Characterization of the interactions of a bifunctional inhibitor with {alpha}-thrombin by molecular modelling and peptide synthesis

A potent thrombin inhibitor, [D-Phe⁴⁵, Arg⁴⁷] hirudin 45-65,that contains an active site-directed sequence D-Phe-Pro-Arg-Pro,an exosite specific fragment hirudin 55-65 (H55-65) and a linkerportion hirudin 49-54, was designed based on the hirudin sequence[DiMaio et al. (1990) J. Biol. Chem., 265, 21698-21798]. A three-dimensionalmodel of the complex between the B-chain of human thrombin andthe inhibitor [D-Phe45, Arg47⁴⁵, Arg⁴⁷] hirudin 45-65 was constructedusing molecular modelling starting from the X-ray Ca coordinatesof the thrombin-hirudin complex and the NMR-derived structureof the thrombin-bound hirudin 55 - 65. The contribution of theH49 -54 fragment to the thrombin - inhibitor interaction wasdeduced by examining a series of analogs containing single glycinesubstitution and analogs with reduced number of residues withinthe linker. The results were consistent with the molecular modellingobservations i.e. the H49-54 fragment serves the role of a spacerin the binding interaction and could be replaced by four glycineresidues. The studies on the interaction of the exosite-directedportion of the inhibitor with thrombin using a series of syntheticH55 -65 analogs demonstrated that residues AspH55 to ProH60play a major role in binding to human thrombin where the sidechains of PheH56, IleH59 and GluH57 showed critical contributions.Molecular modelling suggested that these side chains may contributeto inter- and intramolecular hydrophobic and electrostatic interactions,respectively.     

Three-dimensional structure of protein C inhibitor predicted from structure of {alpha}1-antitrypsin with computer graphics

The three-dimensional structure of a proteolytically modifiedprotein C inhibitor, a member of the serine protease inhibitorsuperfamily, was constructed with computer graphics based onits amino acid sequence homology with that of the modified 1-antitrypsinwhose structure had been elucidated by X-ray crystallography.The intact form of protein C inhibitor was predicted with an-carbon model based on its hydrophilicity and hydrogen bondpattern. Furthermore, a model of its interaction with activatedprotein C was constructed based on the structure of the complexbetween trypsin and its inhibitor, which had been determinedby X-ray crystallography.     

Expression of recombinant {alpha}Ains-crystallin and not {alpha}A-crystallin inhibits bacterial growth

A-Crystallin and A^ins-crystallin are derived from the A-crystallingene via alternative splicing. They are identical except forthe presence of a polypeptide, 23 amino acids long, encodedby the ‘insert’ exon. Evolutionary logic would suggestthat the insertion of a 23 amino acid peptide in the middleof A-crystallin, a protein evolving more slowly than eitherhistone H1, cytochrome c or hemoglobin, would lead to appreciablestructural and functional changes. However, based on physico-chemicalstudies, it is presently believed that A-crystallin and A^ins-crystallinare functionally equivalent and that the presence of the ‘insert’peptide in A^Ins-crystallin is inconsequential. We report herethat the independent expression of recombinant A^Ins-crystallin,and not A-crystallin, inhibits growth of the bacterial host.These observations were confirmed in co-expression experiments,wherein both the proteins were expressed in the same cell. Interestingly,growth inhibition is reversible. Importantly, the data demonstratethat it is catalytic amounts and not the gross accumulationof A^Ins-crystalline which causes growth inhibition. Given theprior knowledge that A-crystallin and A^Ins-crystallin differby a peptide of 23 amino acids, these data suggest that the‘insert peptide’ in A^Ins-crystallin imparts propertieson this protein that are different from A-crystallin.     

Efficient cleavage by {alpha}-thrombin of a recombinant fused protein which contains insulin-like growth factor I

The gene for insulin-like growth factor I (IGF-I) was constructedfrom chemically synthesized deoxyoligonucleotides and expressedin Escherichia coli, under the control of a trp promoter, asa set of fusion proteins which were connected with a portionof human growth hormone through the recognition sequence fora sequence-specific protease, either blood coagulation factorXa or -thrombin. Upon induction with 3-indoleacrylic acid, fusionproteins accumulated with a yield of 10–30% of the totalprotein. A fusion protein connected through a tetradecapeptide(Asp-Asp-Pro-Pro-Thr-Val-Glu-Leu-Gln-Gly-Leu-Val-Pro-Arg) wasefficiently and correctly cleaved by -thrombin, and the purifiedIGF-I possessed somatomedin-like activity, as determined bythe enhancement of sulfation of glycosaminoglycans in culturedcostal chondrocytes from rabbits.     

The {alpha}/{beta} hydrolase fold

We have identified a new protein fold—the /ßhydrolase fold—that is common to several hydrolytic enzymesof widely differing phylogenetic origin and catalytic function.The core of each enzyme is similar: an /ß sheet, notbarrel, of eight ß-sheets connected by -helices. Theseenzymes have diverged from a common ancestor so as to preservethe arrangement of the catalytic residues, not the binding site.They all have a catalytic triad, the elements of which are borneon loops which are the best-conserved structural features inthe fold. Only the histidine in the nucleophile-histidine-acidcatalytic triad is completely conserved, with the nucleophileand acid loops accommodating more than one type of amino acid.The unique topological and sequence arrangement of the triadresidues produces a catalytic triad which is, in a sense, amirror-image of the serine protease catalytic triad. There arenow four groups of enzymes which contain catalytic triads andwhich are related by convergent evolution towards a stable,useful active site: the eukaryotic serine proteases, the cysteineproteases, subtilisins and the /ß hydrolase fold enzymes.     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the {alpha}/{beta}-barred packing)

We present here a spectroscopic structural characterizationof octarellin, a recently reported de novo protein modelledon /ß-barrel proteins [K. Go raj, A.Renard and J.A.Martial(1990) Protein Engng, 3, 259–266]. Infrared and Ramanspectra analyses of octarellin‘s secondary structure revealthe expected percentage of -helices (30%) and a higher ß-sheetcontent (40%) than predicted from the design. When the Ramanspectra obtained with octarellin and native triosephosphateisomerase (a natural /ß-barrel) are compared, similarpercentages of secondary structures are found. Thermal denaturationof octarellin monitored by CD confirms that its secondary structuresare quite stable, whereas its native-like tertiary fold is not.Tyrosine residues, predicted to be partially hidden from solvent,are actually exposed as revealed by Raman and UV absorptionspectra. We conclude that the attempted /ß-barrelconformation in octarellin may be loosely packed. The criteriaused to design octarellin are discussed and improvements suggested.     

Mutational analysis of the N-capping box of the {alpha}-helix of chymotrypsin inhibitor 2

The N-terminus of the helix of the chymotrypsin inhibitor 2from barley (CI2) has an N-capping box (Ser at the first positionin the helix and Glu at position 4) as well as a frequentlyfound Glu at position 3. The energetic importance of this motifhas been studied by determining the free energy of unfoldingof the wild-type and protein mutants derived from those residuesusing guanidinium chloride-induced denaturation and differentialscanning microcalorimetry. Mutating N-cap residue Ser31 to eitherAla or Gly destabilizes CI2 by 0.8-1 kcal mol^–1. Truncationof the box in the mutants SA31EA33EA34 or SG31EA33EA34 destabilizesthe protein by 1.5–2 kcal mol^–1. The N-capping boxis an important motif in stabilizing proteins and delineatingthe beginning of -helices in the pathway of protein folding.     

Structure-function validation of high lysine analogs of {alpha}-hordothionin designed by protein modeling

Cereal grains and legume seeds, which are key protien sourcesfor the vegetarian diet, are generally deficient in essentialamino acids. Maize, in particular, is deficient in lysine. Theinherent lack of lysine-rich protiens in maize has necessitatedthe search for heterologous protiens enriched in this aminoacid, the isolation of the corresponding gene and its ultimateintroduction into maize through plant transformation techniques.However, a rate-limiting step to this strategy has been theavailability of plant-derived lysine-rich protiens. An appealingsolution to the problem is to artificially increase the lysinecontent of a given protein by mutating appropriate residuesto lysine. Here, we expound this strategy, starting with theprotein hordothionin that is derived from barley seeds andconsists of five lysine residues in a total of 45 amino acids(11 % lysine). To facilitate rational substitutions, the 3-Dstructure of the protein has been determined by homology modelingwith crambin. based on this model, we have identified surfaceresidues amenable to substitution with lysine. Furthermore,the acceptability of the mutations has been validated throughthe syntheses and characterization of the derivatives. To thisend, our approach has permitted the creation of a modified -hordothionin protein that has a lysine content of 27 % andretains the antifungal activity of the wild-type protein.     

Composition analysis of {alpha}-helices in thermophilic organisms

We present a statistical comparison of the amino acid compositionin a secondary structure element, the -helix, of proteins stableat high temperatures with those which are less so. This studyhas shown that the temperature-dependent Zimm-Bragg helix propagationvalue s is not a good predictor for the helix-forming tendencyof an amino acid in thermostable proteins. However, we haveshown that s, the change in s from 20 to 60°C, accuratelypredicts the direction of the probability shift for 15 aminoacids in thermostable protein a-helices, although it does notpredict the magnitude of that change. The residues tyrosine,glycine and glutamine show a significant increase in residencyin a-helices for thermostable proteins over their nonthermostablecounterparts. Significant decreases in -helix residency occurfor the residues valine, glutamic acid, histidine, cysteineand aspartic acid in proteins from thermophilic organisms. Aromaticinteractions, hydrogen bonding and a reduction of charge mayexplain the increase observed for tyrosine and glutamine andthe decrease in glutamic acid and aspartic acid, although packingconsiderations cannot be ruled out The only physical explanationfor the increase in glycine would seem to be its positive svalue     

The crystal structure of {alpha}-bungarotoxin at 2.5 ? resolution: relation to solution structure and binding to acetylcholine receptor

We report collection of 2.5 ? resolution X-ray diffraction datafrom newly grown crystals of the rare ‘small unit cell’form of the long snake neurotoxin, -bungarotoxin. The previousmodel of the molecule has been rebuilt, and refined using least-squaremethods to a crystallographic residual of 0.24 at 2.5 ? resolution.-Bungarotoxin's crystal structure is compared with the crystalstructures of two other snake neurotoxins (cobratoxin and erabutoxin),and with its solution structure inferred from spectroscopicstudies. Significant differences include less ß-sheetin bungarotoxm's crystal structure than in solution, or in thecrystal structures of other neurotoxins, and an unusual orientationin the crystal of the invariant tryptophan. The functional,binding surface of bungarotoxin is described; it consists primarilyof hydrophobic and hydrogen-bonding groups and only a few chargedside chains. The structure is compared with experimental bindingparameters for neurotoxins.     

Modeling the intact form of the {alpha}1-proteinase inhibitor

The structure of the intact form of the serpin ₁-proteinaseinhibitor has been modeled based on the assumption that thecentral strand s4A of the six-stranded ß-sheet A ofthe cleaved inhibitor is not incorporated into the sheet ofintact ₁-proteinase inhibitor. This strand was removed fromits position in the center of the sheet by suitable rotationsabout the backbone dihedrals of Lys343 using molecular graphics.The resulting structure was then annealed using molecular dynamics(MD) while applying progressive distance restraints to the reactivepeptide bond (Met358-Ser359) for 50 ps. During this time, thedisrupted ß-sheet reformed to create a five-strandedß-sheet with strands 3 and 5 in a parallel arrangement.This change and accompanying structural rearrangements are largelyconfirmed by the X-ray structure of plakalbumin, whose structurereflects the overall structure of intact serpins. The successfulmodeling experiment demonstrates the utility of MD for makinggross structural predictions based on related structures. Thebinding loop of the intact form is modeled to allow dockingwith serine proteinases, in particular thrombin, which mosthighly constrains the possible conformations of the bindingloop.     

Cloning of rabbit interleukin-1{beta}: differential evolution of IL-1{alpha} and IL-1{beta} proteins

We have cloned the rabbit IL-1ß cDNA, which encodesa 268 amino acid precursor similar in length to other sequencedIL-1 precursors. Comparison of all published IL-1 and IL-1ßsequences respectively indicates that the IL-1 gene family isevolving faster than the IL-1ß family, and that thetwo genes diverged –270 million years ago. Surprisingly,there are differences in the regions preferentially conservedwithin the two families. The IL-1 family is most conserved atthe amino terminus whereas the IL-1ß family is mostconserved in the carboxy-terminal half. This is despite thefact that the carboxy-terminal half encodes the active portionof both molecules and would be expected to adopt a similar ß-sheetstructure in IL-1 as in the published X-ray structure of matureIL-1ß. These findings suggest that differences inthe function and properties of the IL-1 and IL-1ßprecursor molecules may have been conserved. These differencesmay therefore provide an explanation for the existence of twoIL-1 molecules.     

The S variant of human {alpha}1-antitrypsin, structure and implications for function and metabolism

The S variant of the human ₁-antitrysin with E-264 – V,is responsible for a mild ₁-antitrypsin deficiency quite commonin the European population. S protein specifically cleaved atthe susceptible peptide bond was crystallized and its crystalstructure determined and refined to 3.1 Å resolution.The S variant crystallizes isomorphous to the normal M variant.The difference Fourier electron density map shows the E –V change as outstanding residual density. In addition, smallstructural changes of the main polypeptide chain radiate fromthe site of mutation and affect parts far removed from it. Bythe mutation, internal hydrogen bonds and salt linkages of E-264to Y-38 and K-487, respectively, are lost. They cause the far-reachingslight distortions and are probobly related to the reduced thermalstability of the S mutant. They may also be responsible forslower folding of the polypeptide chain and the clinical symptomsof ₁-antitrypsin deficiency. In a theoretical study by moleculardynamics methods simulations of the M and S proteins were madeand the results analysed with respect to structrual and dynamicproperties and compared with the experimental results. Thereis a significant correlation between experimental and theoreticalresults in some respects.     

Family of G protein {alpha} chains: amphipathic analysis and predicted structure of functional domains

The G proteins transduce hormonal and other signals into regulationof enzymes such as adenylyl cyclase and retinal cGMP phosphodiesterase.Each G protein contains an subunit that binds and hydrolyzesguanine nucleotides and interacts with ß subunitsand specific receptor and effector proteins. Amphipathic andsecondary structure analysis of the primary sequences of fivedifferent chains (bovine _s, _t1 and _t2, mouse _i, and rat _o)predicted the secondary structure of a composite chain (_avg).The chains contain four short regions of sequence homologousto regions in the GDP binding domain of bacterial elongationfactor Tu (EF-Tu). Similarities between the predicted secondarystructures of these regions in _avg and the known secondary structureof EF-Tu allowed us to construct a three-dimensional model ofthe GDP binding domain of _avg. Identification of the GDP bindingdomain of _avg defined three additional domains in the compositepolypeptide. The first includes the amino terminal 41 residuesof _avg, with a predicted am phipathic helical structure; thisdomain may control binding of the chains to the ßcomplex. The second domain, containing predicted ßstrands and helices, several of which are strongly amphipathic,probably contains sequences responsible for interaction of chains with effector enzymes. The predicted structure of thethird domain, containing the carhoxy terminal 100 amino acids,is predominantly ß sheet with an amphipathic helixat the carboxy terminus. We propose that this domain is reponsiblefor receptor binding. Our model should help direct further experimentsinto the structure and function of the G protein chain.     

Estimating the twist of {beta}-strands embedded within a regular parallel {beta}-barrel structure

The parallel ß-barrel is a recurrent structural motiffound in a large variety of different enzymes belonging to thefamily of /ß-proteins. It has been shown previouslythat the hyperboloid can be considered as a scaffold describingthe parallel ß-barrel structure. To assess restraintson ß-strand twist imposed by a given scaffold geometry,the notion of scaffold twist, T_s, is introduced. From T_s, theß-strand twist (Tw_ß) expected for a givenscaffold geometry can be derived and it is verified that thiscomputed twist can be used to identify ß-barrels characterizedby good hydrogen bonding. It is noted that Tw_ß isonly slightly affected for ß-barrels differing inthe number (N) of ß-strands, suggesting that restraintson main-chain conformation of ß-strands are not likelyto account for the N = 8 invariability observed in natural parallelß-barrels thereby strengthening previous work rationalizingthis constancy.     

Production and characterization of anti-human interferon {gamma} receptor antibody fragments that inhibit cytokine binding to the receptor

Three single-chain antibody fragments that recognize the extracellularhuman interferon receptor -chain (IFNR), and inhibit the bindingof human IFN, have been produced in Escherichia coli. Thesefragments are derived from murine anti-receptor monoclonal antibodies,and comprise the variable heavy (V_H) domain linked to the variablelight (V_L) chain through a 15 amino acid linker [(GGGGS)₃].Using surface plasmon resonance technology (BIAcore), the solubleproteins were shown to retain a high affinity for recombinantIFNR, and by radioimmunoassay to possess high inhibitory activitytowards IFN-binding to human Raji cells. The antibody fragmentsmost likely recognize epitopes that overlap the cytokine bindingsite on the receptor surface. Attempts to dissect further theantibodies to isolated V_H- and V_L-chains and to synthetic linearand cyclic peptides derived from the individual complementaritydetermining regions failed to afford fragments with significantIFNR binding affinity. Nevertheless, these native-like variableregion fragments and petidomimetics derived from them are ofinterest in the design of novel IFNR antagonists.     

Inhibition of {beta}S-chain dependent polymerization by synergistic complementation of contact site perturbations of {alpha}-chain: application of semisynthetic chimeric {alpha}-chains

Mouse _1–30-horse _31–141 chimeric -chain, a semisyntheticsuper-inhibitory -chain, inhibits ß^S-chain dependent polymerizationbetter than both parent -chains. Although contact site sequencedifferences are absent in the _1–30 region of the chimericchain, the four sequence differences of the region _17-22 couldinduce perturbations of the side chains at ₁₆, ₂₀ and ₂₃, thethree contact sites of the region. A synergistic complementationof such contact site perturbation with that of horse _31–141probably results in the super-inhibitory activity of the chimeric-chain. The inhibitory contact site sequence differences, bythemselves, could also exhibit similar synergistic complementation.Accordingly, the polymerization inhibitory activity of Hb Le-Lamentin(LM) mutation [His20()Gln], a contact site sequence difference,engineered into human–horse chimeric -chain has been investigatedto map such a synergistic complementation. Gln20() has littleeffect on the O₂ affinity of HbS, but in human–horse chimeric-chain it reduces the O₂ affinity slightly. In the chimeric-chain, Gln20() increased sensitivity of the ßßcleft for the DPG influence, reflecting a cross-talk betweenthe ₁ß₁ interface and ßß cleft in this semisyntheticchimeric HbS. In the human -chain frame, the polymerizationinhibitory activity of Gln20() is higher compared with horse_1–30, but lower than mouse _1–30. Gln20() synergisticallycomplements the inhibitory propensity of horse _31–141.However, the inhibitory activity of LM–horse chimeric-chain is still lower than that of mouse–horse chimeric-chain. Therefore, perturbation of multiple contact sites inthe _1–30 region of the mouse–horse chimeric -chainand its linkage with the inhibitory propensity of horse _31–141has been now invoked to explain the super-inhibitory activityof the chimeric -chain. The `linkage-map' of contact sites canserve as a blueprint for designing synergistic complementationof multiple contact sites into -chains as a strategy for generatingsuper-inhibitory antisickling hemoglobins for gene therapy ofsickle cell disease.     

Structure of {alpha}-{alpha}-hairpins with short connections

This paper presents the results of detailed stereochemical analysisof structures and sequences of --hairpins with short connections.It is shown that --hairpins of each given type have very similarpatterns of hydrophobic, hydrophilic and glycine residues intheir amino acid sequences. These results can be used in theprediction of --hairpin conformation as well as in protein designand engineering.     

Recognition of transmembrane {alpha}-helical segments with environmental profiles

A method for assessing the environmental properties of membrane-spanning-helical peptides in proteins has beenproposed. The algorithmemploys a set of environmental preference parameters derivedfor amino acid residues based on the analysis of the 3-D structuresof membrane domains in bacteriorhodopsin and photoreaction centersRhodopseudomonasviridis and Rhodobacter sphaeroides. The resulting3-D–1-D scores for transmembrane segments are significantlydifferent from those derived for -helices in globular proteins.The parameters obtained havebeen used to construct environmentalprofiles for membrane -helices in bacteriorhodopsin and photoreactioncenters. The profiles successfully recognize their own sequencesin several specially designed large databases. The method hasbeenapplied to several membrane proteins with unknown spatial structures.Most of their membrane-spanning peptides were efficiently recognizedby the profiles. The predicted environment of the residues inthe membrane segments fits the experimental data well. The approachis independent of any homology data and can be employed to delineatethe membrane segments of a protein with environmental characteristicsclose to those of bacteriorhodopsin and photoreaction centers.The alignment of these segments with the reference profilesprovides a considerable amount of data about their lipid andprotein exposure.     

Efficient generation of a reshaped human mAb specific for the {alpha} toxin of Clostridium perfringens

We have used the technique of antibody reshaping to producea humanized antibody specific for the a toxin of Clostridiumperfringens. The starting antibody was from a mouse hybridomafrom which variable (V) region nucleo-tide sequences were determined.The complementarity-determining regions (CDRs) from these Vregions were then inserted into human heavy and light chainV region genes with human constant region gene fragments subsequentlyadded. The insertion of CDRs alone into human frameworks didnot produce a functional reshaped antibody and modificationsto the V region framework were required. With minor frameworkmodifications, the affinity of the original murine mAb was restoredand even exceeded. Where affinity was increased, an alteredbinding profile to overlapping peptides was observed. Computermodelling of the reshaped heavy chain V regions suggested thatamino acids adjacent to CDRs can either contribute to, or distort,CDR loop conformation and must be adjusted to achieve high bindingaffinity.