constructed that describe, using a common frame of reference, the interactions that occur within the hydrophobic cores of the left-handed four-helix bundles of interleukin-2, -4, -5, granulocyte/macrophage-, granulocyte-, macrophage-colony-stimulating factor, beta- and gamma-interferon, growth hormone, and leukemia inhibitory factor. Based on the assumption that the topologies of the ligand-receptor complexes are similar, analogous patterns were obtained from these diagrams and lead to the detailed comparison of the hydrophobic cores. These patterns were then used to obtain pairwise rigid-body superpositions among all of the four-helix bundles; values of the root-mean-square deviations (C alpha-atoms) over the four alpha-helices of the bundles range from 1.14 A to 3.22 A. Two distinct groups are formed after clustering based on structural superposition of the ten cytokines.

Conservative hydrophobic interdomain contacts of IFN-gamma remain in P17 matrix protein of HIV-1.

A constructed scheme of the surface layers containing helices C, D, and E' of various polypeptide chains which participate in the interdomain contacts in IFN-gamma demonstrated two sites of localization of the conservative hydrophobic amino acids. An analogous scheme of the interaction of helices B, C, and D in the p17 matrix protein of HIV-1 showed that the majority of the hydrophobic positions are similar. These data confirm the structural similarity between p17 and IFN-gamma.

Dimer structure as a minimum cooperative subunit of small heat-shock proteins.

Recently, it has been shown that small heat-shock proteins (Hsp25, Hsp27) are molecular chaperones. They bind to thermally unfolded proteins and can also assist refolding of denatured proteins. Mammalian small Hsps can form oligomeric structures of about 32 subunits. Until now, no data about cooperativity and stability of the interactions between the subunits of sHsps are available. To analyze these interactions we studied mouse Hsp25 and human Hsp27 by difference adiabatic scanning microcalorimetry (DASM) and circular dichroism (CD). Here we show that, according to DASM data, the minimum cooperatively melting structure is a sHsp-dimer. CD data indicate that Hsp25 major secondary structure, the beta-pleated conformation, is resistant to acidic influence up to pH 4.5 and, at neutral pH values, to heat treatment up to 60 degrees C. The melting pattern of Hsp25/27 bears resemblance to alpha-crystallins. CD data indicate similar secondary, tertiary and quaternary structures of the proteins compared. This finding is in agreement with the revealed homology of primary structure of these proteins and their common chaperone function.

Structural and functional homology between periplasmic bacterial molecular chaperones and small heat shock proteins.

The periplasmic Yersinia pestis molecular chaperone Caf1M belongs to a superfamily of bacterial proteins for one of which (PapD protein of Escherichia coli) the immunoglobulin-like fold was solved by X-ray analysis. The N-terminal domain of Caf1M was found to share a 20% amino acid sequence identity with an inclusion body-associated protein IbpB of Escherichia coli. One of the regions that was compared, was 32 amino acids long, and displayed more than 40% identity, probability of random coincidence was 1.2 x 10(-4). IbpB is involved in a superfamily of small heat shock proteins which fulfil the function of molecular chaperone. On the basis of the revealed homology, an immunoglobulin-like one-domain model of IbpB three-dimensional structure was designed which could be a prototype conformation of sHsp's. The structure suggested is in good agreement with the known experimental data obtained for different members of sHsp's superfamily.

Some new aspects of molecular mechanisms of cyclosporin A effect on immune response.

A few protein targets were found to display a specific high-affinity interaction with the immunosuppressant cyclosporin A (CsA): cytosolic cyclophilins (CyP)A, B, C, D, E containing from 122 to 174 amino acid residues in a polypeptide chain, and secreted forms of CyP; CyP-40, 40-kDa CsA-binding polypeptide complexed with steroid receptor (SR); CyP-related 150-kDa receptor of natural killer (NK) cells; interleukin 8 (IL-8); actin; a family of molecular chaperones hsp70 and P-glycoprotein (P-GP). All CyPs possess peptidyl-prolyl cis-trans isomerase activity (PPIase) and may serve as ATP-independent molecular chaperone proteins. The CsA-CyP complexes are specific inhibitors of Ca(2+)-and calmodulin-dependent protein phosphatase calcineurin (CaN). The inhibition of CaN blocks the activation of genes of IL-2, IL-2R, IL-4, etc. in T cells. In addition, immunosuppressive and/or antiinflammatory activity of CsA can be executed via CyP-40 and hsp 70 complexed with SR, and following the interaction with CyP-related receptor of NK and with IL-8. CsA binding to CyPC, P-GP and actin may throw light on the biochemical events leading to nephrotoxicity and graft vessel disease, two major side effects produced by CsA. The discovery of the interaction of human immunodeficiency virus type 1 (HIV-1) Gag protein with CyP and effective disruption of this interaction by CsA may be important for our understanding of the pathology caused by this immunosuppressive virus and will inspire therapeutic strategies to nip HIV in the bud. Bacterial immunophilins (ImPs) contribute to the virulence of pathogenic microorganisms. Elucidation of molecular mechanisms of microbial ImPs' action in the pathogenesis of bacterial infections may lead to new strategies for designing antibacterial drugs.

Modelling of steric structure of a periplasmic molecular chaperone Caf1M of Yersinia pestis, a prototype member of a subfamily with characteristic structural and functional features.

Steric structure of Caf1M, a periplasmic molecular chaperone of Yersinia pestis, was reconstructed by computer modelling based on a statistically significant primary structure homology between Caf1M and PapD protein from Escherichia coli, and using the known atomic coordinates obtained by the X-ray crystallography for PapD. In the three-dimensional model of Caf1M an accessory sequence between F1 and G1 beta-strands (as compared to PapD) can form a strain-specific part of the binding pocket of surface organell subunits. This accessory sequence decreases the depth of the binding pocket. The characteristic structural feature of the subfamily of periplasmic molecular chaperones with the accessory sequence (Caf1M subfamily) is the existence of exposed to a solvent Cys residues in F1 and G1 beta-strands which can form disulfide bond in the putative binding pocket. The characteristic functional feature of Caf1M subfamily is the chaperoning of more simple compositions of virulence-associated surface organells (in the case of Y. pestis a capsule consists of only F1 protein). Highly conserved R82 and D93, located at the domain surface remote from the putative subunit binding pocket, can participate in direct contacts with the conserved portion of molecular usher proteins.

Cyclophilin A and an antibody against cyclosporin A resemble each other in their binding sites.

The three-dimensional structures of cyclosporin A complexed with cyclophilin A or Fab fragment of a monoclonal antibody were compared. In these two complexes conformations of the fragment D-alanine8-N-methylvaline11 in cyclosporin A were in a good agreement. In addition, cyclophilin A and the Fab fragment had related arrangements of the aromatic amino acids in their binding sites, implying that antibody independently utilizes similar structural themes for binding cyclosporin A as cyclophilin A.

Modelling of interhelical contacts in interferons-beta, -gamma, and dimeric interleukin-5.

On the basis of crystallographic data, the alpha-helices of interferon-beta as well as each domain of interferon-gamma are located in two layers. This results in a different contribution from the amino acids placing in the middle and border alpha-helices of the layer to the hydrophobic core of the molecule. A close analysis of the structure of dimeric interleukin-5 shows related arrangements of alpha-helices. The full schemes of the interhelix contacts of interferons-beta, -gamma, and dimeric interleukin-5 were constructed. Schemes for interleukin-5 show that extension of helices correlates with the dimer topology.

Common structural patterns of cytokine outer surfaces.

Schemes of the four-helix bundle surfaces of interleukin-2, -4, -5, granulocyte/macrophage-, granulocyte-, macrophage-colony-stimulating factor, interferon-beta, -gamma and growth hormone were designed. All cytokines appeared to have the structurally similar "holes" on the surfaces. They were suggested to serve as a part of the main receptor-binding sites.

Structural repeats in cyclosporin A.

The conformation of various dipeptides of free and cyclophilin A-bound cyclosporin A were compared. Cyclosporin A was shown to contain in both states conformationally duplicated fragments in the putative binding sites for cyclophilin A and phosphatase calcineurin.

Comparison of conformations of cyclosporin A and macrolide FK506 fragments: localization of putative binding sites with phosphatase calcineurin.

The three-dimensional structures of two immunosuppressants, cyclosporin A and macrolide FK506, were compared. The sites N-methylglycine3-N-methylleucine4 and valine5-N-methylleucine6 of cyclosporin A were found to be similar to each other (the root-mean-square value was 0.29 A for six reference points of the main chain) and also to the site C17-C22 of FK506 (the root-mean-square values were 0.33 A and 0.13 A, respectively). We suggest these fragments of cyclosporin A and FK506 make a major contribution to the interaction of the immunosuppressants with the phosphatase calcineurin.

Structural similarity of the binding sites of cyclophilin A-cyclosporin A and FKBP-FK506 systems.

Two enzymes, cyclophilin A and FK506-binding protein, with similar cis-trans isomerization catalytic activities but no similarity on the amino acid sequence level were compared in their three-dimensional structure by computer modelling. Cyclophilin A and FK506-binding protein proved to have similar arrangements at nine of the amino acids at their active site pockets. Two inhibitory peptides, cyclosporin A and FK506, also have structural similarities at their contact regions to the active sites. The studied systems may be another example of convergent evolution of enzyme catalytic site.

Protein V, a novel type-II IgG receptor from Streptococcus sp.: sequence, homologies and putative Fc-binding site.

We have cloned and sequenced the Fc-receptor-encoding gene, fcrV, from a group G streptococcus. Considerable similarity was revealed between the FcRV, FcRA76 and M proteins of group A streptococci in their signal sequences and 3' termini, and between the Fc-binding regions of FcRV and FcRA76. The promoter and terminator regions showed no homology with those of the fcrA76 and M protein-encoding genes. The A1-A4 domains of FcrV (protein V) exhibit a heptapeptide repeat motif which is characteristic of alpha-helical coiled-coil proteins. The sequence, Ser-Asn-Arg-Ala-Ala, in the outer position, 'f' of each domain is highly conserved and may be involved in FcR-IgG interactions.

Nonapeptide corresponding to the sequence 27-35 of the mature human IL-2 efficiently competes with rIL-2 for binding to thymocyte receptors [corrected].

Previously it was shown [1] that amino acid substitutions at the region of the first alpha-helix of IL-2 specifically inactivate its reactivity with the intermediate-affinity receptor p70, and mutations in the fifth alpha-helix specifically inactivate the binding to the low-affinity receptor p55. We have synthesized the peptides corresponding to the putative binding site of IL-2 with the intermediate-affinity receptor p70 and found that the nonapeptide corresponding to the sequence 27-35 of the mature IL-2 [2] effectively competes with human rIL-2 for binding to thymocyte receptors. Two types of nonapeptide receptors were revealed: those with Kd1 = 1.84 x 10(-8) M and Kd2 = 1.6 x 10(-7) M. The rIL-2 provides a 100% inhibitory effect on the binding of the 125I-labeled nonapeptide to thymocyte receptors, Ki = 3.5 x 10(-8) M. Low immunoproliferative activity of the peptide allows one to recommend it as a specific antiproliferation drug, IL-2 inhibitor [corrected].