Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the Mc(S71L) b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.     

The expression of bovine microsomal cytochrome b5 in Escherichia coli and a study of the solution structure and stability of variant proteins

The DNA sequence of bovine microsomal cytochrome b₅ has beenamplified from a liver cDNA library using a polymerase chainreaction. The amplified cDNA when cloned into plasmids thatsupport the high-level production of cytochrome b_s in E.colileads to protein overexpression and results in cell coloniesbearing a strong red colouration. Using cassette mutagenesis,truncated versions of the cytochrome b₅ cDNA have been madethat encode the first 90 amino acid residues (Ala1-Lys90), thefirst 104 amino acids (Ala1-Ser104) and the complete protein(Ala1-Asnl33). The location of the overexpressed cytochromebs within prokaryotic cells is dependent on the overall lengthof the protein. Expression of the Ala-Lys90 and Alal-SerlO4variants leads to a location in the cytoplasmic phase of thebacteria whereas the whole protein, Alal-Asnl33, is found withinthe bacterial membrane fraction. The last 30 residues of cytochromebs therefore contain all of the necessary information to insertthe protein into E.coli membranes. The solubility of the Alal-SerlO4variant permits the solution structure and stability of thisprotein to be measured using 1- and 2-D ¹HNMR methods and electronicspectroscopy. 1-D NMR studies show that the chemical shiftsof the haem and haem ligand resonances of the Alal - Ser 104variant exhibit only very slight perturbations to their magneticmicroenvlronments when compared with the tryptic fragment offerricytochrome b₅. These results indicate an arrangement ofresidues in the haem pocket that is very similar in both theAlal-Ser 104 variant and the tryptic fragment and by 2-D NMRit is shown that this similarity extends to the conformationsof the poly peptide backbone and side chains. Electronic spectroscopyof this variant shows absorbance maxima for the Soret peaksat 423 run (reduced) and 413 nm (oxidized). From absorbancespectra the relative thermal stabilities of the Alal-Ser 104variant and the tryptic fragment were measured. In the oxidizedstate the Ala1 - Ser104 variant denatures in a single cooperativetransition with a midpoint temperature (T_m of 73°C thatis significantly higher than that of ‘tryptic’ ferricytochromebs. The reduced form of the protein shows increased transitiontemperatures (T_m 78°C) reflected in the values of H_m, S_mand (G) of 420 kj/mol, 1096 J/mol/K and 12.38 kj/mol respectively,estimated for this variant. The increased stability of the Alal-SerlO4variant and other recombinant forms of cytochrome b_s is correlatedwith the presence of additional residues at the N- and C-termini.The subtle differences in reactivity, stability and targetingbetween variant forms of cytochrome bs and the tryptic fragmentare discussed in terms of the overall structure of the protein.     

Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform

Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 pi-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of approximately 93 degrees C and approximately 104 degrees C, respectively. A 15 degrees C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.     

Homology modeling of a heme protein, lignin peroxidase, from the crystal structure of cytochrome c peroxidase

A 3-dimensional model of lignin peroxidase (LiP) was constructedbased on its sequence homology with other peroxidases, particularlycytochrome c peroxidase, the only protein with a known crystalstructure in the peroxidase family. The construction of initialconformations of insertions and deletions was assisted by secondarystructure predictions, amphipathic helix predictions, and considerationof the specific protein environment. A succession of moleculardynamics simulations of these regions with surrounding residuesas constraints were carried out to relax the bond lengths andangles. Full protein molecular dynamics simulations with explicitconsideration of bound waters were performed to relax the geometryand to identify dynamically flexible regions of the successivemodels for further refinement. Among the important functionallyrelevant structural features predicted are: (i) four disulfidebonds are predicted to be formed between Cys3 and Cys15, Cys14and Cys285, Cys34 and Cys120 and Cys249 and Cys317; (ii) a glycosyla-tionsite, Asn257, was located on the surface; (iii) Glu40 was predictedto form a salt bridge with Arg43 on the distal side of the hemeand was considered as a possible origin for the pH dependenceof compound I formation; and (iv) two candidate substrate bindingsites with a cluster of surface aromatic residues and flexiblebackbones were found in the refined model, consistent with thenature of known substrates of LiP. Based on these predictedstructural features of the model, further theoretical and experimentalstudies are proposed to continue to elucidate the structureand function of LiP.     

Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation

Site-directed mutants were constructed in cytochrome P-450_camto re-engineer the stereochemistry and coupling of ethylbenzenehydroxyiation. The reaction with wild-type (WT) enzyme producesone regioisomer 1-phenylethanol with 5% reduced nicotinamideadenine deoxyribonucleic acid to product conversion of and aratio of 73:27 for the R and S enantiomers respectively. Ethyibenzenewas modeled into the active site of WT P-450_cam in a rigid modeand oriented to optimize either pro-R or pro-S hydrogen abstraction.Residues T101, T185 and V247 make extensive contacts with thesubstrate in the static complexes and were therefore chosenfor site-directed mutagenesis. Single mutants T101M, V247A andV247M are more stereospedik producing 89,87 and 82% (R)-1-phenylethanolrespectively. The coupling of the reaction is doubled for thesingle mutants T185L, T185F and V247M. In an effort to engineerincreased stereospecificity and coupling into a single catalystthe T101M, T185F and V247M mutants were combined in a multiplemutant of P-450_cam.This protein hydroxylates ethyibenzene resultingin an R:S ratio of 87:13 for the 1-phenylethanols and 13% couplingof reducing equivalents to product. The catalytic stereospecificityand stoichiometry with T101M–T185F–V247M does notrepresent a summation of the changes observed for the singlemutants. A portion of the individual effects on substrate recognitionproduced by the single substitutions is either eliminated ordegenerate within the triple mutant.     

A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity

A three-dimensional structure for human cytochrome P450IA1 waspredicted based on the crystal coordinates of cytochrome P450camfrom Pseudomonas putida. As there was only 15% residue identitybetween the two enzymes, additional information was used toestablish an accurate sequence alignment that is a prerequisitefor model building. Twelve representative eukaryotic sequenceswere aligned and a net prediction of secondary structure wasmatched against the known -helices and ß-sheets ofP450cam. The cam secondary structure provided a fixed main-chainframework onto which loops of appropriate length from the humanP450IA1 structure were added. The model-built structure of thehuman cytochrome conformed to the requirements for the segregationof polar and nonpolar residues between the core and the surface.The first 44 residues of human cytochrome P450 could not bebuilt into the model and sequence analysis suggested that residues1–26 formed a single membrane-spanning segment. Examinationof the sequences of cytochrome P450s from distinct gene familiessuggested specific residues that could account for the differencesin substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene,was fitted into the proposed active site and this planar aromaticmolecule could be accommodated into the available cavity. Residuesthat are likely to interact with the haem were identified. Thesequence similarity between 59 eukaryotic enzymes was representedas a dendrogram that in general clustered according to genefamily. Until a crystallographic structure is available, thismodel-building study identifies potential residues in cytochromeP450s important in the function of these enzymes and these residuesare candidates for site-directed mutagenesis.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2),which has specificity for both ATP and GTP, shows significantamino acid sequence similarity to the cyclin-dependent kinase2 (CDK2). We constructed site-directed mutants of CK2 and useda three-dimensional model to investigate the basis for the dualspecificity. Introduction of Phe and Gly at positions 50 and51, in order to restore the pattern of the glycine-rich motif,did not seriously affect the specificity for ATP or GTP. Weshow that the dual specificity probably originates from theloop situated around the position His115 to Asp120 (HVNNTD).The insertion of a residue in this loop in CK2 subunits, comparedwith CDK2 and other kinases, might orient the backbone to interactwith the base A and G; this insertion is conserved in all knownCK2. The mutant N118, the design of which was based on the modelling,showed reduced affinity for GTP as predicted from the model.Other mutants were intended to probe the integrity of the catalyticloop, alter the polarity of a buried residue and explore theimportance of the carboxy terminus. Introduction of Arg to replaceAsn189, which is mapped on the activation loop, results in amutant with decreased k_cat, possibly as a result of disruptionof the interaction between this residue and basic residues inthe vicinity. Truncation at position 331 eliminates the last60 residues of the subunit and this mutant has a reduced catalyticefficiency compared with the wild-type. Catalytic efficiencyis restored in the truncation mutant by the replacement of apotentially buried Glu at position 252 by Lys, probably owingto a higher stability resulting from the formation of a saltbridge between Lys252 and Asp208.     

The protein threading problem with sequence amino acid interaction preferences is NP-complete

In recent protein structure prediction research there has beena great deal of interest in using amino acid interaction preferences(e.g. contact potentials or potentials of mean force) to align(‘thread’) a protein sequence to a known structuralmotif. An important open question is whether a polynomial timealgorithm for finding the globally optimal threading is possible.We identify the two critical conditions governing this question:(i) variable-length gaps are admitted into the alignment, and(ii) interactions between amino acids from the sequence areadmitted into the score function. We prove that if both theseconditions are allowed then the protein threading decision problem(does there exist a threading with a score K?) is NP-complete(in the strong sense, i.e. is not merely a number problem) andthe related problem of finding the globally optimal proteinthreading is NP-hard. Therefore, no polynomial time algorithmis possible (unless P = NP). This result augments existing proofsthat the direct protein folding problem is NP-complete by providingthe corresponding proof for the ‘inverse’ proteinfolding problem. It provides a theoretical basis for understandingalgorithms currently in use and indicates that computationalstrategies from other NP-complete problems may be useful forpredictive algorithms.     

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.     

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment

Europium chelates provide a non-radioactive alternative forsensitive labelling of antibodies for diagnostic immunoassays.Lysine residues at antibody surfaces are ready targets for labellingby an isothiocyanate derivative of the europium chelate (Eu³⁺).Here the labelling efficiency of a recombinant anti-human -fetoprotein(hAFP) Fab fragment has been improved by increasing its lysinecontent by protein engineering. Molecular modelling was usedto identify three light chain constant domain surface arginineresidues, R154, R187 and R210, which were mutated to lysineresidues. The mutations did not influence the affinity of thelysine-enriched Fab fragment and its labelling efficiency wasfound to be 40% higher than that of the wildtype Fab fragmentWith low degree of labelling, the affinities of the two Fabfragments were identical and comparable with that of the originalmonoclonal anti-hAFP IgG. With a higher degree of labellingthe affinities of both Fab fragments decreased more than thatof the intact IgG since more lysine residues are available forlabelling in the additional heavy chain constant domains ofthe larger molecule. Electrostatic adsorption and covalent immobilizationof the Fab fragments were characterized by BIAcore^TM and thelysine-enriched Fab fragment was found to be more efficientlyimmobilized to an activated carboxymethyl surface.     

Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions

By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 8–12 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg²⁺. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 27–31 of wild-type)resulted in enzymes with greater dependence on Mg²⁺ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence.     

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.     

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M·EcoRV) is an -adeninemethyltransferase. We have used two different programs to predictthe secondary structure of M·EcoRV. The resulting consensusprediction was tested by a mutant profiling analysis. 29 neutralmutations of M·EcoRV were generated by five cycles ofrandom mutagenesis and selection for active variants to increasethe reliability of the prediction and to get a secondary structureprediction for some ambiguously predicted regions. The predictedconsensus secondary structure elements could be aligned to thecommon topology of the structures of the catalytic domains ofM·HhaI and M·TaqI. In a complementary approachwe have isolated nine catalytically inactive single mutants.Five of these mutants contain an amino acid exchange withinthe catalytic domain of M·EcoRV (Val20-Ala, Lys81Arg,Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant bindsDNA similarly to wild-type M·EcoRV, but is catalyticallyinactive. Hence this mutant behaves like a bona fide activesite mutant. According to the structure prediction, Trp231 islocated in a loop at the putative active site of M·EcoRV.The other inactive mutants were insoluble. They contain aminoacid exchanges within the conserved amino acid motifs X, IIIor IV in M·EcoRV confirming the importance of these regions.     

Stability effects associated with the introduction of a partial and a complete Ca2+-binding site into human lysozyme

Two mutants of human lysozyme were synthesized. Mutant A92D,in which Ala92 was substituted by Asp, contains a partial Ca²⁺-bindingsite and mutant M4, in which Ala83, Gm86, Asn88 and Ala92 werereplaced by Lys, Asp, Asp and Asp respectively, contains thecomplete Ca -binding site of bovine a-lactalbumin. The Ca²⁺-bindingconstants of wild type human lysozyme and of mutants A92D andM4, measured at 25C and pH 7.5, were 2(1) x 102 M"1, 8(2)x l^M"1 and 9(0.5) x 10* M"1 respectively. Information gatheredfrom mkrocalorimetrk and CD spectro-scopic measurements indicatesthat the conformational changes of the M4 mutant lysozyme, inducedby Ca²⁺ binding, are smaller than those observed for bovinea-lactalbumin and for the Ca²⁺-binding equine lysozyme. At pH4.5, the thermostability of both the apo and Ca²⁺ forms of theA92D human was decreased in comparison with that of native humanlysozyme. In particular, within the apo form of this mutantan a-helix-containing sequence was destabilized. In contrast,at the same pH the thermostability of the apo and Ca²⁺ formsof the M4 mutant lysozyme was increased. The e-ammonium groupof the Lys83 side chain is assumed to be responsible for thestabilization of the apo form of this mutant.     

Directed evolution of O6-alkylguanine-DNA alkyltransferase for applications in protein labeling

The specific reaction of O6-alkylguanine-DNA alkyltransferase (AGT) with O6-benzylguanine (BG) derivatives allows for a specific labeling of AGT fusion proteins with chemically diverse compounds in living cells and in vitro. The efficiency of the labeling depends on a number of factors, most importantly on the reactivity, selectivity and stability of AGT. Here, we report the use of directed evolution and two different selection systems to further increase the activity of AGT towards BG derivatives by a factor of 17 and demonstrate the advantages of this mutant for the specific labeling of AGT fusion proteins displayed on the surface of mammalian cells. The results furthermore identify two regions of the protein outside the active site that influence the activity of the protein towards BG derivatives.     

The role of Glu87 and Trp89 in the lid of Humicola lanuginosa lipase

The importance of Glu87 and Trp89 in the lid of Humicola lanuginosalipase for the hydrolytic activity at the water/lipid interfacewas investigated by site-directed mutagenesis. It was foundthat the effect on the hydrolytic activity upon the replacementof Trp89 with Phe, Leu, Gly or Glu was substrate dependent TheTrp89 mutants displayed an altered chain length specificitytowards triglycerides, with a higher relative activity towardstriacetin and trioctanoin compared with tributyrin. Trp89 wasshown to be lessimportant in the hydrolysis of vinyl esterscompared with ethylesters and triglycerides. An exclusive effecton the acylation reaction rate by the mutation of Trp89 wasconsistent with the data. It is suggested that Trp89 is importantin the process of binding the acyl chain of thesubstrate intothe activesite for optimal acylation reaction rate. The Trp89Phemutation resulted in an increased hydrolytic activity towards2-alkylalkanoic acid esters. This is suggested to be due toreduction of unfavourable van der Waals contacts between Trp89and the 2-substituent of the substrate. Thus, in contrast tonatural substrates, Trp89 has a negative impact on the catalyticefficiencywhen substrates with bulky acyl chains are used. Incontrast to the Trp89 mutations, the effect on the hydrolyticactivity of the Glu87Ala mutation was almost substrate independent,35–70% activity of wild-type lipase. Areduction of boththe acylation and deacylation reaction was consistent with thedata.     

Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking

We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 x 10(11) possible conformations. While cross-links represent only limited structural constraints, the combination of only three experimental cross-links with very basic molecular docking was sufficient to determine the complex structure. The crystal structure of the complex between latexin and carboxypeptidase A4 determined recently allowed us to assess the success of this structure determination approach. Our structure was shown to be within 4 A r.m.s. deviation of Calpha atoms of the crystal structure. The study demonstrates that cross-linking in combination with mass spectrometry can lead to efficient and accurate structural modelling of protein complexes.     

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.     

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.     

An evaluation of the performance of an automated procedure for comparative modelling of protein tertiary structure

A 3-D model of a protein can be constructed from its amino acidsequence and the 3-D structures of one or more homologues byannealing three sets of fragments: the structurally conservedregions, structurally variable regions and the side chains.The method encoded in the computer program COMPOSER was assessedby generating 3-D models of eight proteins whose crystal structuresare already known and for which 3-D structures of homologuesare available. In the structurally conserved regions, differencesbetween modelled and X-ray structures are smaller than the differencesbetween the X-ray structures of the modelled protein and thehomologues used to build the model. When several homologuesare used, the contributions of the known structures are weighted,preferably by the square of sequence similarity; this is especiallyimportant when the similarities of the homologues to the modelledstructure differ greatly. The ‘collar’ extensionapproach, in which a similar region of different length in ahomologue is used to extend the framework, can result in a moreaccurate model. If known homologues comprise more than one relatedgroup of proteins and they are both distantly related to theunknown, then alignment of the sequence to be modelled witheach group of homologues facilitates identification of structurallyconserved regions of the unknown and leads to an improved model.Models have root mean square differences (r.m.s.d.s) with thestructures defined by X-ray analysis of between 0.73 and 1.56Å for all C atoms, for seven of the eight models. Forthe model of mucor pepsin, where the closest homologue has 33%sequence identity and 20% of the residues are in structurallyvariable regions, the r.m.s.d. for the framework region is 1.71Å and the r.m.s.d. for all C atoms is 3.47 Â.