Does a backwardly read protein sequence have a unique native state?

Amino acid sequences of native proteins are generally not palindromic.Nevertheless, the protein molecule obtained as a result of readingthe sequence backwards, i.e. a retro-protein, obviously hasthe same amino acid composition and the same hydrophobicityprofile as the native sequence. The important questions whicharise in the context of retro-proteins are: does a retro-proteinfold to a well defined native-like structure as natural proteinsdo and, if the answer is positive, does a retro-protein foldto a structure similar to the native conformation of the originalprotein? In this work, the fold of retro-protein A, originatedfrom the retro-sequence of the B domain of Staphylococcal proteinA, was studied. As a result of lattice model simulations, itis conjectured that the retro-protein A also forms a three-helixbundle structure in solution. It is also predicted that thetopology of the retro-protein A three-helix bundle is that ofthe native protein A, rather than that corresponding to themirror image of native protein A. Secondary structure elementsin the retro-protein do not exactly match their counterpartsin the original protein structure; however, the amino acid sidechain contact pattern of the hydrophobic core is partly conserved.     

Predicting leucine zipper structures from sequence

The leucine zipper structure is adopted by one family of thecoiled coil proteins. Leucine zippers have a characteristicleucine repeat: Leu–X₆–Leu–X₆–Leu–X₆–Leu(where X may be any residue). However, many sequences have theleucine repeat, but do not adopt the leucine zipper structure(we shall refer to these as non-zippers). We have found andanalyzed residue pair patterns that allow one to identify correctly90% of leucine zippers and 97% of non-zippers. Simpler analyses,based on the frequency of occurrence of residues at certainpositions, specify, at most, 65% of zippers and 80–90%of non-zippers. Both short and long patterns contribute to thesuccessful discrimination of leucine zippers from non-zippers.A number of these patterns involve hydrophobic residues thatwould be placed on the solvent-exposed surface of the helix,were the sequence to adopt a leucine zipper structure. Thus,an analysis of protein sequences has allowed us to improve discriminationbetween leucine zippers and non-zippers, and has provided somefurther insight into the physical factors influencing the leucinezipper structure.     

Model structures and action of interleukin 1 and its antagonist

A comparison has been made between the homology and hydrophobkityprofiles of six interleukin amino add sequences and that ofthe human interleukin 1ß (IL-lß) for whicha crystal structure exists. The resulting sequence alignmentwas used to build model structures for the sequences for threeIL-l, two IL-1ß and an interleukin receptor antagonist.Analysis of these structures demonstrates that the interleukinmolecule has a strong electric dipole which is generated bythe topological position of the amino acids in the sequence.Electrostatic surface calculations implicate a particular residues(Lysl45) as being fundamental to interleukin activity and thissupports site-directed mutation evidence that this residue isrequired for activity.     

Predicted secondary structure and membrane topology of the scrapie prion protein

The integral membrane sialoglycoprotein PrP^Sc is the only identifiablecomponent of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakobdisease in humans are transmissible, degenerative neurologicaldiseases caused by prions. Standard predictive strategies havebeen used to analyze the secondary structure of the prion proteinin conjunction with Fourier analysis of the primary sequencehydrophobicities to detect potential amphipathic regions. Severalhydrophobic segments, a proline- and glycine-rich repeat regionand putative glycosylation sites are incorporated into a modelfor the integral membrane topology of PrP. The complete aminoacid sequences of the hamster, human and mouse prion proteinsare compared and the effects of residue substitutions upon thepredicted conformation of the polypeptide chain are discussed.While PrP has a unique primary structure, its predicted secondarystructure shares some interesting features with the serum amyloidA proteins. These proteins undergo a post-translational modificationto yield amyloid A, molecules that share with PrP the abilityto polymerize into birefringent filaments. Our analyses mayexplain some experimental observations on PrP, and suggest furtherstudies on the properties of the scrapie and cellular PrP isoforms.     

Inverse protein folding by the residue pair preference profile method: estimating the correctness of alignments of structurally compatible sequences

The residue pair preference profile (R3P) method is an inversefolding method that combines environmental profiles and pairpreference profiles. The method uses statistical preferencesfor residue pairs which score the likelihood of finding a profiledresidue to be paired with a residue within its local environmentAll pairs are characterized by their dihedral angles, secondarystructure and number of neighboring residues as a function ofresidue type. Each residue pair preference is expressed forall 20 amino acids of the profiled residue and is weighted bythe compatibility of the environment residue with its own localenvironment The R3P method produces an initial profile-sequencealignment which is then refined by converting the initial profileinto a profile of a target sequence threaded into the structureof the initial profile. We have tested this method by evaluatingalignments of sequences with known 3-D structures using structuralsuperposition alignments as reference. R3P-sequence alignmentsare 50% correct on average for sequences whose 3-D structurepairs superimpose with an r.m.s. deviation of 1.97 Å.The average improvement in correctness during this iterativerefinement is 14%. The R3P-sequence alignments are comparedwith sequence-sequence and 3-D profile-sequence alignments.When all three methods are combined, on average 50% of the alignmentsare correct for pairs of 3-D structures that superimpose within2.12 Å. A 3-D model of HisA is predicted with the combinedmethod.     

The fuzzy-end elimination theorem: correctly implementing the side chain placement algorithm based on the dead-end elimination theorem

Recently it has been shown that the dead-end elimination theoremis a powerful tool in the search for the global minimum energyconformation (GMEC) of a large collection of protein side chainsgiven known backbone coordinates and a library of allowed sidechain conformational states, also known as rotamers. A sidechain placement algorithm based on this theorem iterativelyapplies this theorem to single as well as to pairs of rotamersleading to the identification of rotamers, single or pairs,that are incompatible with the GMEC and that can thus be qualifiedas ‘dead-ending’. Here we formulate a theorem whichproves that contrary to intuition dead-end rotamer pairs cannot simply be discarded from consideration in the iterativeprocess leading to the further elimination of dead-end rotamers.We refer to this theorem as the fuzzy-end elimination theorem.We also describe how the obtained dead-end rotamer pairs cancontribute to the search for the GMEC in the protein side chainplacement problem. Hence the present work forms a theoreticalbasis for the correct implementation of a side chain placementalgorithm based on the dead-end elimination theorem. In addition,possible future perspectives are presented.     

Analysis and prediction of the location of catalytic residues in enzymes

The catalytic residues of an enzyme are defined as the aminoacids directly involved in chemical catalysis. They mainly actas a general acid–base, electrophilic or nucleophiliccatalyst or they polarize and stabilize the transition state.An analysis of the structural features of 36 catalytic residuesin 17 enzymes of known structure and with defined mechanismis reported. Residues that bind metal ions (Zn² and Cu²) areconsidered separately. The features examined are: residue type,location in secondary structure, separation between the residues,accessibility to solvent, intra-protein electrostatic interactions,mobility as evaluated from crystallographic temperature factors,polarity of the environment and the sequence conservation betweenhomologous enzymes of residues that were sequentially or spatiallyclose to the catalytic residue. In general the environment ofcatalytic residues is similar to that of polar side chains thathave low accessibility to solvent. Two algorithms have beendeveloped to identify probable catalytic residues. Scanningan alignment of homologous enzyme sequences for peaks of sequenceconservation identifies 13 out of the 16 catalytic residueswith 50 residues overpredicted. When the conservation of thespatially close residues is used instead, a different set of13 residues are identified with 47 residues overpredicted. Acombination of the two algorithms identifies 11 residues with36 residues overpredicted.     

A novel method for the modelling of peptide ligands to their receptors

A knowledge-based approach to the modelling of enzyme- peptideinhibitor comlexes is described. Given the structure of an enzyme,and knowledge of its bindings site, the method seeks to predictthe binding geometry of a peptide ligand. This novel methodinvolves using examples of sidechain packing derived from proteinsof known three dimensional structure to define possible packingarrangements of a peptide inhibitor group to its bindings site.Asuite of progams, GEMINI, was written and used to predict thepacking of pairs of amino acid groups from three inhibitorscomplexed to their enzymes for which the X-ray strutures wereavailable. These included the Phe group of the inhibitor H142bound to endothiapepsin, the Leu group of CLT complexed to thermolysinand the C-terminus of Gly-L-Tyr bound to carboxypeptidase A.A detailed comparison of the modelled and observed inhibitorcoordinates was made. This approach may be extended to modellingother types of protein interactions.     

Development of pswudoenergy potentials for assessing protein 3-D-1-D compatibility and detecting weak homologies

Recent approaches to the 3-D-l-D compatibility problem ave triedto predict protein 3-D structure from sequence. One of the criticalfactors in this issue is the evaluation of fitness between agiven 3-D structure and any sequence mounted on it. We havedeveloped an evaluation function composed of four terms, sidechain packing, hydration, hydrogen bonding and local conformationpotentials, which were empirically derived from 101 proteinsof known structure. The efficiency of the evaluation functionwas tested hi two ways. In the first test, the sequence of proteinA is mounted (without gaps) on the structure of protein B whichis greater in size than A. For 81 proteins examined, the nativestructure was always detected. In the second test, a standardsequence homology search is performed against the entire database,followed by an assessment of the alignment with its proposedstructure, using the empirical evaluation function. When thistest was applied to the 101 proteins, our evaluation functionsuccessfully discriminated truly homologous sequence pairs fromnon-homologous proteins even when the sequence similaritieswere very weak. This approach was found to have clear advantagesover conventional sequence search methods.     

Cellulase EGZ of Erwinia chrysanthemi: structural organization and importance of His98 and Glu133 residues for catalysis

Biochemical, genetic and primary sequence analyses of the Erwiniachrysanthemi endoglucanase EGZ allowed us to identify two functionaldomains and to locate their boundaries. The catalytic domainextends from residue 1 to 288, while a domain required for EGZto bind to microcrystalline cellulose lies from residues 324to 385. Each domain was found capable of functioning in theabsence of the other. A region rich in Pro, Thr and Ser residueslinks both domains and appeared to be susceptible to proteolyticattack. Based upon predictions derived from a method developedto compare sequences sharing a low level of similarity, e.g.hydrophobic cluster analysis (HCA), we analysed the importanceof either residue His98 or Glul33 in EGZ catalytic activity.Two EGZ-derived proteins were engineered in which either His98or Glul33 amino acid was converted to an Ala residue. Characterizationof the purified proteins showed that no enzymatic activity couldbe detected, by using carboxymethylcellulose (CMC) or paranitrophenyl-cellobioside(pNPC) as substrates, while both mutated proteins retained thecapacity to bind to microcrystalline cellulose. These studies,which to date constitute the first experimental testing of HCA-derivedpredictions, allowed us to identify two particular amino acidsinvolved in cellulolytic activity. By taking into account datafrom chemical modification studies of other cellulases, we speculatethat the His98 residue is involved in the folding of the catalyticdomain while the Glul33 residue intervenes directly in the ß,1–4 glycosidic bond cleavage.     

Knowledge based modelling of homologous proteins, part II: rules for the conformations of substituted sidechains

This paper describes a rapid, automated procedure which canbe used for model building sidechains using (i) spatial informationfrom sidechains in topologically equivalent positions as faras such a correlation is observed, and then (ii) most probableconformations of the sidechains in the respective secondarystructure type. Analysis of topologically equivalent residuesin the structurally conserved regions of a family of proteinsimplies that the spatial positions of the atoms in the sidechainsrather than conformations should be considered when model building.Rules for the modelling of all 20 sidechains from each otherin -helical, ß-Sheet and loop regions—a totalof 1200—are established. Cluster analysis is used on positionaldata from the sidechain atoms of structurally equivalent residuesin an homologous family to guide modelling. The most probableconformation for the sidechain is used for modelling atoms whereno useful guidance is obtainable from equivalent sidechainsof the homologous proteins. In order to test the procedure wehave modelled the sidechains of the residues in the structurallyconserved regions of myo globin from four other globins. Theautomated procedure described here has been incorporated intothe program COMPOSER.     

Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamideacetyltransferase subunits (E2p) of the Escherichia coli pyruvatedehydrogenase complex are connected by linker sequences whichare characteristically rich in alanine and proline residues.By facilitating domain movement these linkers are thought topromote interactions between the three types of active sitethat participate in the catalytic cycle of the complex. To investigatefunctional constraints associated with linker composition andsequence, the natural linker of an E2p subunit containing onelipoyl domain was replaced by shorter sequences containing:mixtures of alanine plus proline residues; mainly alanine; mainlyproline; and mainly charged residues. Each artificial linkerpossessed a central histidine residue for assessing linker flexibilityby ¹H-NMR spectroscopy. The resultant complexes exhibited 181%(proline), 74–79% (alanine plus proline), 63% (alanine)and 7% (charged residues) of parental activity compared witha value of 75% expected for a complex with a comparably shortenedlinker. The ¹H-NMR spectra showed that the alanine plus prolinelinkers are flexible but the alanine linker and the prolinelinker are relatively inflexible. Substantial variations inlinker sequence and composition were tolerated without lossof function, and the enhanced activity conferred by the prolinelinker was attributed to the combined effects of length andrelative inflexibility.     

The structural consequences of exchanging tryptophan and tyrosine residues in B.stearothermophilus lactate dehydrogenase

A mutant Bacillus stearothermophilus lactate dehydrogenase hasbeen prepared in which all three tryptophan residues in thewild-type enzyme have been replaced by tyrosines. In addition,a tyrosine residue has been mutated to a tryptophan, which actsas a fluorescence probe to monitor protein folding. The mutantenzyme crystallizes in the same crystal form as the wild-type.The crystal structure of the mutant has been determined at 2.8Å resolution. Solution studies have suggested that thereis little effect upon the mutant enzyme as judged by its kineticproperties. Comparison of the crystal structures of the mutantand wild-type enzymes confirms this conclusion, and revealsthat alterations in structure in the region of these mutationsare of a similar magnitude to those observed throughout thestructure, and are not significant when compared with the errorsin atomic positions expected for a structure at this resolution.     

Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction

De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction.     

Comparison of conservation within and between the Ser/Thr and Tyr protein kinase family: proposed model for the catalytic domain of the epidermal growth factor receptor

The protein kinase family can be subdivided into two main groupsbased on their ability to phosphorylate Ser/Thr or Tyr substrates.In order to understand the basis of this functional difference,we have carried out a comparative analysis of sequence conservationwithin and between the Ser/Thr and Tyr protein kinases. A multiplesequence alignment of 86 protein kinase sequences was generated.For each position in the alignment we have computed the conservationof residue type in the Ser/Thr, in the Tyr and in both of thekinase subfamilies. To understand the structural and/or functionalbasis for the conservation, we have mapped these conservationproperties onto the backbone of the recently determined structureof the cAMP–dependent Ser/Thr kinase. The results showthat the kinase structure can be roughly segregated, based uponconservation, into three zones. The inner zone contains residueshighly conserved in all the kinase family and describes thehydrophobic core of the enzyme together with residues essentialfor substrate and ATP binding and catalysis. The outer zonecontains residues highly variable in all kinases and representsthe solvent–exposed surface of the protein. The thirdzone is comprised of residues conserved in either the Ser/Thror Tyr kinases or in both, but which are not conserved betweenthem. These are sandwiched between the hydrophobic core andthe solvent-exposed surface. In addition to analyzing overallconservation hi the kinase family, we have also looked at conservationof its substrate and ATP binding sites. The ATP site is highlyconserved throughout the kinases, whereas the substrate bindingsite is more variable. The active site contains several positionswhich differ between the Ser/Thr and Tyr kinases and may beresponsible for discriminating between hydroxyl bearing sidechains. Using this information we propose a model for Tyr substratebinding to the catalytic domain of the epidermal growth factorreceptor (EGFR).     

Fusion of a recombinant antibody fragment with a homo-amino-acid polymer: effects on biophysical properties and prolonged plasma half-life

Chemical conjugation of small recombinant proteins with polyethylene glycol (PEG) is an established strategy to extend their typically short circulation times to a therapeutically useful range. We have investigated the production of a genetic fusion with a glycine-rich homo-amino-acid polymer (HAP) as an alternative way to attach a solvated random chain with large hydrodynamic volume. The anti-HER2 Fab fragment 4D5 was used as a model system and fused with either 100 or 200 residue polymers of the repetitive sequence (Gly(4)Ser)(n) to its light chain. Both fusion proteins were successfully produced in the periplasm of Escherichia coli and obtained as homogeneous preparations after two-step affinity chromatography via the His(6) tag fused to the heavy chain and the Strep-tag II fused to the extended light chain. Both modified Fab fragments showed binding activity towards the HER2 antigen indistinguishable from the conventional recombinant Fab fragment. When compared with the unfused Fab fragment, a significantly increased hydrodynamic volume, by ca. 120%, was observed during gel filtration for the 200 residue HAP fusion protein and, to a lesser extent, in the case of the 100 residue HAP. Difference CD measurements revealed a characteristic random coil spectrum for the 100 and 200 residue HAP fusion moieties. Finally, pharmacokinetic experiments were carried out in mice after radioiodination of the recombinant Fab fragments. Although the 100 residue HAP fusion showed a behavior very similar to the unfused Fab fragment, with a terminal plasma half-life of ca. 2 h, the 200 residue HAPylated Fab fragment gave rise to a significantly prolonged half-life of ca. 6 h. While this moderate effect may so far be most beneficial for specialized medical applications, such as in vivo imaging, the genetic engineering of optimized HAP sequences should yield pharmacokinetic properties similar to PEGylation, yet without necessitating in vitro modification steps.     

Families of proteins with similar total structure identified using statistical geometry

Two quantities, herein defined as the displacement and the uniqueness,describe quantitatively the total structural difference betweentwo proteins. All possible pairs of protein chains in the Brookhavendatabase are characterized in terms of these quantities. Pairsof proteins with small values of both displacement and uniqueness,in secondary and super-secondary configuration spaces, havesimilar total structure. Proteins related in this fashion aregrouped into 158 families of similar total structure. The radialdisplacement function is herein defined to characterize therelative displacement of a residue from the center of the massof its protein. In addition, the residue backbone structurefunction is also defined to characterize the local configurationof the protein main chain in the vicinity of the residue. Thevalues of polynomial convolutions of these two functions arecharacteristic of a particular tertiary structure type. Thesepolynomial convolutions, together with other structural parameters,are used to verify the structural similarity of proteins belongingto the families indicated above. Variations in these polynomialconvolutions illustrate the amount and sequence location ofstructural deviations between proteins of the same family.     

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.     

Quantifying the local reliability of a sequence alignment

We present a method for attributing a measure of reliabilityto a residue pair in an optimal alignment of two protein sequences.Validation based on a database of structurally correct alignments[Pascarella and Argos (1992) Protein Engng, 5, 121–137]shows that correctly aligned parts of a sequence alignment systematicallyreceive high scores in this measure. The higher the sequencesimilarity between two sequences, the larger is the fractionfound of the correct parts of the alignment. We used these observationsto design a program that draws a reliability curve along anoptimal alignment reflecting the chances for each residue pairto be aligned correctly.