Functional diversity of the phosphoglucomutase superfamily: structural implications

Three-dimensional structural models of three members of thephosphoglucomutase (PGM) superfamily, parafusin, phosphoglucomutase-relatedprotein and sarcoplasmic reticulum phosphoglucomutase, wereconstructed by homology modeling based on the known crystalstructure of rabbit muscle phosphoglucomutase. Parafusin, phosphoglucomutase-relatedprotein and sarcoplasmic reticulum phosphoglucomutase each have50% or more identity with rabbit muscle phosphoglucomutase atthe amino acid level and all are reported to exhibit no or minorphosphoglucomutase activity. There are four major insertionsand two deletions in the parafusin sequence relative to PGM,all of which are located in surface-exposed loops connectingsecondary structural elements. The remaining amino acid substitutionsare distributed throughout the sequence and are not predictedto alter the polypeptide fold. Parafusin contains a putativeprotein kinase C site located on a surface loop in domain IIthat is not present in the homologs. Although the general domainstructure and the active site of rabbit muscle phosphoglucomutaseare preserved in the model of phosphoglucomutase-related protein,a major structural difference is likely to occur in domain 1due to the absence of 55 amino acid residues in PGM-RP. Thisdeletion predicts the loss of three -helices and one ß-strandfrom an anti-parallel ß-sheet in this domain as comparedwith the rabbit muscle phosphoglucomutase.     

Relationships between sequence and structure for the four-{alpha}-helix bundle tertiary motif in proteins

The sequences of four--helical bundle proteins are characterizedby a pattern of hydrophilic and hydrophobic amino acids whichis repeated every seven residues. At each position of the heptadrepeat there are specific constraints on the amino acid propertieswhich result from the topology of the tertiary motif. Theseconstraints give rise to patterns of amino acid distributionwhich are distinct from those of other proteins. The distributionsin each of the heptad positions have been determined by a statisticalanalysis of structural and sequence data derived from sevenfamines of aligned protein sequences. The constitution of eachposition is dominated by a very small number of different aminoacids, with the core positions consisting overwhelmingly ofLeu and Ala. The positional preferences of the individual aminoacids can be generally interpreted in terms of residue propertiesand topological constraints. The potential for four-a-helixbundle folding is reflected primarily in the pattern of residueoccurrence in the heptad and not in the overall amino acid compositionof the protein. Possible applications of this analysis in structurepredictions, sequence alignments and in the rational designand engineering of four-a-helkal bundle proteins are discussed.     

Sequence divergence analysis for the prediction of seven-helix membrane protein structures: I. Comparison with bacteriorhodopsin

A method using protein sequence divergence to predict the three-dimensionalstructure of the transmembrane domain of seven-helix membraneproteins is described. The key component in the multistep procedureis the calculation of a hydrophilic and lipophilic variabilityindex for each amino acid in an alignment of a family of homologousproteins. The variability profile, a plot of the calculatedvariability index versus alignment position, can be used topredict a tertiary model of the backbone conformation of thetransmembrane domain. This method was applied to bacteriorhodopsin(BR) and the model obtained was compared with the known structureof this protein. Using an alignment of the amino acid sequencesof BR and closely related (20% identity) proteins, the boundariesof the transmembrane regions, their secondary structures andorientations inside the membrane bilayer were predicted basedon the variability profile. Additional information about theshape of the helix bundle was also obtained from the averagevariability of each transmembrane helix with the assumptionthat the helices are packed sequentially and form a closed helixbundle. Correct features of the known structure of BR were foundin the model structure, suggesting that a similar strategy canbe used to predict transmembrane helices and the packing shapeof other membrane proteins with seven transmembrane helices,such as the opsins and other G-protein coupled receptors.     

Binding of the globular domain of linker histones H5/H1 to the nucleosome: a hypothesis

The amino acid sequence of the central globular domain of histoneH1/H5 family members is highly homologous. Twenty-four suchsequences have been compared to establish the conserved andvariable residues. Fitting this to the tertiary structure ofthe H5 globular domain shows which of the conserved and variableresidues are peripheral and which internal. Particular attentionis paid to conserved basic residues on the surface, which wetake to be DNA binding. Variable regions and conserved acidicresidues are assumed not to be sites of contact with DNA. Weconclude that one face of the domain, containing a cluster ofbasic residues, is the principal DNA binding site whilst twoopposing faces, orthogonal to the principal site and also containingconserved basic residues, are subsidiary DNA binding sites.Since the DNA binding surface of the domain covers a full 180°arc, we propose that it contacts a ‘cage’ of threeDNA strands on the 2-fold axis of the chromatosome.     

Prediction of hypervariable CDR-H3 loop structures in antibodies

The structure of the most variable antibody hypervariable loop,CDR-H3, has been predicted from amino acid sequence alone. Incontrast to other approaches predictions are made for loop lengthsup to 17 residues. The predictions have been achieved usingartificial neural networks which are trained on a large setof loops from the Brookhaven Protein Databank which have structuressimilar to CDRH3. The loop structures are described by the twobackbone dihedral angles and for each residue. For 21 CDR-H3loops unique to the neural network, the prediction of dihedralangles leads to an average root mean square deviation in theCartesian coordinates of 2.65 Å. The present method, whencombined with existing modelling protocols, provides an importantaddition to the structural prediction of the complementaritydetermining regions of antibodies.     

Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumour necrosis factor beta (lymphotoxin)

Single amino acid substitutions were generated in predictedhydrophilic loop regions of the human tumour necrosis factorbeta (TNF-ß) molecule, and the mutant proteins wereexpressed in Escherichia coli and purified. Mutants with singleamino acid changes at either of two distinct loop regions, atpositions aspartic acid 50 or tyrosine 108, were found to havegreatly reduced receptor binding and cytotoxic activity. Thesetwo regions in TNF-ß correspond to known loop regionswhere mutations also result in loss of biological activity ofTNF–, a related cytokine which shares the same cellularreceptors with TNF-ß. The two distinct loops at positions31-34 and 84-89 in the known three-dimensional structure ofTNF- (equivalent to positions 46–50 and 105–110respectively in TNF-ß), lie on opposite sides of theTNF- monomer. When the TNF-a monomer forms a trimer, the twoloops, each from a different subunit of the trimer, come togetherand lie in a cleft between adjacent subunits. Together, thesefindings suggest that a TNF receptor binds to a cleft betweensubunits via surface loops at amino acid residues 31–34and 84–89 in TNF–, and similarly via surface loopsincluding amino acids aspartic acid 50 and tyrosine 108 in TNF–ß.     

Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1

Many proteins have been predicted to contain domains with immunoglobulin-Iikefolds and hence to be members of the immunoglobulin superfamily(IgSF). However, several members lack the Cys residues capableof forming the disulphide bond that forms a characteristic bridgebetween the ß sheets in the Ig fold, e.g. domain 1of the lymphocyte antigen CD2. The assignment of ßstrands in CD2 by sequence analysis was tested by attemptingto introduce a disulphide bridge between the ß sheetsby mutating two residues in the relevant positions to Cys. Mutant,soluble forms of CD2 were expressed in Chinese hamster ovarycell lines and amino add sequencing showed that a disulphidebond was formed as predicted, but not in the control where oneCys residue was misplaced by four residues. Evidence that bothmutated molecules folded correctly is given by the indistinguishablebinding of three monoclonal antibodies recognising differentepftopes on CD2. The 3-D structure of rat CD2 domain 1 has beendetermined by NMR spectroscopy and X-ray crystallography, confirmingthe predictions from the sequence. Applications of this methodof insertion of disulphide residues for probing protein structuresare discussed, together with other structures of IgSF domainslacking the typical inter-sheet disulphide bond.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

Prediction and analysis of SH2 domain-phosphopeptide interactions

Src homology 2 (SH2) domains are small protein modules of -100amino acids that are found in many proteins involved in intracellularsignal transduction. They mediate protein-protein interactionsand modulate enzyme activity by their ability to bind to specificsequence patterns that contain a phosphorylated tyrosine. Asthe three-dimensional structures of the phosphatidylinositol(PI) 3-kinase, Lck, Src and Abl SH2 domains have been shownto be similar, we have modelled other SH2 domains that showdistinct sequence specificity to allow comparative analysisof SH2-phosphopeptide interactions. The SH2 domains of PLC-Nterm.,Nck, Grb2, GAP and Abl have been model-built with high-affinityphosphopeptides fitted into the putative binding sites. Foreach SH2 domain a detailed analysis of the peptide-protein interactionwas performed. It is apparent that specificity is mainly conferredby three to five residues downstream from the phosphotyrosineresidue (Y*), especially, although not exclusively, peptideposition Y* + 3. The SH2 pocket that binds the Y* + 3 residueis mainly composed of three sections: part of strand (ßEgoing into loop EF, part of B and loop BG. The residues thatconstitute the Y* +3 binding pocket show variability that seemsto determine which amino acid binds preferentially. Residueposition ßE4 seems to play a vital role in the SH2specificity. This study shows that the development of modellingprotocols for SH2 domains whose structure has not been determinedcan prove very useful in predicting which residues are involvedin conferring the affinity and binding specificity of thesedomains towards distinct phosphotyrosine-containing sequences.     

Modeling the three-dimensional structure of the monocyte chemo-attractant and activating protein MCAF/MCP-1 on the basis of the solution structure of interleukin-8

A model of the three-dimensional structure of the monocyte chemo-attractantand activating protein MCAF/MCP-1 is presented. The model ispredicted based on the previously determined solution structureof interleukin-8 (IL-8/NAP-1) [Clore, G.M., Appella, E., Yamada,M., Matsushima, K. and Gronenborn, A.M. (1990) Biochemistry29, 1689–1696]. Both proteins belong to a superfamilyof cytokine proteins involved in cell-specific chemotaxis, hostdefense and the inflammatory response. The amino acid sequenceidentity between the two proteins is 24%. It is shown that theregular secondary structure elements of the parent structurecan be retained in the modeled structure, such that the backbonehydrogen bonding pattern is very similar in the two structures.The polypeptide backbone is superimposable with an atomic r.m.s.difference of 0.9 Å and all side chains can be modeledby transferring the parent side chain conformation to the newstructure. Thus, the deduced structure, like the parent one,is a dimer and consists of a six-stranded antiparallel /3-sheet,formed by two three-stranded Greek keys, one from each monomer,upon which lie two symmetry-related antiparallel a-helices,24 Å long and separated by 14 Å. All amino acidsequence changes can be accommodated within the parent polypeptideframework without major rearrangements. This is borne out bythe fact that the IL-8/NAP-1 and modeled MCAF/MCP-1 structureshave similar non-bonding energies. These results strongly suggestthat both proteins and all other members of the superfamilymost likely have the same tertiary structure. Analysis of thedistribution of the solvent-exposed residues can be interpretedin the context of the different receptors involved in mediatingthe specific responses to both proteins and suggests that thedifferent activities of the two proteins, namely neutrophil(IL-8) versus monocyte (MCAF/MCP-1) activation and chemotaxis,reside in the specific arrangements of amino acid side chainspointing outwards from and lying in the cleft between the twoexposed long a-helices.     

Selection and characterization of HER2/neu-binding affibody ligands

Affibody® (affibody) ligands that are specific for the extracellulardomain of human epidermal growth factor receptor 2 (HER2/neu)have been selected by phage display technology from a combinatorialprotein library based on the 58 amino acid residue staphylococcalprotein A-derived Z domain. The predominant variants from thephage selection were produced in Escherichia coli, purifiedby affinity chromatography, and characterized by biosensor analyses.Two affibody variants were shown to selectively bind to theextracellular domain of HER2/neu (HER2-ECD), but not to controlproteins. One of the variants, denoted His₆-Z_HER2/neu:4, wasdemonstrated to bind with nanomolar affinity (     

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.     

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 stability and unfolding of an IgG binding protein based upon the B domain of protein A from Staphylococcus aureus probed by tryptophan substitution and fluorescence spectroscopy

The stability and unfolding of an immunoglobulin (Ig) G bindingprotein based upon the B domain of protein A (SpAB) from Staphylococcusaureus were studied by substituting tryptophan residues at strategiclocations within each of the three a-helical regions (al-a3)of the domain. The role of the C-terminal helix, a3, was investigatedby generating two protein constructs, one corresponding to thecomplete SpAB, the other lacking a part of ct3; the Trp substitutionswere made in both one-and two-domain versions of each of theseconstructs. The fluorescence properties of each of the single-tryptophanmutants were studied in the native state and as a function ofguanidine-HCl-mediated unfolding, and their IgG binding activitieswere determined by a competitive enzyme-linked immunosorbentassay. The free energies of folding and of binding to IgG foreach mutant were compared with those for the native domains.The effect of each substitution upon the overall structure andupon the IgG binding interface was modelled by molecular graphicsand energy minimization. These studies indicate that (i) 3 contributesto the overall stability of the domain and to the formationof the IgG binding site in l and 2, and (ii) al unfolds first,followed by 2 and 3 together.     

Site-directed mutagenesis of the putative active site residues of 3C proteinase of coxsackievirus B3: evidence of a functional relationship with trypsin-like serine proteinases

Picornavirus 3C proteinases (3C^pro) are cysteine proteinasesbut recent sequence analyses have shown that they are relatedto trypsin-like serine proteinases. Two models of 3C^pro structurehave been presented. Both models indicate that residues His40and Cysl47 are members of the catalytic triad but the modelsdiffer in the designation of the third member of the catalytictriad, which is assigned as either Glu71 or Asp85. To test theimportance of these four residues in the catalytic activityof 3C^pro of coxsackievirus B3, a member of the enterovirus subgroupof the picornavirus family, single amino acid substitutionswere introduced at each of the four sites. All of these mutationsresulted in the reduction or inactivation of autocatalytic cleavageof the 3C precursor protein expressed in Escherichia coli, suggestingthat all of these residues are essential for the proteolyticreaction. The substitution of Cysl47 with Ala abolished 3C^proactivity while the mutant in which Cysl47 was replaced withSer retained reduced proteolytic activity both in cis and intrans. Our results strongly support the proposal that Cysl47of 3C^pro functions as a nucleophile analogous to Serl95 of trypsin-likeserine proteinases.     

Position-dependent protein mutant profile based on mean force field calculation

The application of the mean force field in protein mutant stabilityprediction is explored. Based on protein main chain characteristics,including polar fraction, accessibility and dihedral angles,the mean force field was constructed to evaluate the compatibilitybetween an amino acid residue and its environment, from whicha position-dependent protein mutant profile was constructed.At each position along a protein sequence, the native residuewas replaced by the other 19 types of amino acid residues. Thematches were evaluated by energies from mean force field calculation,from which a mutant profile along the protein sequence was derived.General characteristics of such a profile were analyzed. Mutantstabilities for two sets of mutants in two proteins were foundto be reasonable compared with experimental data, which indicatesthat the present method can act as a guide in protein engineeringand as an effective scoring matrix in protein sequence–structurealignment studies.     

Structure--activity relationships in human interleukin-1{alpha}: identification of key residues for expression of biological activities

To identify the sites important for the different biologicalactivities of human interleukin-l (hIL-1), 56 single-amino acid-substitutedmutants of hIL-l were produced in Escherichia coli using site-directedmutagenesis, and were examined for their biological activitiessuch as mouse lymphocyte activating factor activity (LAF activity),cytostatic activity against human melanoma cells A-375 (A375activity) and prostaglandin E2 (PGE2) inducing activity in humanosteosarcoma cells MG-63 (PEI activity). Two amino acid residues,Asp26 and Asp151, were found to be important for these activities.The replacement of Asp26 by Val caused a decrease in LAF andA375 activities by one or two orders of magnitude and a slightdecrease in A375 activity. The Tyr or Phe substitution for Asp151caused decreases in LAF and A375 activities by one or two ordersof magnitude and complete loss of PEI activity. The change fromAsp151 to Lys or Arg resulted in marked decrease in LAF activityand complete loss of A375 and PEI activities. Since Asp26 andAsp151 are close to each other in the three-dimensional structure,the region involving these amino acids seems to be importantfor the biological activities of hIL-1.     

A multivariate analysis method for discriminating protein secondary structural segments

Using discriminant analysis, three types of protein secondarystructure segments—helices, ß-strands and coils—arediscriminated by amino acid sequence information alone. A variablein the discriminant analysis is defined by the amino acid indexused to represent the sequence data and by the calculation methodused to extract a feature in this representation. Thus, thethree types of secondary structure segments derived from a setof non-homologous proteins from the Protein Data Bank are analyzedby 888 variables, which correspond to the mean, standard deviation,3.6-residue periodicity and 2-residue periodicity for the numericalprofiles determined from 222 published amino acid indices. Thesevariables are combined to obtain best discrimination of thethree types of segments. When up to three variables are combined,the best discrimination rate was 75%. The variables selectedconsist of the mean of propensity (or turn propensity), themean of ß propensity, and the 3.6-residue periodicityof hydrophobicity. This variable selection procedure can alsobe applied to other types of discrimination problem, once groupsof sequence data are properly organized.     

Main-chain complementarity in protein-protein recognition

We present a statistical analysis of protein structures basedon interatomic C_a distances. The overall distance distributionsreflect in detail the contents of sequence-specific substructuresmaintained by local interactions (such as -helixes) and longerrange interactions (such as disulfide bridges and ß-sheets).We also show that a volume scaling of the distances makes distancedistributions for protein chains of different length superimposable.Distance distributions were also calculated specifically foramino acids separated by a given number of residues. Specificfeatures in these distributions are visible for sequence separationsof up to 20 amino acid residues. A simple representation, whichpreserves most of the information in the distance distributions,was obtained using six parameters only. The parameters giverise to canonical distance intervals and when predicting coarse-graineddistance constraints by methods such as data-driven artificialneural networks, these should preferably be selected from theseintervals. We discuss the use of the six parameters for determiningor reconstructing 3-D protein structures.     

Alternating charge clusters of side chains: new surface structural invariants observed in calf eye lens gamma-crystallins

A detailed stereochemical analysis of the oppositely chargedside chains of amino acid residues on the surface of calf eyelens protein gamma-crystallin B has been carried out. The refinedstructural data of very high quality obtained at 1.47 Åresolution have been used. Charge–charge interactionswere considered to be valuable for all the charged oxygen andnitrogen atoms situated at distances, d, between 2.4 and 7.0Å. This means we consider short contact ion pairs as thosewith interchange distances 2.4 < d 4.0 Å and distantcontact ion pairs as those with distances 4.0 < d 7.0 Å.Hydrogen bonding of the charged atomic groups with the structuralwater molecules also has been considered. We have not lookedat the side groups of histidines which are charged only partiallyat neutral pH. Five clusters of charged side chains which werelarge enough were observed. The clusters are comprised of fourto six residues which compose 543% of the total charged residuesin the protein. The clusters contain from eight to 12 chargedatoms and look like the bent chains of oppositely charged atoms.All clusters are of plane geometry and their maximal lineardimensions are from 11 to 18 Å. The root mean square deviationsof charged atoms from the cluster plane varied from 0.63 to0.86 Å for four clusters and was only 1.85 Å forthe largest cluster. All clusters include a number of watermolecules situated on the cluster boundary and grouped nearthe cluster plane. It was shown that the amino acid sequencepositions of charged residues are conservative for all the proteinsof the gamma-crystallin family of vertebrates including fish,frog, mouse, rat, calf and human. The cluster properties werediscussed both in their functional aspect for gamma-crystallinsand in other aspects common for globular proteins. As a result,the alternating charge clusters should be considered as newlyrecognized surface structural invariants. The importance ofthe charged side chain clusters is claimed for the updated conceptof the protein surface.