Conserved mode of peptidomimetic inhibition and substrate recognition of human cytomegalovirus protease

Human cytomegalovirus (HCMV) protease belongs to a new class of serine proteases, with a unique polypeptide backbone fold. The crystal structure of the protease in complex with a peptidomimetic inhibitor (based on the natural substrates and covering the P4 to P1' positions) has been determined at 2.7 A resolution. The inhibitor is bound in an extended conformation, forming an anti-parallel beta-sheet with the protease. The P3 and P1 side chains are less accessible to solvent, whereas the P4 and P2 side chains are more exposed. The inhibitor binding mode shows significant similarity to those observed for peptidomimetic inhibitors or substrates of other classes of serine proteases (chymotrypsin and subtilisin). HCMV protease therefore represents example of convergent evolution. In addition, large conformational differences relative to the structure of the free enzyme are observed, which may be important for inhibitor binding.     

Simultaneous binding of basic peptides at intracellular sites on a large conductance Ca2+-activated K+ channel. Equilibrium and kinetic basis of negatively coupled ligand interactions

The homologous Kunitz inhibitor proteins, bovine pancreatic trypsin inhibitor (BPTI) and dendrotoxin I (DTX-I), interact with large conductance Ca2+-activated K+ channels (maxi-KCa) by binding to an intracellular site outside of the pore to produce discrete substate events. In contrast, certain homologues of the Shaker ball peptide produce discrete blocking events by binding within the ion conduction pathway. In this study, we investigated ligand interactions of these positively charged peptide molecules by analysis of single maxi-KCa channels in planar bilayers recorded in the presence of DTX-I and BPTI, or DTX-I and a high-affinity homologue of ball peptide. Both DTX-I (Kd, 16.5 nM) and BPTI (Kd, 1, 490 nM) exhibit one-site binding kinetics when studied alone; however, records in the presence of DTX-I plus BPTI demonstrate simultaneous binding of these two molecules. The affinity of BPTI (net charge, +6) decreases by 11.7-fold (Kd, 17,500 nM) when DTX-I (net charge, +10) is bound and, conversely, the affinity of DTX-I decreases by 10.8-fold (Kd, 178 nM) when BPTI is bound. The ball peptide homologue (BP; net charge, +6) exhibits high blocking affinity (Kd, 7.2 nM) at a single site when studied alone, but has 8.0-fold lower affinity (Kd, 57 nM) for blocking the DTX-occupied channel. The affinity of DTX-I likewise decreases by 8.4-fold (Kd, 139 nM) when BP is bound. These results identify two types of negatively coupled ligand-ligand interactions at distinct sites on the intracellular surface of maxi-KCa channels. Such antagonistic ligand interactions explain how the binding of BPTI or DTX-I to four potentially available sites on a tetrameric channel protein can exhibit apparent one-site kinetics. We hypothesize that negatively coupled binding equilibria and asymmetric changes in transition state energies for the interaction between DTX-I and BP originate from repulsive electrostatic interactions between positively charged peptide ligands on the channel surface. In contrast, there is no detectable binding interaction between DTX-I on the inside and tetraethylammonium or charybdotoxin on the outside of the maxi-KCa channel.     

Ultrastructural study of anionic sites in glomerular basement membranes at different perfusion pressures by quick-freezing and deep-etching method

In a previous large scale screen for differentially expressed genes in pancreatic cancer, we identified a gene highly overexpressed in cancer encoding a novel putative transmembrane protein with two Kunitz-type serine protease inhibitor domains. The identified gene named kop (Kunitz domain containing protein overexpressed in pancreatic cancer) was assigned to chromosome 19 in the region 19q13.1. Kop was detected at high levels in pancreatic cancer cell lines and was overexpressed in pancreatic cancer tissues as compared to both, normal pancreas and chronic pancreatitis tissues. Being a member of the Kunitz-type serine protease inhibitor family, this new gene may participate in tumour cell invasion and metastasis and in the development of the marked desmoplastic reaction typical for human pancreatic cancer tissues. In this context, the fact that kop has a putative transmembrane domain may have functional implications of particular interest.     

Activating a zymogen without proteolytic processing: mutation of Lys15 and Asn194 activates trypsinogen

The zymogen and mature enzyme forms of trypsin-like serine proteases exhibit a wide range of activities. The prototypical trypsinogen-trypsin system is an example of a minimally active zymogen and a maximally active mature protease. The present work identifies several features of trypsinogen which govern its activity. Our results indicate that rat trypsin is 10(8)-fold more active than rat trypsinogen. Rat trypsinogen appears to be less active than bovine trypsinogen. His40 is believed to be an important determinant of zymogen activity. We are unable to verify this role for His40 in trypsinogen since the mutation of His40 to Phe appears to change the trypsin-substrate interface. Deletion of the N-terminal Ile16 from trypsin is expected to produce a trypsinogen-like protein since the Ile16-Asp194 salt bridge cannot form. Such mutants have higher activity and BPTI affinity than trypsinogen, which indicates that the activation peptide stabilizes the inactive trypsinogen conformation. The mutation of Lys15 to Ala increases the BPTI affinity and activity of trypsinogen to an even greater extent; thus, removal of Lys15 can account for the effect of the loss of the activation peptide. These results suggest that Lys15 is an important determinant of zymogen activity. The mutation of Asp194 to Asn also increases the BPTI affinity and activity of trypsinogen. This result suggests that in addition to stabilizing the active conformation of trypsin via the Ile16-Asp194 salt bridge, Asp194 also maintains the inactive conformation of trypsinogen. A correlation exists between the values of kcat/Km and BPTI affinity of mutant trypsinogens and trypsins. However, the slope of this correlation is 0.64, which indicates that different "active" conformations are involved in BPTI binding and substrate hydrolysis. DeltaI16V17 trypsinogen is the lone outlier; its BPTI affinity is higher than would be expected based on the value of kcat/Km. We show that the rate of BPTI association is slower for DeltaI16V17 trypsinogen than for a mutant trypsinogen with a similar BPTI affinity. This observation suggests that BPTI binds to an "active" trypsinogen conformation that is not kinetically accessible to substrates.     

Structure of extracellular tissue factor complexed with factor VIIa inhibited with a BPTI mutant

The event that initiates the extrinsic pathway of blood coagulation is the association of coagulation factor VIIa (VIIa) with its cell-bound receptor, tissue factor (TF), exposed to blood circulation following tissue injury and/or vascular damage. The natural inhibitor of the TF.VIIa complex is the first Kunitz domain of tissue factor pathway inhibitor (TFPI-K1). The structure of TF. VIIa reversibly inhibited with a potent (Ki=0.4 nM) bovine pancreatic trypsin inhibitor (BPTI) mutant (5L15), a homolog of TFPI-K1, has been determined at 2.1 A resolution. When bound to TF, the four domain VIIa molecule assumes an extended conformation with its light chain wrapping around the framework of the two domain TF cofactor. The 5L15 inhibitor associates with the active site of VIIa similar to trypsin-bound BPTI, but makes several unique interactions near the perimeter of the site that are not observed in the latter. Most of the interactions are polar and involve mutated positions of 5L15. Of the eight rationally engineered mutations distinguishing 5L15 from BPTI, seven are involved in productive interactions stabilizing the enzyme-inhibitor association with four contributing contacts unique to the VIIa.5L15 complex. Two additional unique interactions are due to distinguishing residues in the VIIa sequence: a salt bridge between Arg20 of 5L15 and Asp60 of an insertion loop of VIIa, and a hydrogen bond between Tyr34O of the inhibitor and Lys192NZ of the enzyme. These interactions were used further to model binding of TFPI-K1 to VIIa and TFPI-K2 to factor Xa, the principal activation product of TF.VIIa. The structure of the ternary protein complex identifies the determinants important for binding within and near the active site of VIIa, and provides cogent information for addressing the manner in which substrates of VIIa are bound and hydrolyzed in blood coagulation. It should also provide guidance in structure-aided drug design for the discovery of potent and selective small molecule VIIa inhibitors.     

Structure and multiple conformations of the kunitz-type domain from human type VI collagen alpha3(VI) chain in solution

BACKGROUND: The Kunitz-type inhibitor motif is found at the C terminus of the human collagen alpha3(VI) chain. This 76-residue module (domain C5) was prepared in recombinant form and showed high stability against proteases; however, it lacked any inhibitory activity against trypsin, thrombin, kallikrein and several other proteases. We have undertaken the determination of the three-dimensional (3D) structure of domain C5 in solution, by nuclear magnetic resonance (NMR), in order to establish the structural basis for the properties of this protein. RESULTS: The 7 N-terminal and 12 C-terminal residues of domain C5 are disordered in the solution structure. The 55-residue core, which shows high homology to bovine pancreatic trypsin inhibitor, retains the characteristic fold of all members of the Kunitz-type inhibitor family. 24 residues of this main structural body show more than one resonance, symptomatic of multiple conformations slowly exchanging on the NMR time scale. In addition, significant proton chemical exchange line broadening is observed for residues in the vicinity of the disulfide bridge between residues 20 and 44: this indicates interconversion, on the micro- to millisecond time scale, between multiple conformations. CONCLUSION: The NMR study demonstrates that domain C5 is a highly dynamic molecule at temperatures studied (between 10 and 30 degrees C). Indeed, some 44% of the main body structure of C5 showed multiple conformations. The existence of multiple conformations was not necessarily expected in view of the conformational constraints imposed by the 3D structure of proteins as rigid as C5; it should therefore be considered in the interpretation of its structural and dynamical properties. The accessibility of the inhibitory binding loop (Gly18 [P4] to Leu25 [P4']) should be relatively unaffected by this conformational exchange and thus would not explain the unusual specificity of C5. Most serine proteinase inhibitors that, like C5, have an arginine at the P1 position inhibit trypsin; the lack of trypsin inhibition of C5 must therefore arise from the amino-acid side-chain composition of the adjoining positions in the binding loop.     

The crystal structure of bikunin from the inter-alpha-inhibitor complex: a serine protease inhibitor with two Kunitz domains

Bikunin is a serine protease inhibitor found in the blood serum and urine of humans and other animals. Its sequence shows internal repetition, suggesting that it contains two domains that resemble bovine pancreatic trypsin inhibitor (BPTI). A fragment of bikunin has been crystallised, its structure solved and subsequently refined against 2.5 A data. The two BPTI-like domains pack closely together and are related by an approximate 60 degrees rotation combined with a translation. These domains are very similar to each other and other proteins with this fold. The largest variations occur in the loops responsible for protease recognition. The loops of the first domain are unobstructed by the remaining protein. However, the loops of the second domain are close to the first domain and it is possible that protease binding may be affected or, in some cases, abolished by the presence of the first domain. Thus, cleavage of the two domains could alter the substrate specificity of domain II. Bikunin has a hydrophobic patch close to the N terminus of domain I, which is the most likely site for cell-surface receptor binding. In addition, there is a basic patch at one end of domain II that may be responsible for the inhibition of calcium oxalate crystallization in urine.     

Structures of native and complexed complement factor D: implications of the atypical His57 conformation and self-inhibitory loop in the regulation of specific serine protease activity

Factor D is a serine protease essential for the activation of the alternative pathway of complement. The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at resolution of 2.3 and 1.5 A, respectively, in an isomorphous monoclinic crystal form containing one molecule per asymmetric unit. The native structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site conformations. The current structure shows greater similarity with molecule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differences with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the residues shown to be critical for factor D catalytic activity. The conformational flexibility associated with these regions is proposed to provide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution structure of the factor D/isatoic anhydride complex reveals the binding mode of the mechanism-based inhibitor. The higher specificity towards factor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg218 that is salt bridged with Asp189 at the bottom of the primary specificity (S1) pocket. Comparison of factor D structural variants with other serine protease structures revealed the presence of a unique "self-inhibitory loop". This loop (214-218) dictates the resting-state conformation of factor D by (1) preventing His57 from adopting active tautomer conformation, (2) preventing the P1 to P3 residues of the substrate from forming anti-parallel beta-sheets with the non-specific substrate binding loop, and (3) blocking the accessibility of Asp189 to the positive1y charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the single macromolecular substrate, C3b-bound factor B. This self-inhibitory mechanism is highly correlated with the unique functional properties of factor D, which include high specificity toward factor B, low esterolytic activity toward synthetic substrates, and absence of regulation by zymogen and serpin-like or other natural inhibitors in blood.     

Analysis of electrostatic interactions and their relationship to conformation and stability of bovine pancreatic trypsin inhibitor

The modified Tanford-Kirkwood electrostatic theory has been employed to evaluate pK values for all charge sites in the bovine pancreatic trypsin inhibitor (BPTI). 13C NMR titration data were obtained for all titrating groups except arginine residues in BPTI at nearly constant ionic strength in 0.1 M NaCl, at 41 degrees C. The chemical shifts of 46 resonances were found to be sensitive to pH. The pK values of these titrating resonances compared well with those computed by the modified Tanford-Kirkwood electrostatic theory. A conformational change involving the NH2- and COOH-terminal and nearby residues is shown to be partly electrostatically driven by the formation of a salt bridge between the alpha-amino and alpha-carboxyl groups at mid-pH values. The computed total electrostatic free energy of the molecule is found to be stabilizing at neutral pH despite the substantial net positive charge borne by the protein under such conditions.     

Hydrophobic interactions control zymogen activation in the trypsin family of serine proteases

Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.     

The catalytic domain of lambda site-specific recombinase

The Escherichia coli phage lambda integrase protein (Int) belongs to the large Int family of site-specific recombinases. It is a heterobivalent DNA binding protein that makes use of a high energy covalent phosphotyrosine intermediate to catalyze integrative and excisive recombination at specific chromosomal sites (att sites). A 293-amino acid carboxy-terminal fragment of Int (C65) has been cloned, characterized, and used to further dissect the protein. From this we have cloned and characterized a 188-amino acid, protease-resistant, carboxy-terminal fragment (C170) that we believe is the minimal catalytically competent domain of Int. C170 has topoisomerase activity and converts att suicide substrates to the covalent phosphotyrosine complexes characteristic of recombination intermediates. However, it does not show efficient binding to att site DNA in a native gel shift assay. We propose that lambda Int consists of three functional and structural domains: residues 1-64 specify recognition of "arm-type" DNA sequences distant from the region of strand exchange; residues 65-169 contribute to specific recognition of "core-type" sequences at the sites of strand exchange and possibly to protein-protein interactions; and residues 170-356 carry out the chemistry of DNA cleavage and ligation. The finding that the active site nucleophile Tyr-342 is in a uniquely protease-sensitive region complements and reinforces the recently solved C170 crystal structure, which places Tyr-342 at the center of a 17-amino acid flexible loop. It is proposed that C170 is likely to represent a generic Int family domain that thus affords a specific route to studying the chemistry of DNA cleavage and ligation in these recombinases.     

Hemodynamic and inotropic effects of milrinone after heart transplantation in the setting of recipient pulmonary hypertension

The complementary fragments of human Hb alpha, alpha1-30, and alpha31-141 are spliced together by V8 protease in the presence of 30% n-propanol to generate the full-length molecule (Hb alpha-semisynthetic reaction). Unlike the other protease-catalyzed protein/peptide splicing reactions of fragment complementing systems, the enzymic condensation of nonassociating segments of Hb alpha is facilitated by the organic cosolvent induced alpha-helical conformation of product acting as the "molecular trap" of the splicing reaction. The segments alpha24-30 and alpha31-40 are the shortest complementary segments that can be spliced by V8 protease. In the present study, the chemistry of the contiguous segment (product) alpha24-40 has been manipulated by engineering the amino acid replacements to the positions alpha27 and alpha31 to delineate the structural basis of the molecular trap. The location of Glu27 and Arg31 residues in the contiguous segment alpha24-40 (as well as in other larger segments) is ideal to generate (i, i + 4) side-chain carboxylate-guanidino interaction in its alpha-helical conformation. The amino acid residue replacement studies have confirmed that the side chains at alpha27 and alpha31 facilitate the semisynthetic reaction. The relative influence of the substitute at these sites on the splicing reaction depends on the chemical nature of the side chain and the location. The gamma-carboxylate guanidino side-chain interaction appears to contribute up to a maximum of 85% of the thermodynamic stability of the molecular trap. The studies also demonstrate that the thermodynamic stability of the molecular trap is determined by two interdependent conformational aspects of the peptide. One is an amino acid-sequence-specific event that facilitates the induction of an alpha-helical conformation to the contiguous segment in the presence of organic cosolvent that imparts some amount of protease resistance to Glu30-Arg31 peptide bond. The second structural aspect is a site-specific event, an i, i + 4 side-chain interaction in the alpha-helical conformation of the peptide which imparts an additional thermodynamic stability to the molecular trap. The results suggest that conformationally driven "molecular traps" of protease-mediated ligation reactions of peptides could be designed into products to facilitate the modular assembly of peptides/proteins.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

BACKGROUND: Trimeresurus stejnejeri venom plasminogen activator (TSV-PA) is a snake venom serine proteinase that specifically activates plasminogen. Snake venom serine proteinases form a subfamily of trypsin-like proteinases that are characterised by a high substrate specificity and resistance to inhibition. Many of these venom enzymes specifically interfere with haemostatic mechanisms and display a long circulating half-life. For these reasons several of them have commercial applications and are potentially attractive pharmacological tools. RESULTS: The crystal structure of TSV-PA has been determined to 2.5 A resolution and refined to an R factor of 17.8 (R free, 24.4). The enzyme, showing the overall polypeptide fold of trypsin-like serine proteinases, displays unique structural elements such as the presence of a phenylalanine at position 193, a C-terminal tail clamped via a disulphide bridge to the 99-loop, and a structurally conserved Asp97 residue. The presence of a cis proline at position 218 is in agreement with evolutionary relationships to glandular kallikrein. CONCLUSIONS: We postulate that Phe 193 accounts for the high substrate specificity of TSV-PA and renders it incapable of forming a stable complex with bovine pancreatic trypsin inhibitor and other extended substrates and inhibitors. Mutational studies previously showed that Asp97 is crucial for the plasminogenolytic activity of TSV-PA, here we identify the conservation of Asp97 in both types of mammalian plasminogen activator - tissue-type (tPA) and urokinase-type (uPA). It seems likely that Asp97 of tPA and uPA will have a similar role in plasminogen recognition. The C-terminal extension of TSV-PA is conserved among snake venom serine proteinases, although its function is unknown. The three-dimensional structure presented here is the first of a snake venom serine proteinase and provides an excellent template for modelling other homologous family members.     

Determinants of backbone dynamics in native BPTI: cooperative influence of the 14-38 disulfide and the Tyr35 side-chain

15Nitrogen relaxation experiments were used to characterize the backbone dynamics of two modified forms of bovine pancreatic trypsin inhibitor (BPTI). In one form, the disulfide between Cys14 and Cys38 in the wild-type protein was selectively reduced and methylated to generate an analog of the final intermediate in the disulfide-coupled folding pathway. The second form was generated by similarly modifying a mutant protein in which Tyr35 was replaced with Gly (Y35G). For both selectively reduced proteins, the overall conformation of native BPTI was retained, and the relaxation data for these proteins were compared with those obtained previously with the native wild-type and Y35G proteins. Removing the disulfide from either protein had only small effects on the observed longitudinal relaxation rates (R1) or heteronuclear cross relaxation rates (nuclear Overhauser effect), suggesting that the 14-38 disulfide has little influence on the fast (ps to ns) backbone dynamics of either protein. In the wild-type protein, the pattern of residues undergoing slower (micros to ms) internal motions, reflected in unusually large transverse relaxation rates (R2), was also largely unaffected by the removal of this disulfide. It thus appears that the large R2 rates previously observed in native wild-type protein are not a direct consequence of isomerization of the 14-38 disulfide. In contrast with the wild-type protein, reducing the disulfide in Y35G BPTI significantly decreased the number of backbone amides displaying large R2 rates. In addition, the frequencies of the backbone motions in the modified protein, estimated from R2 values measured at multiple refocusing delays, appear to span a wider range than those seen in native Y35G BPTI. Together, these observations suggest that the slow internal motions in Y35G BPTI are more independent in the absence of the 14-38 disulfide and that formation of this bond may lead to a substantial loss of conformational entropy. These effects may account for the previous observation that the Y35G substitution greatly destabilizes the disulfide. The results also demonstrate that the disulfide and the buried side-chain influence the dynamics of the folded protein in a highly cooperative fashion, with the effects of removing either being much greater in the absence of the other.     

Crystal structure of human RhoA in a dominantly active form complexed with a GTP analogue

The 2.4-A resolution crystal structure of a dominantly active form of the small guanosine triphosphatase (GTPase) RhoA, RhoAV14, complexed with the nonhydrolyzable GTP analogue, guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), reveals a fold similar to RhoA-GDP, which has been recently reported (Wei, Y., Zhang, Y., Derewenda, U., Liu, X., Minor, W., Nakamoto, R. K., Somlyo, A. V., Somlyo, A. P., and Derewenda, Z. S. (1997) Nat. Struct. Biol. 4, 699-703), but shows large conformational differences localized in switch I and switch II. These changes produce hydrophobic patches on the molecular surface of switch I, which has been suggested to be involved in its effector binding. Compared with H-Ras and other GTPases bound to GTP or GTP analogues, the significant conformational differences are located in regions involving switches I and II and part of the antiparallel beta-sheet between switches I and II. Key residues that produce these conformational differences were identified. In addition to these differences, RhoA contains four insertion or deletion sites with an extra helical subdomain that seems to be characteristic of members of the Rho family, including Rac1, but with several variations in details. These sites also display large displacements from those of H-Ras. The ADP-ribosylation residue, Asn41, by C3-like exoenzymes stacks on the indole ring of Trp58 with a hydrogen bond to the main chain of Glu40. The recognition of the guanosine moiety of GTPgammaS by the GTPase contains water-mediated hydrogen bonds, which seem to be common in the Rho family. These structural differences provide an insight into specific interaction sites with the effectors, as well as with modulators such as guanine nucleotide exchange factor (GEF) and guanine nucleotide dissociation inhibitor (GDI).     

Structural and energetic aspects of protein-protein recognition

Specific recognition between proteins plays a crucial role in a great number of vital processes. In this review different types of protein-protein complexes are analyzed on the basis of their three-dimensional structures which became available in recent years. The complexes which are analyzed include: those resulting from different types of recognition between proteinase and protein inhibitor (canonical inhibitors of serine proteinases, hirudin, inhibitors of cysteine proteinases, carboxypeptidase inhibitor), barnase-barstar, human growth hormone-receptor and antibody-antigen. It seems obvious that specific and strong protein-protein recognition is achieved in many different ways. To further explore this question, the structural information was analyzed together with kinetic and thermodynamic data available for the respective complexes. It appears that the energy and rates of specific recognition of proteins are influenced by many different factors, including: area of interacting surfaces; complementarity of shapes, charges and hydrogen bonds; water structure at the interface; conformational changes; additivity and cooperativity of individual interactions, steric effects and various (conformational, hydration) entropy changes.     

The atomic structure of protein-protein recognition sites

The non-covalent assembly of proteins that fold separately is central to many biological processes, and differs from the permanent macromolecular assembly of protein subunits in oligomeric proteins. We performed an analysis of the atomic structure of the recognition sites seen in 75 protein-protein complexes of known three-dimensional structure: 24 protease-inhibitor, 19 antibody-antigen and 32 other complexes, including nine enzyme-inhibitor and 11 that are involved in signal transduction.The size of the recognition site is related to the conformational changes that occur upon association. Of the 75 complexes, 52 have "standard-size" interfaces in which the total area buried by the components in the recognition site is 1600 (+/-400) A2. In these complexes, association involves only small changes of conformation. Twenty complexes have "large" interfaces burying 2000 to 4660 A2, and large conformational changes are seen to occur in those cases where we can compare the structure of complexed and free components. The average interface has approximately the same non-polar character as the protein surface as a whole, and carries somewhat fewer charged groups. However, some interfaces are significantly more polar and others more non-polar than the average. Of the atoms that lose accessibility upon association, half make contacts across the interface and one-third become fully inaccessible to the solvent. In the latter case, the Voronoi volume was calculated and compared with that of atoms buried inside proteins. The ratio of the two volumes was 1.01 (+/-0.03) in all but 11 complexes, which shows that atoms buried at protein-protein interfaces are close-packed like the protein interior. This conclusion could be extended to the majority of interface atoms by including solvent positions determined in high-resolution X-ray structures in the calculation of Voronoi volumes. Thus, water molecules contribute to the close-packing of atoms that insure complementarity between the two protein surfaces, as well as providing polar interactions between the two proteins.     

The immunoglobulin superfamily: an insight on its tissular, species, and functional diversity

The immunoglobulin superfamily (IgSF) is a heterogenic group of proteins built on a common fold, called the Ig fold, which is a sandwich of two beta sheets. Although members of the IgSF share a similar Ig fold, they differ in their tissue distribution, amino acid composition, and biological role. In this paper we report an up-to-date compilation of the IgSF where all known members of the IgSF are classified on the basis of their common functional role (immune system, antibiotic proteins, enzymes, cytokine receptors, etc.) and their distribution in tissue (neural system, extracellular matrix, tumor marker, muscular proteins, etc.), or in species (vertebrates, invertebrates, bacteria, viruses, fungi, and plants). The members of the family can contain one or many Ig domains, comprising two basic types: the constant domain (C), with seven strands, and the variable domain (V), with eight, nine, or ten strands. The different overviews of the IgSF led to the definition of new domain subtypes, mainly concerning the C type, based on the distribution of strands within the two sheets. The wide occurrence of the Ig fold and the much less conserved sequences could have developed from a common ancestral gene and/or from a convergent evolutionary process. Cell adhesion and pattern recognition seem to be the common feature running through the entire family.     

Methods for studying microvascular barrier function in ischemia-reperfusion injury

Mutants of an archaeon Halobacterium halobium, resistant to the universal inhibitor of translation, pactamycin, were isolated. Pactamycin resistance correlated with the presence of mutations in the 16 S rRNA gene of H. halobium single rRNA operon. Three types of mutations were found in pactamycin resistant cells, A694G, C795U and C796U (Escherichia coli 16 S rRNA numeration) located distantly in rRNA primary structure but probably neighboring each other in the three-dimensional structure. Pactamycin resistance mutations either overlapped (C795U) or were located in the immediate vicinity of nucleotides protected by the drug in E. coli and H. halobium 16 S rRNA indicating that corresponding rRNA sites might be directly involved in pactamycin binding. Ribosomal functions were not affected significantly either by mutation of C795 (one of the positions protected by the P-site-bound tRNA), or by mutations of A694 and C796 (which neighbor nucleotides protected by tRNA) suggesting that tRNA-dependent protections of C795 and G693 are explained by a conformational change in the ribosome induced by the P-site-bound tRNA. A novel mode of pactamycin action is proposed suggesting that pactamycin restricts structural transitions in 16 S rRNA preventing the ribosome from adopting a functional conformation induced by tRNA binding.