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.     

Biosynthesis of winter flounder antifreeze proprotein in E.coli

A semisynthetic winter flounder antifreeze proprotein (proAFP)coding region was constructed and inserted into a lacZ expressionvector. ProAFP was produced from the vector in Escherichia colias a C-terminal fusion to the first 289 amino acids of ß-galactosidase(ß-gal). The proAFP and ß-gal domains ofthe ß-gal–proAFP fusion protein were separatedby the recognition signal for the blood coagulation protease,factor X_a. Upon induction with isopropylthio-ß-D-galactosidethe fusion protein accumulated to levels of 15% of the totalprotein. The ß-gal–proAFP fusion protein waspartially purified by differential centrifugation, but requiredsolubilization prior to factor X_a digestion. The solubilizedfusion protein was efficiently and correctly cleaved by factorX_a, after which the proAFP was purified by gel permeation. BacterialproAFP was indistinguishable from natural proAFP by the criteriaof antifreeze activity, amino-terminal sequence (15 cycles),reverse-phase HPLC and SDS–polyacrylamide gel electrophoresis.Circular dichroism measurements showed that proAFP is a compositeof random coil and -helical secondary structure, with an -helicalcontent of 44% at 0°C. It seems probable that the C-terminalregion of proAFP, which corresponds to the mature AFP protein,is mainly -helical, and that the N-terminal pro-segment is randomcoiled.     

Investigation of the solution structure of chymotrypsin inhibitor 2 using molecular dynamics: comparison to X-ray crystallographic and NMR data

The native solution structure and dynamics of chymotrypsin inhibitor2 (CI2) have been studied using a long (5.3 ns) molecular dynamics(MD) simulation without any imposed restraints. The majorityof the experimentally observed spin–spin coupling constants,short– and long–range nuclear Overhauser effect(NOE) cross peaks and the amide hydrogen exchange behavior werereproduced by the MD simulation. This good correspondence suggeststhat the major structural features of the protein during thesimulation are representative of the true protein structurein solution. Two water molecules formed hydrogen bond bridgesbetween ß₂ and ß₃, in agreement with X–raycrystallographic data and a recent reassessment of the solutionstructure using time averaged NMR restraints during MD refinement.The active–site loop of the protein displayed the greateststructural changes and the highest mobility. When this loopregion was excluded, the average C r.m.s. deviation of the simulatedsolution structures from the crystal structure was 1.5 from0.5 to 5 3 ns. There is structural heterogeneity in particularregions of the NMR–derived solution structures, whichcould be a result of imprecision or true internal motion. Astudy of the distribution of mobility through the protein allowsus to distinguish between these two alternatives. In particular,deviations in the active–site loop appear to be a resultof heightened mobility, which is also supported by good correspondencebetween calculated and experimental S² N–H order parameters.On the other hand, other ill–defined regions of the NMR-derivedstructures are well defined in the simulation and are probablythe result of a lack of structural restraints (i.e. NOEs), asopposed to reflecting the true mobility.     

The correlation between protein stability and dipole moment: a critical test

Improving the stability of proteins is a major aim in basic and applied protein science. Querol and coworkers calculated changes in the quasi-electric dipole moment of a protein and used it as a simple criterion to predict stabilizing charge mutations. They employed this method to propose for the bacterial cold shock protein Bc-Csp a number of charge mutations that should have a strong influence on stability. We produced eight variants of Bc-Csp with such mutations and measured their stabilities experimentally. However, we could not find a correlation between the stability and the quasi dipole moment of these variants. Possibly, the quasi dipole moment reflects only a secondary aspect of the changes that are caused by charge mutations in a protein.     

Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance

Both freezing tolerance and NaCI tolerance are improved whenantifreeze proteins are expressed as fusion proteins with twodomains of staphylococcal protein A (SPA) in Escherichia coli.To characterize these properties further we created a randomlymutated expression library in E.coli, based on the winter flounderantifreeze protein HPLC-8 component gene. Low-fidelity PCR productsof this gene were fused to the spa gene encoding two domainsof the SPA. The library was screened for enhanced NaCl toleranceand four clones were selected. The freezing tolerance of eachof the selected clones was enhanced to varying extents. DNAsequencing of the isolated mutants revealed that the amphiphilicproperties of the native antifreeze protein were essentiallyconserved. Furthermore, by studying the primary sequence ofthe randomly mutated clones, in comparison with the degree offreezing tolerance, we have identified clues which help in understandingthe relationship between salt and freezing tolerance.     

Comparative molecular dynamics simulation studies of salmon and bovine trypsins in aqueous solution

The flexibility and conformational behaviour of salmon and bovinetrypsins were modelled with a 300 ps molecular dynamics simulationin aqueous solution. Trajectories from both trypsins were analysedto eventually detect differences in mobility that could explainobserved variations in stability and activity. The simulationswere performed at 300 K with all the acidic groups deprotonatedand the basic groups protonated. The radius of gyration, theoverall r.m.s. deviation from the starting structure as a functionof time, together with the r.m.s. deviation from the startingstructures as a function of residue number, demonstrated thatthe simulations were stable and representative of the X-raystructures of both enzymes. Isotropic Debye-Waller factors werecalculated from the fluctuations for mainchain atoms and werein good agreement with experimental values. The overall dynamicproperties of the two enzymes were similar. Based on the present300 ps molecular dynamics simulation, it cannot be concludedthat either of the two enzymes is more 'flexible' than the other.However, there are clearly differences in mobility on a moredetailed level and for particular regions.     

Refinement of protein cores and protein-peptide interfaces using a potential scaling approach

Refinement of side chain conformations in protein model structures and at the interface of predicted protein-protein or protein-peptide complexes is an important step during protein structural modelling and docking. A common approach for side chain prediction is to assume a rigid protein main chain for both docking partners and search for an optimal set of side chain rotamers to optimize the steric fit. However, depending on the target-template similarity in the case of comparative protein modelling and on the accuracy of an initially docked complex, the main chain template structure is only an approximation of a realistic target main chain. An inaccurate rigid main chain conformation can in turn interfere with the prediction of side chain conformations. In the present study, a potential scaling approach (PS-MD) during a molecular dynamics (MD) simulation that also allows the inclusion of explicit solvent has been used to predict side chain conformations on semi-flexible protein main chains. The PS-MD method converges much faster to realistic protein-peptide interface structures or protein core structures than standard MD simulations. Depending on the accuracy of the protein main chain, it also gives significantly better results compared with the standard rotamer search method.     

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.     

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.     

Physicochemical factors for discriminating between soluble and membrane proteins: hydrophobicity of helical segments and protein length

The average hydrophobicity of a polypeptide segment is consideredto be the most important factor in the formation of transmembranehelices, and the partitioning of the most hydrophobic (MH) segmentinto the alternative nonpolar environment, a membrane or hydrophobiccore of a globular protein may determine the type of proteinproduced. In order to elucidate the importance of the MH segmentin determining which of the two types of protein results froma given amino acid sequence, we statistically studied the characteristicsof MH helices, longer than 19 residues in length, in 97 membraneproteins whose three-dimensional structure or topology is known,as well as 397 soluble proteins selected from the Protein DataBank. The average hydrophobicity of MH helices in membrane proteinshad a characteristic relationship with the length of the protein.All MH helices in membrane proteins that were longer than 500residues had a hydrophobicity greater than 1.75 (Kyte and Doolittlescale), while the MH helices in membrane proteins smaller than100 residues could be as hydrophilic as 0.1. The possibilityof developing a method to discriminate membrane proteins fromsoluble ones, based on the effect of size on the type of proteinproduced, is discussed.     

L3 loop-mediated mechanisms of pore closing in porin: a molecular dynamics perturbation approach

L3 loop-mediated mechanisms for pore closing in porin are investigatedwith molecular dynamics simulation, using an approach that canbe related to the phenomenon of voltage gating. Voltage gatingis seen as a perturbation of the electrostatic screening insidethe porin pore where, by the influence of the potential gradient,water and counter-ion distribution can be slightly displacedfrom their equilibrium distribution. This is simulated by perturbingthe screening electrostatics of ionizable groups inside thepore. Under these conditions, a localized conformational changetakes place, involving 12 (Ile102–Alall3) out of the 44residues of the loop. The pore is reduced to a sixth of itsopen state size. The conformational change can be achieved witha small perturbation and it is reversible once the perturbationis switched off (relaxation process). Other types of behaviourpredominating at higher simulation temperatures are found forthe loop, involving an extra conformational change in the Thr92–Asp96loop segment. This conformational change completely closes thepore, but is not reversible under the simulation conditions.Both zones involved in the conformational changes contain oroverlap the zones which were described previously, using othertechniques, to be the most flexible zones of the loop.     

Computational analysis of chain flexibility and fluctuations in Rhizomucor miehei lipase

We have performed molecular dynamics simulation of Rhizomucormiehei lipase (Rml) with explicit water molecules present. Thesimulation was carried out in periodic boundary conditions andconducted for 1.2 ns in order to determine the concerted proteindynamics and to examine how well the essential motions are preservedalong the trajectory. Protein motions are extracted by meansof the essential dynamics analysis method for different lengthsof the trajectory. Motions described by eigenvector 1 convergeafter approximately 200 ps and only small changes are observedwith increasing simulation time. Protein dynamics along eigenvectorswith larger indices, however, change with simulation time andgenerally, with increasing eigenvector index, longer simulationtimes are required for observing similar protein motions (alonga particular eigenvector). Several regions in the protein showrelatively large fluctuations and in particular motions in theactive site lid and the segments Thr57–Asn63 and the activesite hinge region Pro101–Gly104 are seen along severaleigenvectors. These motions are generally associated with glycineresidues, while no direct correlations are observed betweenthese fluctuations and the positioning of prolines in the proteinstructure. The partial opening/closing of the lid is an exampleof induced fit mechanisms seen in other enzymes and could bea general mechanism for the activation of Rml.     

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.     

Correlation between occupancy and B factor of water molecules in protein crystal structures

An empirical relationship between occupancy and the atomic displacementparameter of water molecules in protein crystal structures hasbeen found by comparing a set of well refined sperm whale myoglobincrystal structures. The relationship agrees with a series ofindependent structural features whose impact on water occupancycan easily be predicted as well as with other known data andis independent of the protein fold. The estimation of the wateroccupancy in protein crystal structures may help in understandingthe physico-chemical properties of the protein–solventinterface and can allow the monitoring of the accuracy of theprotein crystal structure refinement.     

Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose binding surface

A total of 23 fungal cellulose-binding domain (CBD) sequenceswere aligned. Structural models of the cellulosebinding domainof an exoglucanase (CBHII) and of three endoglucanases (EGI,EGII and EGV) from Trichoderma reesei cellulases were homologymodelled based on the NMR structure of the fungal cellobiohydrolaseCBHI, from the same organism. The completed models and the knownstructure of the CBHI cellulose-binding domain were refinedby molecular dynamics simulations in water. All four modelswere found to be very similar to the structure of the CBHI cellulose-bindingdomain and sequence comparison indicated that in general thethree-dimensional structures of fungal cellulose-binding domainsare very similar. In all the CBDs studied, two disulphide bridgesapparently stabilize the polypeptide fold. From the models,an additional disulphide bridge was predicted in EGI and CBHII,and in eight further CBDs from other organisms. Three highlyconserved aromatic residues on the hydrophilic side of the wedgemake this surface flat This surface is expected to make contactwith the substrate. Three invariant amino acids, Gln7, Asn29and Gln34, on this flat face are in suitable positions for hydrogenbonding with the cellulose surface. Analysis of the differencesin the protein surface properties indicated that the endoglucanasestend to be more hydrophilic than the exoglucanases. The largeststructural variation was found around positions 12-16. The fungalCBD sequences are discussed in relation to variations in functionand pH dependence. Comparison of the modelled structures withexperimental binding data for the CBHI and EGI allowed the formulationof a qualitative relationship to cellulose affinity. This relationshipwas used to predict the cellulose affinities for 21 CBDs.     

Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence

Statistical analysis of 12 unstable and 32 stable proteins revealedthat there are certain dipeptides, the occurrence of which issignificantly different in the unstable proteins compared withthose in the stable ones. Based on the impact of these dipeptideson the unstable proteins over the stable ones, a weight valueof instability is assigned to each of the dipeptides. For agiven protein the summation of these weight values normalizedto the length of its sequence helps to distinguish between unstableand stable proteins. Results suggest that the in vivo instabilityof proteins is possibly determined by the order of certain aminoacids in its sequence. An attempt is made to correlate metabolicstability of proteins with features of their primary sequencewhere weight values of instability for a protein of known sequencecould thus be used as an index for predicting its stabilitycharacteristics.     

The determination of the three-dimensional structure of barley serine proteinase inhibitor 2 by nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution structure of the 64 residue structured domain (residues20–83) of barley serine proteinase inhibitor 2 (BSPI-2)is determined on the basis of 403 interproton distance, 34 øbackbone torsion angle and 26 hydrogen bonding restraints derivedfrom n.m.r. measurements. A total of 11 converged structureswere computed using a metric matrix distance geometry algorithmand refined by restrained molecular dynamics. The average rmsdifference between the final 11 structures and the mean structureobtained by averaging their coordinates is 1.4±0.2 Åfor the backbone atoms and 2.1±0.1 A for all atoms. Theoverall structure, which is almost identical to that found byX-ray crystallography, is disc shaped and consists of a centralfour component mixed parallel and antiparallel ß-sheetflanked by a 13 residue helix on one side and the reactivesite loop on the other.     

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.     

Substrate mobility in thiocamphor-bound cytochrome P450cam: an explanation of the conflict between the observed product profile and the X-ray structure

Thiocamphor is an unusual substrate for P450_cam in that in theX-ray structure it binds in the active site pocket in two distinctorientations and neither of these orientations are consistentwith the 5-alcohol being the primary product. Other camphoranalogs such as norcamphor or camphane bind in a single orientationconsistent with the 5-alcohol being a major product. We presentan analysis of four 175 ps molecular dynamics trajectories ofthiocamphor-bound cytochrome P450_cam. The first two trajectorieswere calculated for cytochrome P450_cam with thiocamphor boundin both its major and minor crystallographic orientations. Inthe second set of simulations, a single oxygen atom was addedas a distal ligand to the heme group in order to model the putativeferryl oxygen reaction intermediate. Trajectories were againcalculated starting with thiocamphor in its major and minororientations. While the protein dynamics were quite similarin all four trajectories, the substrate showed distinctly differentmotions in each of the trajectories. In particular, the preferredsubstrate orientations were very different in the presence ofthe ferryl oxygen than in the absence of that oxygen. The preferredorientations in the absence of the distal oxygen were consistentwith the 3-akohol being the major product, while the preferredorientations in the presence of the distal oxygen were consistentwith the 5-alcohol being a major product. These simulationsoffer an explanation for the inconsistency between the X-raydata and the product profile.     

Analysis of the catalyic mechanism of a fungal lipase using computer-aided design and structural mutants

Both an active enzyme conformation and stabilization of tetrahedraltransition states are essential for the catalysis of ester bondhydrolysis by lipases. X-ray structural data and results fromsite-directed mutagenesis experiments with proteases have beenused as a basis for predictions of amino acid residues likelyto have key functions in lipases. The gene encoding a lipasefrom Rhizopus oryzae was cloned and expressed in Escherichiacoli. Site-directed mutagenesis of this gene was used to testthe validity of computer-aided predictions of the functionalroles of specific amino acids in this enzyme. Examination ofthe kinetic constants of the Rhizopus oryzae lipase variantsallowed us to identify amino acid residues which are directlyinvolved in the catalytic reaction or which stabilize the activegeometry of the enzyme. The combination of these results withmolecular mechanics simulations, based on a homology-derivedstructural model, provided new information about structure-functionrelationships. The interpretation of the data is consistentwith results obtained with other hydrolases, such as proteases.