Identifying the mechanism of protein loop closure: a molecular dynamics simulation of the Bacillus stearothermophilus LDH loopin solution

The ‘loop’ involving residues 98–110 in Bacillusstearothermophilus lactate dehydrogenase (BSLDH) is of greatinterest as substrate-induced ‘loop’ closure isthought to berate-limiting and essential in catalyzing the reactionand in determining substrate specificity. Consequently, we haveexplored the mechanism underlying ‘loop’ openingin BSLDH through a molecular dynamics simulation at high temperature(1000 K) in the presence of explicit solvent, starting fromthe X-ray structure of BSLDH complexed with the co-enzyme NAD+and oxamate at 2.5 Å. During the simulation, a significantconformational change occurred, as evidenced by sharp dihedralangle transitions, hydrogen bond breaking and formation andlarge root mean square deviations from the starting structure;these changes define the criteria for ‘loop’ opening.The mechanical elements responsible for ‘loop’ opening,i.e. ‘loop’ hinges and flap, are defined througha combination of order parameters, dihedral angle changes andtheir correlations and the dynamical cross-correlation map ofatomic displacements for the ‘loop’ residues. Theresults indicate that the ‘loop’ consists of twoflexible hinge regions on either side of a relatively rigidthree-residue segment that undergoes a significant spatial displacementduring ‘loop’opening. ‘Loop’ openingis made possible through an array of correlated dihedral anglechanges and intra-& ‘loop’ rearrangements ofhydrogen-bond interactions. The presentfindings are comparedto previous work related to ‘loop’ opening and site-directedmutagenesis experiments.     

The role of heat-shock and chaperone proteins in protein folding: possible molecular mechanisms

Recently some heat-shock proteins have been linked to functionsof ‘chaperoning’ protein folding in vivo. Here currentexperimental evidence is reviewed and possible requirementsfor such an activity are discussed. It is proposed that onemode of chaperone action is to actively unfold misfolded orbadly aggregated proteins to a conformation from whkh they couldrefold spontaneously; that improperly folded proteins are recognizedby excessive stretches of solvent-exposed backbone, rather thanby exposed hydrophobic patches; and that the molecular mechanismfor unfolding is either repeated binding and dissociation (‘plucking’)or translocation of the protein backbone through a binding cleft(‘threading’), allowing the threaded chain to refoldspontaneously. The observed hydrolysis of ATP would providethe energy for active unfolding. These hypotheses can be appliedto both monomeric folding and oligomeric assembly and are sufficientlydetailed to be open to directed experimental verification.     

Geometric and energy parameters in lysine-retinal chromophores

The parameters used in the computer program ECEPP (EmpiricalConformational Energy Program for Peptides) have been expandedto cover some key elements in retinal-containing proteins. Theseelements are ‘all-trans retinal lysine with unprotonatedimine’, ‘all-trans retinal lysine with protonatedimine’, ‘13-cis retinal lysine with unprotonatedimine’ and ‘13-cis retinal lysine with protonatedimine’ respectively. The geometric parameters of thesefour new ‘amino acid residues’ were derived by optimizingtheir molecular structures with the AMI Hamiltonian includedin MOPAC (Molecular Orbital PACkage), and their partial atomiccharges were determined with a CNDO/2 (Complete Neglect of DifferentialOverlap) calculation. The parameters for nonbonded interactionsand torsional potentials were obtained from the existing ECEPPparameters through a logical extension. The augmented ECEPPsystem thus obtained can be employed to investigate the conformationof bacteriorhodopsin and its proton-pumping mechanism from anenergetic point of view. The computer modeling study on bacteriorhodopsinand other seven-helix membrane proteins, e.g. serotonin receptorand dopamine receptor, is under way in the Upjohn Laboratories.     

When awaiting 'Bio' Champollion: dynamic programming regularization of the protein secondary structure predictions

Predictions of protein secondary structure using current methodsare often unrealistic, i.e. the predicted -helices or ß-strandsare too short. To improve the realism, various heuristic ‘filtering’or ‘smoothing’ methods are used. They are more orless intuitive and are based on ad hoc corrections. We presenta regularization method to obtain a realistic secondary structurefrom predicted propensities. It is based on the known dynamicprogramming algorithm and is quite objective. It can be usedwith any prediction method which yields propensities. The regularizedpredictions conserve well the overall prediction accuracy andimprove the ‘protein-likeness’ of the prediction.     

Molecular recognition at the active site of subtilisin BPN': crystallographic studies using genetically engineered proteinaceous inhibitor SSI (Streptomyces subtilisin inhibitor)

Unlike trypsin-like serine proteases having only one conspicuousbinding pocket in the active site, subtilisin BPN' has two suchpockets, the S1 and S4 pockets, which accommodate the P1 andP4 residues of ligands (after Schechter and Berger notation)respectively. Using computer graphics, the geometrical natureof the two pockets was carefully examined and strategies forsite-directed mutagenesis studies were set up against a proteinSSI (Streptomyces subtilisin inhibitor), which is a strong proteinaceousinhibitor (or a substrate analogue) of subtilisin BPN'. It wasdecided to convert the P1 residue, methionine 73, into lysine(M73K) with or without additional conversion of the P4 residue,methionine 70, into glycine (M70G). The crystal structures ofthe two complexes of subtilisin BPN', one with the single mutantSSI (M73K) and the other with the double mutant SSI (M73K, M70G)were solved showing that (i) small ‘electrostatic induced-fitmovement’ occurs in the S1 pocket upon introducing theterminal plus charge of the lysine side chain, and (ii) large‘mechanical induced-fit movement’ occurs in theS4 pocket upon reducing the size of the P4 side chain from methionineto glycine. In both (i) and (ii), the induced-fit movement occurredin a concerted fashion involving both the enzyme and ‘substrate’amino acid residues. The term ‘substrate-assisted stabilization’was coined to stress the cooperative nature of the induced-fitmovements.     

Detection of internal cavities in globular proteins

We have undertaken a study of internal cavities in five proteinstructure groups, each containing different crystallographicstructure determinations of the same protein, to understandbetter the nature of packing defects in protein tertiary architectures.Our results show that cavity detection and consistency of detectionare highly dependent on probe and cavity size, cavity positionwithin the globular protein and the local ‘quality’(r.m.s. deviation) of structural consistency within the group.The consistency of solvent placement within cavities has alsobeen examined. We provide guidelines for estimating the likelihoodof a given cavity to be an actual packing defect or to be aresult of experimental error.     

Structural basis for the extreme thermostability of D-glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: analysis based on homology modelling

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a hyperthermophiliceubacterium, Thermotoga maritima, is remarkably heat stable(T_m = 109°C). In this work, we have applied homology modellingto predict the 3-D structure of Th.maritima GAPDH to revealthe structural basis of thermostability. Three known GAPDH structureswere used as reference proteins. First, the rough model of onesubunit was constructed using the identified structurally conservedand variable regions of the reference proteins. The holoenzymewas assembled from four subunits and the NAD molecules. Thestructure was refined by energy minimization and molecular dynamicssimulated annealing. No errors were detected in the refinedmodel using the 3-D profile method. The model was compared withthe structure of Bacillus stearothermophilus GAPDH to identifystructural details underlying the increased thermostability.In all, 12 extra ion pairs per subunit were found at the proteinsurface. This seems to be the most important factor responsiblefor thermostability. Differences in the non-specific interactions,including hydration effects, were also found. Minor changeswere detected in the secondary structure. The model predictsthat a slight increase in a-helical propensities and helix-dipoleinteractions also contribute to increased stability, but toa lesser degree.     

Folding of chains with random and edited sequences: similarities and differences

We have investigated the process of protein folding by Monte-Carlosimulation of folding occurring in a simple 3D lattice modelof a protein globule. We have found the range of ‘optimal’temperatures where the native fold is achieved by the Monte-Carloprocess much faster than that by exhaustive sorting of all thechain folds. The ‘optimal’ temperatures are essentiallythe same for different random and lsquo;edited’ sequences(for the latter, the native fold energy is separated by a considerablegap from the energies of other low-energy folds; for randomsequences, this gap is negligible). At the ‘optimal’temperatures, the ‘edited’ chains attain their nativefold faster than the random ones. However, the essence is thatthe native folds of ‘edited’ chains are thermodynamicallystable at temperatures optimal for fast folding, while the nativefolds of random chains are unstable at the temperatures optimalfor fast folding; also, at low temperatures where the nativefolds of random chains are stable, folding kinetics is veryslow. Consequently, stable native folds are formed slowly byrandom sequences and rapidly by the ‘edited’ ones     

Co-enzyme specificity of 3-isopropylmalate dehydrogenase from Thermits thermophilus HB8

The co–enzyme specificity of 3–isopropylmalate dehydrogenasefrom an extreme thermophile, Thermus thermophilus HB8, was changedfrom NAD to NADP by site–directed mutagenesis Based onsequence comparison of 3–isopropylmalate dehydrogenasesfrom various organisms with NAD– and NADP–dependentisocitrate dehydrogenases, Ser226, Ser253 and De279 of 3–isopropylmalatedehydrogenase were suggested as determining the co–enzymespecificity. These residues were replaced with the correspondingresidues of NADP–dependent isocitrate dehydrogenases;Arg, Gly and Tyr respectively. The single–mutated enzymes,S226R and I279Y, enhanced the activities towards NADP 10–and 3–fold respectively, whereas S253G reduced the activity.Among the multiple–mutated enzymes, the double–mutatedS226R/I279Y increased the catalytic efficiency against NADP( fold) and shifted the specificity for NAD towards NADP mostsignificantly ( 173–fold).     

Engineering the aggregation properties of dodecameric glutamine synthetase: a single amino acid substitution controls 'salting out'

Escherichia coli glutamine synthetase (GS) is a dodecamer ofidentical subunits which are arranged as two face-to-face hexamericrings. In the presence of 10% ammonium sulfate, wild type GSexhibits a pH-dependent ‘salting out’ with a pK_aof 4.51. Electron micrographs indicate that the pH-dependentaggregation corresponds to a highly specific self-assembly ofGS tubules, which result from stacking of individual dodecamers.This stacking of dodecamers is similar to the metal ion-inducedGS tubule formation previously described. Site-directed mutagenesisexperimentsindicate that the N-terminal helix of each subunit is involvedin the salting out reaction, as it is in the metal-induced stacking.A single substitution of alanine for His4 completely abolishesthe (NH₄₂SO₄-induced aggregation. However, the H4C mutant proteindoes nearly completely precipitate under the same salting outconditions. Mutations at other residues within the helix haveno effect on the stacking reaction. Differential catalyticactivityof unadenylylated GS versus adenylylated GS has been used todetermine whether wild type dodecamers ‘complement’the H4A mutant in the stacking reaction. The complementationexperiments indicate that His4 residues on bothsides of thedodecamer-dodecamer interfaces are not absolutely required forsalting out, although the wild type dodecamers clearly stackpreferentially with other wild type dodecamers. Approximately20% of the protein precipitated fromthe mixtures containingthe wild type GS and the H4A mutant is the mutant. The implicationsof these results for protein engineering are discussed.     

On the choice of reference mutant states in the application of the double-mutant cycle method

Pairwise interactions in proteins can be detected and, in certaincircumstances, their strength measured by applying the methodof double-mutant cycles. Such cycles comprise wild type protein,two single mutants and the corresponding double mutant The analysisof double-mutant cycles is most straightforward when the mutationsare to alanine since interactions are mostly removed withoutnew interactions being formed. Here, ‘not-to-alanine’double-mutant cycles are analysed. It is shown that a ‘not-to-alanine’double-mutant cycle can be decomposed into four double-mutantcycles with mutations only to alanine. The coupling energy correspondingto the ‘not-to-alanine’ double-mutant cycle is expressedas a function of the coupling energies of these four cycles.     

Computer-aided gene design

A computer program, which runs on MS-DOS personal computers,is described that assists in the design of synthetic genes codingfor proteins. The goal of the program is the design of a genewhich (0 contains as many unique restriction sites as possibleand (ii) uses a specific codon usage. The gene designed accordingto the criteria above is (i) suitable for ‘modular mutagenesis’experiments and (ii) optimized for expression. The program 'reverse-translates'protein sequences into degenerated DNA sequences, generatesa map of potential restriction sites and locates sequence positionswhere unique restriction sites can be accommodated. The nucleicacid sequence is then ‘refined’ according to a specificcodon usage to remove any degeneration. Unique restriction sites,if potentially present, can be ‘forced’ into thedegenerated nucleic acid sequence by using 'priority codes'assigned to different restriction sequences.     

Directing phage selections towards specific epitopes

It is possible to direct selections from antibody repertoiresdisplayed on filamentous phage towards unique epitopes on proteinantigens by competing with related molecules. A phage displayrepertoire of human single chain Fvs (scFvs) was panned threetimes against foetal haemoglobin (HbF). The selection was dominatedby one clone with a K_d of 10 nM but yielded at least 17 others,all of which bound HbF but crossreacted with adult haemoglobin(HbA). To direct selection towards HbF-specific epitopes, therepertoire was preincubated with HbA in solution before eachpanning. Crossreactive scFvs can form complexes with the solubleHbA and thereby be prevented from binding the immobilized HbF.Four clones with preferential binding to HbF emerged under theseconditions. One of these (Hb-1), with a K_d of 6 µM, hadexquisite specificity for HbF and could distinguish cells expressingHbF from those expressing HbA by immunocytochemistry and flowcytometry. This antibody has an affinity that is 600-fold lowerthan the dominant crossreactive clone, and so only emerged underconditions of ‘competitive deselection’. Thus, competitivedeselection is a viable means for directing selections towardsuseful epitopes. It permits a more effective ‘search’of phage display repertoires and allows the emergence of loweraffinity clones with useful specificities. These clones maybe useful in themselves or may serve as leads for in vitro affinitymaturation.     

A library of organic landscapes on filamentous phage

A billion-clone library of filamentous phage with differentsurface structures (‘landscapes’) was generatedby fusing random octapeptides to the N-terminus of all 4000copies of the major coat protein. Such a ‘landscape library’might include clones exhibiting emergent properties that inherein the entire surface architecture, not in the peptides by themselves.Because the diverse surface landscapes are displayed on viablephage, they can be surveyed for exceedingly rare functions usingmicrobiological selection methods. Clones with several emergentproperties of the sort envisioned were successfully selected,suggesting that landscape libraries have promise as a novelsource of nanomaterials with exploitable surface properties.     

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphatedehydrogenase, Ser¹⁴⁸ – Ala, was produced byoligonucleotide-directedmutagenesis. The study of the catalytic properties of this mutanthas shown that this mutation significantly affects the Michaelisconstant of inorganic phosphate and to a lesser extent thatof 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate.This result is consistent with model-building studies whichshow that, for the phosphorylation step of catalysis, inorganicphosphate must bind to the anion recognition site designatedP_i with the C(3) phosphate of the acyl-enzyme intermediate inthe alternative anion site P_s. Studies of the enantiomeric specificityusing D- and L-glyceraldehyde as substrates show that the hydroxylgroup of Ser¹⁴⁸, combined with the presence of the C(3) phosphateof the substrate, enhances stereospecificity as well as catalysis.However, the stereospecific effect cannot be a consequence ofthe direct interaction of Ser¹⁴⁸ with the C(2)-hydroxyl of thesubstrate. The changed K_m for glyceraldehyde-3-phosphate suggeststhat the initial step of hemithioacetal formation may take placewith its C(3) phosphate bound in the P_i site. This supportsthe molecular mechanism proposed by Moody (1984). Therefore,catalysis could be enhanced through interactions of the serinehydroxyl group not only with inorganic phosphate but also withthe C(3) phosphate of glyceraldehyde-3-phosphate.     

Automated protein crystallization and a new crystal form of a subtilisin:eglin complex

A procedure is described for automating labour-intensive stepsof the ‘hanging drop’ protein crystallization method.An automatic sample changer is employed to fill the wells ina multi-well plate so that concentration gradients in variouscomponents are obtained. The sample changer is also used forpreparing droplets on a second multi-well plate. Subse quently,this second plate is manually turned around and placed on topof the first multi-well plate such that a large number of chamberswith different conditions is obtained simultaneously. Duringinitial trials a new crystal form of a subtilisin:eglin complexwas obtained. The crystals have space group P2₁ contain twoenzyme inhibitor complexes per asymmetric unit and diffractbeyond 2.2 Å.     

'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization

‘Knobs-into-holes’ was originally proposed by Crickin 1952 as a model for the packing of amino acid side chainsbetween adjacent -helices. ‘Knobs-into-holes’ isdemonstrated here as a novel and effective design strategy forengineering antibody heavy chain homodimers for heterodimerization.In this approach a ‘knob’ variant was first obtainedby replacement of a small amino acid with a larger one in theC_H3 domain of a CD4-IgG immuno-adhesin: T366Y. The knob wasdesigned to insert into a ‘hole’ in the C_H3 domainof a humanized anti-CD3 antibody created by judicious replacementof a large residue with a smaller one: Y407T. The anti-CD3/CD4-IgGhybrid represents up to 92% of the protein A purified proteinpool following co-expression of these two different heavy chainstogether with the anti-CD3 light chain. In contrast, only upto 57% of the anti-CD3/CD4-IgG hybrid is recovered followingco-expression in which heavy chains contained wild-type C_H3domains. Thus knobs-into-holes engineering facilitates the constructionof an antibody/ immunoadhesin hybrid and likely other Fc-containingbifunctional therapeutics including bispecific immuno-adhesinsand bispecific antibodies.     

Protein fragment variability analysis and some principles of protein engineering of vaccines

Based on protein sequence databank (PIR), the ‘variablefragment’ bank, comprising pairs of closely-related proteins,containing one or more strongly differing sites of primary structures,was formed. The bank includes 465 ‘variable fragments’in 383 protein pairs. The amino acid composition of ‘variablefragments’ was examined and indices of potential aminoacid residue variability were formed. An analysis of the interchangeabilityof amino acid fragments depending on the substitution site (N-or C-terminal, or middle part of a chain), the fragment lengthdifferences and physico-chemical properties of residues, suchas volume, hydrophobicity, polarity and isoelectric point, wascarried out. Based on this analysis some general empirical rulesof peptide insertions in carrier proteins were created. Therules are directed at performing modifications leaving the generalstructure and function of the carrier protein molecule unchanged.The selection scheme for the regions suitable for modificationand the criteria for determination of the range of acceptablevariations in these regions were suggested. The use of the potentialvariability profile for detecting regions suitable for peptideinsertion was considered using surface protein of hepatitisB virus as an example.     

Knowledge based modelling of homologous proteins, part I: three-dimensional frameworks derived from the simultaneous superposition of multiple structures

An approach is described for modelling the three-dimensionalstructure of a protein from the tertiary structures of severalhomologous proteins that have been determined by X-ray analysis.A method is developed for the simultaneous superposition ofseveral protein molecules and for the calculation of an ‘averagestructure’ or ‘framework’. Investigation ofthe convergence properties of this method, in the case of bothweighted and unweighted least squares, demonstrates that bothgive a unique answer and the latter is robust for an homologousfamily of proteins. Multi-dimensional scaling is used to subgroupthe proteins with respect to structural homology. The frameworkcalculated on the basis of the family of homologous proteins,or of an appropriate subgroup, is used to align fragments ofthe known protein structures of high sequence homology withthe unknown. This alignment provides a basis for model buildingthe tertiary structure. Different techniques for using the frameworkto model the mainchain of various globins and an immunoglobulindomain in the structurally conserved regions are in vestigated.