Topology of the stable serpin-protease complexes revealed by an autoantibody that fails to react with the monomeric conformers of antithrombin

Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P1 and P'1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of beta-sheet A, which becomes strand 5 of beta-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P14 to P6 of the RCL are inserted into beta-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex.     

Role of the P6-P3' region of the serpin reactive loop in the formation and breakdown of the inhibitory complex

Serpins have a large external peptide loop known as the reactive loop. Part of the reactive loop functions as the primary recognition site for target proteases; however, the complete role of the reactive loop in determining serpin specificity is unclear. In the current study, we investigated the reactive loop region that could potentially interact with the extended binding site of target proteases; the P6-P3' region. We utilized a reactive loop switching strategy to determine the extent to which the inhibitory activity of alpha-1-protease inhibitor (PI) against human neutrophil elastase (HNE) could be transferred to alpha-1-antichymotrypsin (ACT), a serpin that does not inhibit HNE. A series of ACT-PI chimeras were constructed in which segments of increasing length taken from the P6-P3' region of PI replaced the corresponding residues of ACT. The effectiveness of each chimera as an inhibitor of HNE was assessed by measuring (1) the rate of inhibitory complex formation and (2) the rate of complex breakdown (complex stability). Although all the ACT-PI chimeras were fully functional against chymotrypsin-like proteases, the series of chimeras showed no consistent progress toward the production of an inhibitor with the inhibitory properties of PI. The most rapid complex formation and most stable complexes were observed for chimeras with the P3-P1 residues of PI, whereas extending the replacement region to the P6 residue resulted in a considerable decrease in both inhibitory parameters. In order to study two additional features of the PI reactive loop that may play a role in the presentation of the P6-P3' region to HNE, we constructed variants that contained a P4' proline and deleted the P6'-P9' residues. Changes on the prime side appeared to have little effect on rates of inhibition or complex stability. Overall, even the most effective chimeras demonstrated an inhibition rate constant at least 60-fold less than that observed for PI inhibition of HNE and the most long lived chimera-HNE complexes broke down more rapidly than PI-HNE complexes. These results indicate that residues in the reactive loop region predicted to contact a specific target protease cannot fully transfer inhibitory activity from one serpin to another, suggesting that specific reactive loop-serpin body and serpin body-protease body interactions play a significant role in determining serpin inhibitory activity against target proteases.     

Characterization of a human alpha1-antitrypsin variant that is as stable as ovalbumin

The metastability of inhibitory serpins (serine proteinase inhibitors) is thought to play a key role in the facile conformational switch and the insertion of the reactive center loop into the central beta-sheet, A-sheet, during the formation of a stable complex between a serpin and its target proteinase. We have examined the folding and inhibitory activity of a very stable variant of human alpha1-antitrypsin, a prototype inhibitory serpin. A combination of seven stabilizing single amino acid substitutions of alpha1-antitrypsin, designated Multi-7, increased the midpoint of the unfolding transition to almost that of ovalbumin, a non-inhibitory but more stable serpin. Compared with the wild-type alpha1-antitrypsin, Multi-7 retarded the opening of A-sheet significantly, as revealed by the retarded unfolding and latency conversion of the native state. Surprisingly, Multi-7 alpha1-antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of inhibition as the wild type, indicating that enhanced A-sheet closure conferred by Multi-7 does not affect the complex formation. It may be that the stability increase of Multi-7 alpha1-antitrypsin is not sufficient to influence the rate of loop insertion during the complex formation.     

Biological implications of a 3 A structure of dimeric antithrombin

BACKGROUND: Antithrombin, a member of the serpin family of inhibitors, controls coagulation in human plasma by forming complexes with thrombin and other coagulation proteases in a process greatly accelerated by heparin. The structures of several serpins have been determined but not in their active conformations. We have determined the structure of intact antithrombin in order to study its mechanism of activation, particularly with respect to heparin, and the dysfunctions of this mechanism that predispose individuals to thrombotic disease. RESULTS: The crystal structure of a dimer of one active and one inactive molecule of antithrombin has been determined at 3 A. The first molecule has its reactive-centre loop in a predicted active conformation compatible with initial entry of two residues into the main beta-sheet of the molecule. The inactive molecule has a totally incorporated loop as in latent plasminogen activator inhibitor-1. The two molecules are linked by the reactive loop of the active molecule which has replaced a strand from another beta-sheet in the latent molecule. CONCLUSION: The structure, together with identified mutations affecting its heparin affinity, allows the placement of the heparin-binding site on the molecule. The conformation of the two forms of antithrombin demonstrates the extraordinary mobility of the reactive loop in the serpins and provides insights into the folding of the loop required for inhibitory activity together with the potential modification of this by heparin. The mechanism of dimerization is relevant to the polymerization that is observed in diseases associated with variant serpins.     

Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length

The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction. Mutations within this region have been used to generate novel potentially therapeutic inhibitors. In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors. The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively. AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them. ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition. The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1. These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR). AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1. These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation. This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin.     

A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism

A mutant recombinant plasminogen activator inhibitor 1 (PAI-1) was created (Ser-338-->Cys) in which cysteine was placed at the P9 position of the reactive center loop. Labeling this mutant with N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) provided a molecule with a fluorescent probe at that position. The NBD-labeled mutant was almost as reactive as wild type but was considerably more stable. Complex formation with tissue or urokinase type plasminogen activator (tPA or uPA), and cleavage between P3 and P4 with a catalytic concentration of elastase, all resulted in identical 13-nm blue shifts of the peak fluorescence emission wavelength and 6.2-fold fluorescence enhancements. Formation of latent PAI showed the same 13-nm spectral shift with a 6.7-fold fluorescence emission increase, indicating that the NBD probe is in a slightly more hydrophobic milieu. These changes can be attributed to insertion of the reactive center loop into the beta sheet A of the inhibitor in a manner that exposes the NBD probe to a more hydrophobic milieu. The rate of loop insertion due to tPA complexation was followed using stopped flow fluorimetry. This rate showed a hyperbolic dependence on tPA concentration, with a half-saturation concentration of 0.96 microM and a maximum rate constant of 3.4 s-1. These results demonstrate experimentally that complexation with proteases is presumably associated with loop insertion. The identical fluorescence changes obtained with tPa.PAI-1 and uPA.PAI-1 complexes and elastase-cleaved PAI-1 strongly suggest that in the stable protease-PAI-1 complex the reactive center loop is cleaved and inserted into beta sheet A and that this process is central to the inhibition mechanism.     

Inhibitory mechanism of serpins: loop insertion forces acylation of plasminogen activator by plasminogen activator inhibitor-1

Serpin inhibitors are believed to form an acyl enzyme intermediate with their target proteinases which is stabilized through insertion of the enzyme-linked part of the reactive center loop (RCL) as strand 4 in beta-sheet A of the inhibitor. To test critically the role and timing of these steps in the reaction of the plasminogen activator inhibitor PAI-1, we blocked the vacant position 4 in beta-sheet A of this serpin with an octapeptide. The peptide-blocked PAI-1 was a substrate for both tissue-type plasminogen activator (tPA) and trypsin and was hydrolyzed at the scissile bond. The reactivity of the peptide-blocked substrate PAI-1 was compared to that of the unmodified inhibitor by rapid acid quenching as well as photometric techniques. With trypsin as target, the limiting rate constants for enzyme acylation were essentially the same with inhibitor and substrate PAI-1 (21-23 s-1), as were also the associated apparent second-order rate constants (2.8-2.9 microM-1 s-1). With tPA, inhibitor and substrate PAI-1 reacted identically to form a tightly bound Michaelis complex (Kd approximately Km approximately 20 nM). The limiting rate constant for acylation of tPA, however, was 57 times faster with inhibitor PAI-1 (3.3 s-1) than with the substrate form (0.059 s-1), resulting in a 5-fold difference in the corresponding second-order rate constants (13 vs 2.5 microM-1 s-1). We attribute the ability of tPA to discriminate between the two PAI-1 forms to exosite bonds that cannot occur with trypsin. The exosite bonds retain specifically the distal part of the PAI-1 RCL in the substrate pocket, which favors a reversal of the acylation step. Acylation of tPA becomes effective only by separating the products of the acylation step. With substrate PAI-1, this depends on passive displacement of bonds, whereas with inhibitor PAI-1, separation is accomplished by loop insertion that pulls tPA from its docking site on PAI-1, resulting in faster acylation than with substrate PAI-1.     

Virtual colonoscopy: what will the issues be?

To determine whether formation of the stable complex between a serpin and a target proteinase involves a major translocation of the proteinase from its initial position in the noncovalent Michaelis complex, we have used fluorescence resonance energy transfer to measure the separation between fluorescein attached to a single cysteine on the serpin and tetramethylrhodamine conjugated to the proteinase. The interfluorophore separation was determined for the noncovalent Michaelis-like complex formed between alpha 1-proteinase inhibitor (Pittsburgh variant) and anhydrotrypsin and for the stable complex between the same serpin and trypsin. A difference in separation between the two fluorophores of approximately 21 A was found for the two types of complex. This demonstrates a major movement of the proteinase in going from the initial noncovalent encounter complex to the kinetically stable complex. The change in interfluorophore separation is most readily understood in terms of movement of the proteinase from the reactive center end of the serpin toward the distal end, as the covalently attached reactive center loop inserts into beta-sheet A of the serpin.     

The crystal structure of zinc-containing ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7

The crystal structure of ferredoxin from the thermoacidophilic archaeon Sulfolobus sp. strain 7 was determined by multiple isomorphous replacement supplemented with anomalous scattering effects of iron atoms in the Fe-S clusters, and refined at 2.0 A resolution to a crystallographic R value of 0.173. The structural model contains a polypeptide chain of 103 amino acid residues, 2 [3Fe-4S] clusters, and 31 water molecules; in this model, the cluster corresponding to cluster II in bacterial dicluster ferredoxins loses the fourth iron atom although it may originally be a [4Fe-4S] cluster. The structure of the archaeal ferredoxin consists of two parts: the core fold part (residues 37-103) and the N-terminal extension part (residues 1-36). The "core fold" part has an overall main-chain folding common to bacterial dicluster ferredoxins, containing two clusters as the active center, two alpha-helices near the clusters, and two sheets of two-stranded antiparallel beta-sheet (the terminal and central beta-sheets). The "N-terminal extension" part is mainly formed by a one-turn alpha-helix and a three-stranded antiparallel beta-sheet. The beta-sheet in the N-terminal extension is hydrogen-bonded with the terminal beta-sheet in the core fold to form a larger beta-sheet. The distinct structural feature of this archaeal ferredoxin lies in the zinc-binding center where the zinc ion is tetrahedrally ligated by four amino acid residues (His 16, His 19, and His 34 from the N-terminal extension, and Asp 76 from the core fold). The zinc ion in the zinc-binding center is located at the interface between the core fold and the N-terminal extension, and connects the beta-sheet in the N-terminal extension and the central beta-sheet in the core fold through the zinc ligation. Thus, the zinc ion plays an important role in stabilizing the structure of the present archaeal ferredoxin by connecting the N-terminal extension and the core fold, which may be common to thermoacidophilic archaeal ferredoxins.     

Context-dependent nature of destabilizing mutations on the stability of FKBP12

The context-dependent nature in which mutations affect protein stability was investigated using the FK506-binding protein, FKBP12. Thirty-four mutations were made at sites throughout the protein, including residues located in the hydrophobic core, the beta-sheet, and the solvent-exposed face of the alpha-helix. Urea-induced denaturation experiments were used to measure the change in stability of the mutants relative to that of the wild type (Delta DeltaGU-F). The results clearly show that the extent of destabilization, or stabilization, is highly context-dependent. Correlations were sought in order to link Delta DeltaGU-F to various structural parameters. The strongest correlation found was between Delta DeltaGU-F and N, the number of methyl(ene) groups within a 6 A radius of the group(s) deleted. For mutations of buried hydrophobic residues, a correlation coefficient of 0.73 (n = 16,where n is the number of points) was obtained. This increased to 0.81 (n = 24) on inclusion of mutations of partially buried hydrophobic residues. These data could be superimposed on data obtained for other proteins for which similarly detailed studies have been performed. Thus, the contribution to stability from hydrophobic side chains, independent of the extent to which a side chain is buried, can be estimated quantitatively using N. This correlation appears to be a general feature of all globular proteins. The effect on stability of mutating polar and charged residues in the alpha-helix and beta-sheet was also found to be highly context-dependent. Previous experimental and statistical studies have shown that specific side chains can stabilize the N-caps of alpha-helices in proteins. Substitutions of Ile56 to Thr and Asp at the N-cap of the alpha-helix of FKBP12, however, were found to be highly destabilizing. Thus, the intrinsic propensities of an amino acid for a particular element of secondary structure can easily be outweighed by tertiary packing factors. This study highlights the importance of packing density in determining the contribution of a residue to protein stability. This is the most important factor that should be taken into consideration in protein design.     

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).     

The X-ray structure of Escherichia coli enoyl reductase with bound NAD+ at 2.1 A resolution

Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.     

Improved contractility and coronary flow in isolated hearts after 1-day hypothermic preservation with isoflurane is not dependent on K(ATP) channel activation

S8 is one of the core ribosomal proteins. It binds to 16 S RNA with high affinity and independently of other ribosomal proteins. It also acts as a translational repressor in Escherichia coli by binding to its own mRNA. The structure of Thermus thermophilus S8 has been determined by the method of multiple isomorphous replacement at 2.9 A resolution and refined to a crystallographic R-factor of 16.2% (Rfree 27.5%). The two domains of the structure have an alpha/beta fold and are connected by a long protruding loop. The two molecules in the asymmetric unit of the crystal interact through an extensive hydrophobic core and form a tightly associated dimer, while symmetry-related molecules form a joint beta-sheet of mixed type. This type of protein-protein interaction could be realized within the ribosomal assembly. A comparison of the structures of T. thermophilus and Bacillus stearothermophilus S8 shows that the interdomain loop is eight residues longer in the former and reveals high structural conservation of an extensive region, located in the C-terminal domain. From mutational studies this region was proposed earlier to be involved in specific interaction with RNA. On the basis of these data and on the comparison of the two structures of S8, it is proposed that the three-dimensional structure of specific RNA binding sites in ribosomal proteins is highly conserved among different species.     

Core mutants of the immunoglobulin binding domain of streptococcal protein G: stability and structural integrity

A library of core mutants of the GB1 domain of streptococcal protein G was created, and the structure and stability of selected members was assessed by 1H-15N heteronuclear correlation NMR spectroscopy and fluorescence. All mutants comprised changes in beta-sheet residues, with sidechains at positions 5 (Leu), 7 (Leu), 52 (Phe) and 54 (Val) forming the beta-sheet side of the sheet-helix core interface. A solvent exposed position Ile-6 was chosen as a control. Randomization of bases at codon positions 1 and 3 with thymine at position 2 introduces five possible hydrophobic amino acids, namely Leu, Val, Ile, Phe, and Met. The distribution of encoded amino acids at all five positions is approximately as expected theoretically and indicates that no major bias was introduced towards particular residues. The overall structural integrity of several mutants, as assessed by NMR, ranges from very close to wild type to fully unfolded. Interestingly, the stability of the mutants is not strictly correlated with the number of changes or residue volume.     

Structural basis of the RNA-binding specificity of human U1A protein

The RNP domain is a very common eukaryotic protein domain involved in recognition of a wide range of RNA structures and sequences. Two structures of human U1A in complex with distinct RNA substrates have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning the origin of binding specificity. The beta-sheet of the domain provides an extensive RNA-binding platform for packing aromatic RNA bases and hydrophobic protein side chains. However, many interactions between functional groups on the single-stranded nucleotides and residues on the beta-sheet surface are potentially common to RNP proteins with diverse specificity and therefore make only limited contribution to molecular discrimination. The refined structure of the U1A complex with the RNA polyadenylation inhibition element reported here clarifies the role of the RNP domain principal specificity determinants (the variable loops) in molecular recognition. The most variable region of RNP proteins, loop 3, plays a crucial role in defining the global geometry of the intermolecular interface. Electrostatic interactions with the RNA phosphodiester backbone involve protein side chains that are unique to U1A and are likely to be important for discrimination. This analysis provides a novel picture of RNA-protein recognition, much closer to our current understanding of protein-protein recognition than that of DNA-protein recognition.     

Polyalanine-based peptides as models for self-associated beta-pleated-sheet complexes

The occurrence of beta-sheet motifs in a number of neurodegenerative disorders has brought about the need for the de novo design of soluble model beta-sheet complexes. Such model complexes are expected to further the understanding of the interconversion processes that occur from cellular allowed random coil or alpha-helical conformation into insoluble cell-deleterious beta-pleated-sheet motifs. In the present study, polyalanine-based peptides (i.e., derived from Ac-KA14K-NH2) were designed that underwent conformational changes from monomeric random coil conformations into soluble, macromolecular beta-pleated-sheet complexes without any covalent modification. The interconversion was found to be length-, environment-, and concentration-dependent and to be driven by hydrophobic interactions between the methyl groups of the alanine side chains. A series of substitution analogs of Ac-KA14K-NH2 was used to study the amino acid acceptability within the hydrophobic core of the complex, as well as at both termini. The formation of amyloid plaques in a number of amyloidogenic peptides could be related to the presence of amino acids within their sequences that were found to have a high propensity to occur in these model beta-sheet complexes.     

Events in the kinetic folding pathway of a small, all beta-sheet protein

The folding of cardiotoxin analogue III (CTX III), a small (60 amino acids), all beta-sheet protein from the venom of the Taiwan Cobra (Naja naja atra) is here investigated. The folding kinetics is monitored by using a variety of techniques such as NMR, fluorescence, and circular dichroism spectroscopy. The folding of the protein is complete within a time scale of 200 ms. The earliest detectable event in the folding pathway of CTX III is the formation of a hydrophobic cluster, which possess strong affinity to bind to nonpolar dye such as 1-anilino-8-napthalene-sulfonic acid. Quenched-flow deuterium-hydrogen exchange experiments indicate that the segment spanning residues 51-55 along with Lys23, Ile39, Val49, Tyr51 and Val52 could constitute the "hydrophobic cluster." Folding kinetics of CTX III based on the amide-protection data reveals that the triple-stranded, antiparallel beta-sheet segment, which is located in the central core of the molecule, appears to fold faster than the double-stranded beta-sheet segment.     

Hydrogen bonding at the active site of delta 5-3-ketosteroid isomerase

The solution secondary structure of the highly active Y55F/Y88F "Tyr-14-only" mutant of delta 5-3-ketosteroid isomerase complexed with 19-nortestosterone hemisuccinate has been shown to consist of three helices, a six-stranded mixed beta-sheet, and five turns. The steroid binds near the general acid, Tyr-14, on helix 1, near the general base, Asp-38, on the first strand of the beta-sheet, and on the hydrophobic face of the beta-sheet [Zhao, Q., Abeygunawardana, C., & Mildvan, A. S. (1997) Biochemistry 36, 3458-3472]. On this hydrophobic face, Asp-99 is the only polar residue. Free isomerase shows a deshielded exchangeable proton resonance at 13.1 ppm assigned to the N epsilon H of neutral His-100. Its fractionation factor (phi = 0.79) and slow exchange with solvent suggest it to be buried or involved in an H-bond. The binding of dihydroequilenin or estradiol to isomerase induces the appearance of two additional deshielded proton resonances, one at 18.2 ppm assigned to the gamma-carboxyl proton of Asp-99, and the other, at 11.6 ppm, assigned to the zeta-OH proton of Tyr-14. While mutation of Asp-99 to Ala results in the disappearance of only the resonance near 18 ppm [Wu, R. W., Ebrahemian, S., Zwrotny, M. E., Thornberg, L. D., Perez-Alverado, G. C., Brothers, P., Pollack, R. M., & Summers, M. F. (1997) Science 276, 415-418], both of these resonances disappear in mutants lacking Tyr-14, suggesting an H-bonded catalytic diad, Asp-99-COOH--Tyr14-OH--O-steroid enolate. The catalytic diad is further supported by NOEs from the beta 1 and beta 2 protons of Asp-99 to the epsilon protons of Tyr-14, and from the zeta-OH proton of Tyr-14 to the gamma-carboxyl proton of Asp-99, indicating close proximity of these two residues, and by other data from the literature. A strong, low-barrier H-bond between Asp-99 and Tyr-14 is indicated by the 6.2 ppm deshielding, low fractionation factor (phi = 0.34) and slow exchange of the resonance at 18.2 ppm. A normal H-bond between Tyr-14 and the steroid is indicated by the 1.8 ppm deshielding, fractionation factor of 0.97 and the slow exchange of the resonance at 11.6 ppm. It is suggested that the 10(4.7)-fold contribution of Tyr-14 to catalysis is made possible by strong H-bonding from Asp-99 in the catalytic diad which strengthens general acid catalysis by Tyr-14. It is also noted that highly deshielded proton resonance on enzymes between 15 and 20 ppm, assigned to low-barrier H-bonds, generally involve carboxyl groups.     

Three-dimensional structure of an Fab-peptide complex: structural basis of HIV-1 protease inhibition by a monoclonal antibody

F11.2.32, a monoclonal antibody raised against HIV-1 protease (Kd = 5 nM), which inhibits proteolytic activity of the enzyme (K(inh) = 35(+/-3)nM), has been studied by crystallographic methods. The three-dimensional structure of the complex between the Fab fragment and a synthetic peptide, spanning residues 36 to 46 of the protease, has been determined at 2.2 A resolution, and that of the Fab in the free state has been determined at 2.6 A resolution. The refined model of the complex reveals ten well-ordered residues of the peptide (P36 to P45) bound in a hydrophobic cavity at the centre of the antigen-binding site. The peptide adopts a beta hairpin-like structure in which residues P38 to P42 form a type II beta-turn conformation. An intermolecular antiparallel beta-sheet is formed between the peptide and the CDR3-H loop of the antibody; additional polar interactions occur between main-chain atoms of the peptide and hydroxyl groups from tyrosine residues protruding from CDR1-L and CDR3-H. Three water molecules, located at the antigen-antibody interface, mediate polar interactions between the peptide and the most buried hypervariable loops, CDR3-L and CDR1-H. A comparison between the free and complexed Fab fragments shows that significant conformational changes occur in the long hypervariable regions, CDR1-L and CDR3-H, upon binding the peptide. The conformation of the bound peptide, which shows no overall structural similarity to the corresponding segment in HIV-1 protease, suggests that F11.2.32 might inhibit proteolysis by distorting the native structure of the enzyme.     

The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits

The D4/S4-5 interhelical region plays a role in sodium channel fast inactivation. Examination of S4-5 primary structure in all domains suggests a possible amphipathic helical conformation in which a conserved group of small hydrophobic residues occupies one contiguous surface with a more variable complement of nonpolar and polar residues on the opposite face. We evaluated this potential structure by replacing each residue in D4/S4-5 of the rat SkM1 skeletal muscle sodium channel with substitutions having different side chain properties. Of the 63 mutations analyzed, 44 produced functional channels. P1473 was intolerant of substitutions. Nonpolar substitutions in the conserved hydrophobic region were functionally similar to wild type, while charged mutations in this region before P1473 were nonfunctional. Charged mutations at F1466, M1469, M1470, and A1474, located on the opposite surface of the predicted helix, produced functional channels with pronounced slowing of inactivation, shifted voltage dependence of steady-state inactivation, and increased rate of recovery from inactivation. The substituted-cysteine-accessibility method was used to probe accessibility at each position. Residues L1465, F1466, A1467, M1469, M1470, L1472, A1474, and F1476C were easily accessible for modification by sulfhydryl reagents; L1464, L1468, S1471, and L1475 were not accessible within the time frame of our measurements. Molecular dynamics simulations of residues A1458 to N1477 were then used to explore energetically favorable local structures. Based on mutagenesis, substituted-cysteine-accessibility method, and modeling results, we suggest a secondary structure for the D4/S4-5 region in which the peptide chain is alpha-helical proximal to P1473, bends at this residue, and may continue beyond this point as a random coil. In this configuration, the entire resultant loop is amphipathic; four residues on one surface could form part of the binding site for the inactivation particle.