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.     

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.     

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.     

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.     

Modelling the polypeptide backbone with 'spare parts' from known protein structures

An automatic procedure for building a protein polyalanine backbonefrom C positions and ‘spare parts’ retrieved froma data base of 66 high-resolution protein structures is described.Protein backbones are constructed from over-lapping fragmentsof variable length, which allows the backbone of regular secondarystructure elements to be built in one block. The procedure isshown to yield backbones which compare very favourably withthose from highly refined X-ray structures (r.m.s. deviationbetween generated and crystal structures <lÅ). Themethod is furthermore quite insensitive to experimental errorsin C positions as well as to the size of the data base, andis seen to yield valuable insight into the relationships betweensequence and 3-D structure: one example on triose phosphateisomerase, a ß-barrel protein, shows that ßloops can be considered as structurally more uncommon than ßloops. The ‘spare parts’ approach is also foundto be useful for general-purpose modelling of local structuralchanges produced by insertion or deletion of residues. It should,however, be used with caution. Crude selection criteria basedsolely on fragment length and geometric fit to the loop baseregions yield realistic backbones in about two-thirds of thetest cases (r.m.s. deviations from refined crystal structure{small tilde}lÅ). In the remaining cases, sequence information,in particular the presence of glycine residues which tend toadopt more unusual backbone conformations, must be consideredto obtain comparable results.     

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.     

A combinatorial library of an {alpha}-helical bacterial receptor domain

The construction and characterization of a combinatorial libraryof a solvent-exposed surface of an -helical domain derived froma bacterial receptor is described. Using a novel solid-phaseapproach, the library was assembled in a directed and successivemanner utilizing single-stranded oligonucleotides containingmultiple random substitutions for the variegated segments ofthe gene fragment The simultaneous substitution of 13 residuesto all 20 possible amino acids was carried out in a region spanning81 nucleotides. The randomization was made in codons for aminoacids that were modelled to be solvent accessible at a surfacemade up from two of the three a-helices of a monovalent Fc-bindingdomain of staphylococcal protein A. After cloning of the PCR-amplifiedlibrary into a phagemid vector adapted for phage display ofthe mutants, DNA sequencing analysis suggested a random distributionof codons in the mutagenized positions. Four members of thelibrary with multiple substitutions were produced in Escherichiacoli as fusions to an albumin-binding affinity tag derived fromstreptococcal protein G. The fusion proteins were purified byhuman serum albumin affinity chromatography and subsequentlycharacterized by SDSelectrophoresis, CD spectroscopy and biosensoranalysis. The analyses showed that the mutant protein A derivativescould all be secreted as soluble full-length proteins. Furthermore,the CD analysis showed that all mutants, except one with a prolineintroduced into helix 2, have secondary structures in closeagreement with the wild-type domain. These results proved thatmembers of this -helical receptor library with multiple substitutionsin the solvent-exposed surface remain stable and soluble inE.coli. The possibility of using this library for a phenotypicselection strategy to obtain artificial antibodies with novelfunctions is discussed.     

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.     

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.     

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     

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.     

An 8-fold {beta}{alpha} barrel protein with redundant folding possibilities

Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)₁₀proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)₈ protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)₁₀variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)₈ protein. The datasuggest that the folding of the (ß)₁₀ variant is controlledthermodynamically both in vivo and in vitro.     

Stability, activity and flexibility in {alpha}-lactalbumin

-Lactalbumins and the type-c lysozymes are homologues with similarfolds that differ in function and stability. To determine ifthe lower stability of -lactalbumin results from specific substitutionsrequired for its adaptation to a new function, the effects oflysozyme-based and other substitutions on thermal stabilitywere determined. Unblocking the upper cleft in -lactalbuminby replacing Tyr103 with Ala, perturbs stability and structurebut Pro, which also generates an open cleft, is compatible withnormal structure and activity. These effects appear to reflectalternative enthalpic and entropic forms of structural stabilizationby Tyr and Pro. Of 23 mutations, only three, which involve substitutionsfor residues in flexible substructures adjacent to the functionalsite, increase stability. Two are lysozyme-based substitutionsfor Leu110, a component of a region with alternative helix andloop conformations, and one is Asn for Lys114, a residue whosemicroenvironment changes when -lactalbumin interacts with itstarget enzyme. While all substitutions for Leu110 perturb activity,a Lys114 to Asn mutation increases T_m by more than 10°Cand reduces activity, but two other destabilizing substitutionsdo not affect activity. It is proposed that increased stabilityand reduced activity in Lys114Asn result from reduced flexibilityin the functional site of -lactalbumin.     

Automating the identification and analysis of protein {beta}-barrels

ßBarrels are widespread and well-studied featuresof a great many protein structures. In this paper an unsuper-visedmethod for the detection of P-barrels is developed based ontechniques from graph theory. The hydrogen bonded connectivityof ß-sheets is derived using standard pattern recognitiontechniques and expressed as a graph. Barrels correspond to topologicalrings in these connectivity graphs and can thus be identifiedusing ring perception algorithms. Following from this, the characteristictopological structure of a barrel can be expressed using a novelform of reduced nomenclature that counts sequence separationsbetween successive members of the ring set These techniquesare tested by applying them to the detection of barrels in anon-redundant subset of the Brookhaven database. Results indicatethat topological rings do seem to correspond uniquely to ß-barrelsand that the technique, as implemented, finds the majority ofbarrels present in the dataset.     

Molecular dynamics simulations of the whey protein {beta}-lactoglobulin

Molecular dynamics simulations have been used to model the motionsand conformational behavior of the whey protein bovine ß-lactoglobulin.Simulations were performed for the protein by itself and complexedto a single retinol ligand located in a putative interior bindingpocket. In the absence of the retinol ligand, the backbone loopsaround the opening of this ulterior pocket shifted inward topartially close off this cavity, similar to the shifts observedin previously reported molecular dynamics simulations of theuncomplexed form of the homologous retinol binding protein.The protein complexed with retinol does not exhibit the sameconformational shifts. Conformational changes of this type couldserve as a recognition signal allowing in vivo discriminationbetween the free and retinol complexed forms of the 3-lactoglobulinmolecule. The unusual bending of the single a-helix observedin the simulations of retinol binding protein were not observedin the present calculations     

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.     

Comparison of solvent-inaccessible cores of homologous proteins: definitions useful for protein modelling

The three-dimensional structures of 41 homologous proteins (belongingto eight families) were compared by pairwise superposition.A subset of "core" residues was defined as those whose sidechains have <7% of their surface exposed to solvent. Thissubset has significantly higher sequence identity and lowerroot mean square (RMS) carbon separation than for all topologicallyequivalent residues in the structure, when members of a proteinfamily are superposed. For such superpositions the relationshipbetween RMS distance and percentage sequence identity of thissubset of residues is similar to that for all equivalent residues,although some variation is observed between families of proteinswhich are predominantly ß sheet and those which aremainly helix. The definition of a structurally more conservedcore may be ueful in model building proteins from an homologousfamily. The RMS differences of coordinates of structures ofproteins with identical sequences are found to be related tothe resolutions of the structures.     

Characterization of His-X3-His sites in {alpha}-helices of synthetic metal-binding bovine somatotropin

Variants of bovine somatotropin have been engineered to containsynthetic metal-binding sites consisting of two solvent-exposedhistidines separated by a single turn of an -helix (His-X₃-Hisvariants). The affinities of these proteins for Cu(II) werecharacterized by measuring their partitioning coefficients inan aqueous two-phase polymer system. The partition coefficientswere used to generate binding constants for formation of a complexbetween the engineered metal-binding site and Cu(II) chelatedto an iminodiacetic acid derivative of polyethylene glycol.For three His-X₃-His variants described here, these constantsrange from 2x104 to 1.6x106 M-1. The metal affinity of a His-X₃-Hissite depends on the rigidity of the helix into which the siteis engineered. The affinities of the His-X₃-His sites for Cu(II)are large enough to dramatically increase not only the partitioningof these proteins in aqueous two-phase systems, but also theirretention times on a metal-affinity chromatography column. Boththese features can greatly facilitate the purification of engineeredproteins. Criteria for choosing positions for incorporatingmetal-binding sites are discussed.     

The elusive role of the N-terminal extension of {beta}A3- and {beta}Al-crystallin

ß-Crystallins are structural lens proteins with aconserved two-domain structure and variable N- and C-terminalextensions. These extensions are assumed to be involved in quaternaryinteractions within the ß-crystallin oligomers orwith other lens proteins. Therefore, the production of ßA3-and ßAl-crystallin from the single ßA3/A1mRNA by dual translation initiation is of interest. These crystallinsare identical, except that ßAl has a much shorterN-terminal extension than ßA3. This rare mechanismhas been conserved for over 250 million years during the evolutionof the ßA3/A1 gene, suggesting that the generationof different N-terminal extensions confers a selective advantage.We therefore compared the stability and association behaviourof recombinant ßA3- and ßAl-crystallin.Both proteins are equally stable in urea- and pH-induced denaturationexperiments. Gel filtration and analytical ultracentrifugationestablished that ßA3 and ßA1 both form homodimers.In the water-soluble proteins of bovine lens, ßA3and ßA1 are present in the same molecular weight fractions,indicating that they oligomerize equally with other ß-crystallins.¹H-NMR spectroscopy showed that residues Met1 to Asn22 of theN-terminal extension of ßA3 have great flexibilityand are solvent exposed, excluding them from protein interactionsin the homodimer. These results indicate that the differentN-terminal extensions of ßA3 and ßA1 donot affect their homo- or heteromeric interactions.