The T {leftrightharpoons} R structural transition of insulin; pathways suggested by targeted energy minimization

The transition of insulin between its crystallographically definedstates T and R is connected with considerable change even ofbackbone structure: the N-terminal B chain (residues B1 –B8) refolds from extended conformation in T into helical inR, and vice versa. Although hitherto observed only in hexamersthe transition of the monomer was adequate for developing andtesting the method of ‘targeted energy minimization’(TEM), capable of coping with conformational changes of suchextent at moderate computational expenditure. The simulationis performed in a predetermined number of steps consisting oftwo atomic displacements each, one by force in the directionof the target structure, the second by energy minimization releasingthe constraint caused in the first. The transition pathway isrepresented by the string of energy minimized transient structures.Due to the directedness of the algorithm the simulated pathwayfor R T is not the reversal of that for T R. It is, therefore,not pretended that the minimum energy pathway was identified.In the T R direction the N-terminal B chain first swivels whileremaining largely stretched and then winds up extending thepre-existing helix B9–B19. The A chain advances into thespace abandoned and withdraws from it in the R T simulation.In the latter the extended helix first kinks at B8/B9, and thenthe B1 B8 segment is unwound and stretched. The helical H-bondsof that segment are formed late in T R and are maintained duringalmost half of R T. The A_N helix is less stable and more involvedin the transitions than helix A_C. The two pathways seem plausiblefrom both the energetic and geometric points of view. Knowledgeof them will be of value to suggest mutations to test them byexperimental evidence.     

Simulations of the T {leftrightarrow} R conformational transition in aspartate transcarbamylase

Aspartate transcarbamylase (ATCase) from Escherichia coli isone of the best known allosteric enzymes. In spite of numerousexperiments performed by biochemists, no consensus model forthe cooperative transition between the tensed (T) and the relaxed(R) forms exists. It is hypothesized, however, that changesin the quaternary structure play a key role in the allostericproperties of oligomeric proteins such as ATCase. Previous normalmode calculations of the two states of ATCase illustrated thetype of motions that could be important in initiating the transition.In this work four pathways for the transition were calculatedusing the targeted molecular dynamics (TMD) method without constrainton the symmetry of the system. The most important quaternarystructure changes are the relative rotation and translationof the catalytic trimers and the rotations of the regulatorydimers. The simulations show that these quaternary changes startimmediately and finish when about 70% of the transition is completedwhereas there are tertiary changes throughout the transition.In agreement with the work of Lipscomb et al., it was foundthat the relative translation between the catalytic trimersappears to play a central role in allowing the transition tooccur. In all the simulations differences are observed in theopening and closing behaviours of the domains in the catalyticand regulatory chains that could provide a structural interpretationfor the results of certain site-directed mutagenesis experiments.Overall the motions of the subunits are concerted even thoughthe constraint imposed on the TMD method does not explicitlyrequire that this be so.     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.     

Energetic approach to the folding of four {alpha}-helices connected sequentially

The packing of four -helices, which each consist of 12 Ala residuesand are sequentially connected to each other by a segment of10 Ala residues, has been investigated by means of energy minimizations.For the lowest energy structure thus obtained, the followingfeatures have been found: (i) the four -helices are intimatelypacked to form an assembly with an approximately square section;(ii) the distances of closest approach between two adjacentinterhelix axes are 7.7±0.2 Å and those betweentwo diagonal interhelix axes are 11.2±0.2 Å; (iii)the adjacent interhelix angles are -163±2°; and (iv)the diagonal interhelix angles are 24±4°. These resultsindicate that the polypeptide chain, driven by energetics (nonbondedand electrostatic interactions), is folded into a typical left-handedtwisted four-helix bundle with an {small tilde}4-fold symmetricarray, as observed in most four -helix proteins. Furthermore,it has been found that the interaction between the loops formedby the connecting segments and the other part of molecule playsa significant role in stabilizing such a bundle structure. Thetechnology developed here and the relevant knowledge obtainedthrough this study are very useful for the study of modelingfour-helix bundle proteins.     

Improvement of turn structure prediction by molecular dynamics: a case study of {alpha}1-purothionin

Because of the problems in predicting a correct conformationfor loop regions in homology-based prediction, disagreementsare often found between the predicted models and the refinedX-ray structures of the same protein in loop regions. Such asituation has been encountered for ₁-purothionin (₁-PT). Hence,attempts have been made to improve the predicted model of ₁PTby limited molecular dynamics using both AMBER and XPLOR. Withmolecular dynamics, the previously predicted incorrect turnregion reverts to the correct conformation as seen in the X-rayrefined structure. In contrast to the model which is not subjectedto molecular dynamics, the improved model refines with the X-raydata of ₁PT in fewer cycles, without any manual rebuilding andwith comparable or better refinement statistics. Also, the improvedmodel serves as a better starting model in the determinationof the structure with the molecular replacement methods.     

Cloning of rabbit interleukin-1{beta}: differential evolution of IL-1{alpha} and IL-1{beta} proteins

We have cloned the rabbit IL-1ß cDNA, which encodesa 268 amino acid precursor similar in length to other sequencedIL-1 precursors. Comparison of all published IL-1 and IL-1ßsequences respectively indicates that the IL-1 gene family isevolving faster than the IL-1ß family, and that thetwo genes diverged –270 million years ago. Surprisingly,there are differences in the regions preferentially conservedwithin the two families. The IL-1 family is most conserved atthe amino terminus whereas the IL-1ß family is mostconserved in the carboxy-terminal half. This is despite thefact that the carboxy-terminal half encodes the active portionof both molecules and would be expected to adopt a similar ß-sheetstructure in IL-1 as in the published X-ray structure of matureIL-1ß. These findings suggest that differences inthe function and properties of the IL-1 and IL-1ßprecursor molecules may have been conserved. These differencesmay therefore provide an explanation for the existence of twoIL-1 molecules.     

Molecular dynamics studies on the thermostability of family 11 xylanases

Twelve members of the family 11 xylanases, including both mesophilic and thermophilic proteins, were studied using molecular dynamics (MD). Simulations of xylanases were carried out in an explicit water environment at four different temperatures, 300, 400, 500 and 600 K. A difference in thermotolerance between mesophilic and thermophilic xylanases became clear: thermophilic xylanases endured heat in higher simulation temperatures better than mesophilic ones. The unfolding pathways seemed to be similar for all simulations regardless of the protein. The unfolding initiates at the N-terminal region or alternatively from the alpha-helix region and proceeds to the 'finger region'. Unfolding of these regions led to denaturated structures within the 4.5 ns simulation at 600 K. The results are in agreement with experimental mutant studies. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. The MD analysis suggests regions in the protein structure which are more unstable and thus potential targets for mutation experiments to improve thermostability.     

The structure of a designed peptidomimetic inhibitor complex of {alpha}-thrombin

Thrombin displays remarkable specificity, effecting the removalof fibrinopeptides A and B of fibrinogen through the selectivecleavage of two Arg–Gly bonds between the 181 Arg/Lys–Xaabonds in fibrinogen. Significant advances have been made inrecent years towards understanding the origin of the specificityof cleavage of the Argl6–Gly17 bond of the A-chain ofhuman fibrinogen. We have previously proposed a model for thebound structure of fibrinopeptide A_7–16 (FPA), based uponNMR data, computer-assisted molecular modeling and the synthesisand study of peptidomimetic substrates and inhibitors of thrombin.We now report the structure of the ternary complex of an FPAmimetic (FPAM), hirugen and thrombin at 2.5 Å resolution(R-factor = 0.138) and specificity data for the inhibition ofthrombin and related trypsin-like proteinases by FPAM. The crystallographicstructures of FPA and its chloromethyl ketone derivative boundto thrombin were determined. Although there are differencesbetween these structures in the above modeled FPA structureand that of the crystal structure of FPAM bound to thrombin,the , angles in the critical region of P1–P2–P3in all of the structures are similar to those of bovine pancreatictrypsin inhibitor (BPTI) in the BPTI–trypsin complex andD–Phe–Pro–Arg (PPACK) in the PPACK–thrombinstructure. A comparison between these and an NMR-derived structureis carried out and discussed.     

Simulated annealing approach to the study of protein structures

One of the most difficult problems in predicting the three dimensionalstructure of proteins is how to deal with the local minimumproblem. In many cases of practical interest this problem hasbeen reduced to how to select an appropriate set of startingconformations for carrying out energy minimizations. How thesestarting conformations are selected, however, is often basedon the physical intuition of the person doing the calculations,and hence it is hard to avoid bearing some sort of arbitrariness.To improve such a situation, we introduced the simulated annealingMonte Carlo algorithm to locate the optimal starting conformationsfor energy minimizations. The method developed here is validfor both single and multiple polypeptide chain systems. Theannealing process can be conducted with respect to either theinternal dihedral angles of a polypeptide chain or the externalrotations and translations of various constituent polypeptidechains, and hence is particularly useful for studying the packingarrangements of secondary structures in proteins, such as helix/helixpacking, helix/sheet packing and sheet/sheet packing. It wasshown via a number of comparative calculations that the finalstructures obtained through the annealing process not only hadlower energies than the corresponding energy-minimized structuresreported previously, but also assumed the forms closer to theobservations in proteins. All these results indicate that abetter result can be obtained in search of low-energy structuresof proteins by incorporating the simulated annealing approach.It has been observed during simulated annealing for each ofthese cases that there is a critical temperature T^*, termedas ‘phase transition point’, around which the energyhas a remarkable drop and below which the decrease in energygradually becomes steady and slow.     

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

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

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding

Insulin-like growth factor-1 (IGF-1) is a serum protein whichunexpectedly folds to yield two stable tertiary structures withdifferent disulphide connectivities; native IGF-1 [18–61,6–48,47–52]and IGF-1 swap [18–61,6–47, 48–52]. Here we demonstratein detail the biological properties of recombinant human nativeIGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae.IGF-1 swap had a ~30 fold loss in affinity for the IGF-1 receptoroverexpressed on BHK cells compared with native IGF-1.The parallelincrease in dose required to induce negative cooperativity togetherwith the parallel loss in mitogenicity in NIH 3T3 cells impliesthat disruption of the IGF-1 receptor binding interaction ratherthan restriction of a post-binding conformational change isresponsible for the reduction in biological activity of IGF-1swap. Interestingly, the affinity of IGF-1 swap for the insulinreceptor was ~200 fold lower than that of native IGF-1 indicatingthat the binding surface complementary to the insulin receptor(or the ability to attain it) is disturbed to a greater extentthan that to the IGF-1 receptor. A 1.0 ns high-temperature moleculardynamics study of the local energy landscape of IGF-1 swap resultedin uncoiling of the first A-region -helix and a rearrangementin the relative orientation of the A- and B-regions. The modelof IGF-1 swap is structurally homologous to the NMR structureof insulin swap and CD spectra consistent with the model arepresented. However, in the model of IGF-1 swap the C-regionhas filled the space where the first A-region -helix has uncoiledand this may be hindering interaction of Val44 with the secondinsulin receptor binding pocket.     

Solution conformation of a synthetic bis-headed inhibitor of trypsin and carboxypeptidase A: new structural alignment between the squash inhibitors and the potato carboxypeptidase inhibitor

The trypsin carboxypeptidase peptide inhibitor (TCPI) whichinhibits both trypsin and carboxypeptidase A has been chemicallyengineered by modification of the Ecballium elaterium trypsininhibitor II (EETI-II). The solution conformation of TCPI, studiedby two-dimensional nuclear magnetic resonance, was shown tobe very close to those of squash inhibitors. Only limited deviationsof the trypsin binding loop compared to its location in theEETI-II/trypsin complex were detected. It was also shown thatthe position of the C-terminal tail did not significantly changefrom the position observed in the complex between carboxypeptidaseA and the potato carboxypeptidase inhibitor (PCI). The conformationof TCPI was carefully compared with the PCI one and a new structuralalignment between the two microproteins is proposed. This alignmentpoints out the very good conservation in the two inhibitorsof a subdomain comprising segments 7–15, 19–22 and25–28. Most importantly, the 2–19 disulfide bridgeof TCPI was not structurally conserved in PCI and appeared tobe rather unimportant for the early folding process of thesemolecules. This result agrees with the recent observation thatthe 2–19 bridge is the last to be formed in the foldingof the squash inhibitor EETI-II and suggests that this is alsothe case during the folding of the potato carboxypeptidase inhibitor.     

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

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

Efficient generation of a reshaped human mAb specific for the {alpha} toxin of Clostridium perfringens

We have used the technique of antibody reshaping to producea humanized antibody specific for the a toxin of Clostridiumperfringens. The starting antibody was from a mouse hybridomafrom which variable (V) region nucleo-tide sequences were determined.The complementarity-determining regions (CDRs) from these Vregions were then inserted into human heavy and light chainV region genes with human constant region gene fragments subsequentlyadded. The insertion of CDRs alone into human frameworks didnot produce a functional reshaped antibody and modificationsto the V region framework were required. With minor frameworkmodifications, the affinity of the original murine mAb was restoredand even exceeded. Where affinity was increased, an alteredbinding profile to overlapping peptides was observed. Computermodelling of the reshaped heavy chain V regions suggested thatamino acids adjacent to CDRs can either contribute to, or distort,CDR loop conformation and must be adjusted to achieve high bindingaffinity.     

Can the stability of protein mutants be predicted by free energy calculations?

The use of free energy simulation techniques in the study ofprotein stability is critically evaluated. Results from twosimulations of the thermostability mutation Asn218 to Ser218in Subtilisin are presented. It is shown that components ofthe free energy change can be highly sensitive to the computationaldetails of the simulation leading to the conclusion that freeenergy calculations cannot currently be used to reliably predictprotein stability. The different factors that undermine thereliability are discussed.     

On the stability and plastic properties of the interior L3 loop in R.capsulatus porin. A molecular dynamics study

Structural properties of Rhodobacter capsulatus porin are studiedby molecular dynamics simulation using the GROMOS force field.Unconstrained simulations of the trimer and monome r show thetrimer to be more stable than the isolated monomer. Simulationsof the L3 loop insi de the pore are used to assess its stabilityand plastic properties. Simulated annealing shows that the conformationalspace available to the L3 loop inside the pore is very large.Simulations at different temperatures show that the energy hypersurfacearound the open state is complex and flat. These studies alsoindicate four zones that are more flexible than the rest ofthe loop. Two of these are stabilized by the addition of thedetergent molecule present in the X-ray structure. It is possiblethat the two remaining flexible zones, situated in the halfof the loop facing the extracellular end of the porin molecule,residues Asp93–Gly98 and Arg110–Leu111, are involvedin a mechanism for opening and closing of the pore.     

Search for the most stable folds of protein chains: II. Computation of stable architectures of {beta}-proteins using a self-consistent molecular field theory

In a preceding paper we presented a novel approach to computationof 3-D folds of protein chains from their amino acid sequences.This approach is a physically correct generalization of the‘threading’ methods. It is based on a self-consistentmolecular field theory and on a physical theory of protein foldingpatterns, which make it possible to examine all the varietyof ‘potentially stable’ folding patterns and allthe variety of the chain conformations within each of them andto determine the thermodynamically stable structure. In thispaper, we apply this approach to single out stable folding patternsand conformations for the chains of ß-sandwich proteinsand show that the similarity of the calculated and observedstructures is usually rather close.     

Estimating the twist of {beta}-strands embedded within a regular parallel {beta}-barrel structure

The parallel ß-barrel is a recurrent structural motiffound in a large variety of different enzymes belonging to thefamily of /ß-proteins. It has been shown previouslythat the hyperboloid can be considered as a scaffold describingthe parallel ß-barrel structure. To assess restraintson ß-strand twist imposed by a given scaffold geometry,the notion of scaffold twist, T_s, is introduced. From T_s, theß-strand twist (Tw_ß) expected for a givenscaffold geometry can be derived and it is verified that thiscomputed twist can be used to identify ß-barrels characterizedby good hydrogen bonding. It is noted that Tw_ß isonly slightly affected for ß-barrels differing inthe number (N) of ß-strands, suggesting that restraintson main-chain conformation of ß-strands are not likelyto account for the N = 8 invariability observed in natural parallelß-barrels thereby strengthening previous work rationalizingthis constancy.     

Kinetic identification of a hydrogen bonding pair in the glucoamylase-maltose transition state complex

Molecular recognition and site-directed mutagenesis are usedin combination to identify kinetically, transition state interactionsbetween glucoamylase (GA) and the substrate maltose. Earlierstudies of mutant Glu180 – Gin GA had indicated a rolein substrate binding for Glul80 (Slerks, M.R., Ford, C., Reilly,P.J. and Svensson, B. (1990) Protein Engng, 3, 193–198).Here, changes in activation energies calculated from measuredk_cat/K_m values for a series of deoxygenated maltose analoguesindicate hydrogen bonding between the mutant enzyme and the3-OH group of the reducing end sugar ring. Using the same substrateanalogues and determining activation energies with wild-typeGA an additional hydrogen bond with the 2-OH group of maltoseis attributed to an interaction with the carboxylate Glu180.This novel combination of molecular recognition and site-directedmutagenesis enables an enzyme substrate transition state contactto be identified and characterized even without access to thethree dimensional structure of the enzyme. Given the distantstructural relationships between glucoamylases and several starchhydrolases (Svensson, B. (1988) FEBS Lett., 230, 72–76),such identified contacts may ultimately guide tailoring of theactivity of these related enzymes.