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.     

Comparison of the solution and X-ray structures of barley serine proteinase inhibitor 2

A comparison of the solution n.m.r. structures of barley serineprotease inhibitor 2 (BSPI-2) with the X-ray structures of bothsubtilisin complexed and native BSPI-2 is presented. It is shownthat the n.m.r. and X-ray structures are very similar in termsof overall shape, size, polypeptide fold and secondary structure.The average atomic rms difference between the 11 restraineddynamics structures on the one hand and the two X-ray structureson the other is 1.9±0.2 Å for the backbone atomsand 3.0±0.3 Å for all atoms. The cor respondingvalues for the restrained energy minimized mean dynamics structureare 1.5 and 2.4 Å, respectively.     

Methods of translating NMR proton distances into their corresponding heavy atom distances for protein structure prediction with limited experimental data

This paper proposes a strategy to translate experimental 1H NMR proton distance restraints into their corresponding heavy atom distance restraints for the purpose of protein structure prediction. The relationships between interproton distances and the corresponding heavy atom distances are determined by studying well-resolved X-ray protein structures. The data from the interproton distances of amide protons, alpha-protons, beta-protons and side chain methyl protons are plotted against the corresponding heavy atoms in scatter plots and then fitted with linear equations for lower bounds, upper bounds and optimal fits. We also transform the scatter plots into two-dimensional heat maps and three-dimensional histograms, which identify the regions where data points concentrate. The common interproton distances between amide protons, alpha-protons, beta-protons in alpha-helices, anti-parallel beta-sheets and parallel beta-sheets are also tabulated. We have found several patterns emerging from the distance relationships between heavy atom pairs and their corresponding proton pairs. All our upper bound, lower bound and optimal fit results for translating the interproton distance into their corresponding heavy atom distances are tabulated.     

Expression, purification and characterization of recombinant crambin

Crambin, a small hydrophobic protein (4.7 kDa and 46 residues),has been successfully expressed in Escherichia coli from anartificial, synthetic gene. Several expression systems wereinvestigated. Ultimately, crambin was successfully expressedas a fusion protein with the maltose binding protein, whichwas purified by affinity chromatography. Crambin expressed asa C-terminal domain was then cleaved from the fusion proteinwith Factor Xa protease and purified. Circular dichroism spectroscopyand amino acid analysis suggested that the purified materialwas identical to crambin isolated from seed. For positive identificationthe protein was crystallized from an ethanol–water solution,by a novel method involving the inclusion of phospholipids inthe crystallization buffer, and then subjected to crystallographicanalysis, Diffraction data were collected at the Brookhavensynchrotron (beamline-X12C) to a resolution of 1.32 Åat 150 K. The structure, refined to an R value of 9.6%, confirmedthat the cloned protein was crambin. The availability of clonedcrambin will allow site-specific mutagenesis studies to be performedon the protein known to the highest resolution.     

DNA recognition by a {beta}-sheet

DNA recognition by a ß-sheet is discussed in the lightof crystal structures of the MetJ and Arc repressors. The DNAbinding geometry of a ß-sheet can be understood interms of (i) close fitting of the two surfaces and (ii) matchingof residue and base positions. A ß-sheet is not entirelyflat but has a curvature. A ß-sheet of the Met-Arcfamily faces the DNA major groove with its convex surface; thelocal DNA major groove is deepest at the centre. The ß-sheetfollows 6 bp; every two residues face the DNA and the firstand fifth residues, which are separated by 13.2 Å, bind,respectively, to the third and sixth bases, which are separatedby 13.5 Å, on the same DNA strand.     

Analysis and prediction of inter-strand packing distances between {beta}-sheets of globular proteins

Any two ß-strands belonging to two different ß-sheetsin a protein structure are considered to pack interactivelyif each ß-strand has at least one residue that undergoesa loss of one tenth or more of its solvent contact surface areaupon packing. A data set of protein 3-D structures (determinedat 2.5 Å resolution or better), corresponding to 428 proteinchains, contains 1986 non-identical pairs of ß-strandsinvolved in interactive packing. The inter-axial distance betweenthese is significantly correlated to the weighted sum of thevolumes of the interacting residues at the packing interface.This correlation can be used to predict the changes in the inter-sheetdistances in equivalent ß-sheets in homologous proteinsand, therefore, is of value in comparative modelling of proteins.     

The 2 A crystal structure of subtilisin E with PMSF inhibitor

Using enzyme prepared by the DNA recombination technique, subtilisinE from Bacillus subtilis was crystallizedin space group P2₁2₁2₁with two molecules in an asymmetric unit. The crystal structureof PMSF-inhibited subtilisin E was solved by molecular replacementfollowed by refinement with the X-PLOR program. This resultedin the 2.0 Å structure of subtilisin E with an R-factorof 0.191 for 8–2 Å data and r.m.s. deviations fromideal values of 0.021 Å and 2.294° for bond lengthsand bond angles respectively. The PMSF group covalently boundto Ser221 appeared very clearly in the electron density map.Except for the active site disturbed by PMSF binding, the structuralfeatures of subtilisin E are almost the same as in other subtilisins.The calcium-binding sites are different in detail in the twoindependent molecules of subtilisin E. Based on the structure,the remarkably enhanced heat stability of mutant N118S of subtilisinE is discussed. It is very likely that there is an additionalwater molecule in the mutant structure, which is hydrogen bondedto side chains of Serll8 and its neighbouring residues Lys27and Asp 120.     

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.     

The structure of iron superoxide dismutase from Pseudomonas ovalis complexed with the inhibitor azide

The 2.9 Å resolution structure of iron superoxide dismutase(FeSOD) (EC 1.15.1.1 [EC] ) from Pseudomonas ovalis complexed withthe inhibitor azide was solved. Comparison of this structurewith free enzyme shows that the inhibitor is bound at the opencoordination position of the iron, with a bond length of 2.0Å. The metal moves by 0.4 Å into the trigonal planeto produce an orthogonal geometry at the iron. Binding of theinhibitor also causes a movement of the axial ligand (histidine26) away from the metal, a lengthening of the iron—histidinebond, and a rotation of the histidine 74 ring. The inhibitorpossesses contacts in the binding pocket with a pair of conservedtryptophan residues and with the side chains of tyrosine 34and glutamine 70. This glutamine is conserved between all FeSODs,but is absent in MnSOD. Comparisons with MnSOD show that a differentglutamine which possesses the same interactions in the activesite as Gln70 in FeSOD is conserved at position 154 in the overallSOD sequence, implying that while manganese and FeSODs are structuralhomologues in a global sense, their functional and evolutionaryrelationship is that of second-site mutation revertants.     

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 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.     

Confirmation of the predicted source of a slow folding reaction: proline 8 of bovine pancreatic trypsin inhibitor

A major question in protein structural analysis concerns theapplicability of results from model systems to other proteins.Theoretical approaches seem the best manner of transferringinformation from one system to another, but their accuracy inthe model systems must first be tested with results from experiment.Since bovine pancreatic trypsin inhibitor (BPTI) is a modelsystem for the evaluation of energy minimization and moleculardynamics routines, we can use folding and stability measurementsto examine the reliability of these methods. All two-disulfidemutants of BPTI investigated thus far have two very slow foldingreactions which have characteristics of proline isomerization.These reactions may occur because the non-native cis form oftwo of the four prolines in BPTI significantly destabilizesthe folded state of the protein. Previous energy minimizationstudies of wild-type BPTI suggested that the cis form of Pro8was the most destabilizing of the four prolines [Levitt,M. (1981)J. Mol. Biol., 145, 251–263]. In this paper, we show thatmutation of Pro8 - Gln in the two-disulfide bond mutant Val30–Ala51results in a loss of the slowest folding reaction, consistentwith Levitt's prediction.     

1.85 A structure of anti-fluorescein 4-4-20 Fab

The crystal complex of fluorescein bound to the high-affinityanti-fluorescein 4-4-20 Fab {K_a = 10¹⁰ M^–1 at 2°C)has been determined at 1.85 Å. Isomorphous crystals oftwo isoelectric forms (p1 = 7.5 and 7.9) of the antifluorescein4-4-20 Fab, an IgG2A [Gibson et al (1988)Proteins: Struct. FunctGenet., 3, 155–160], have been grown. Both complexes crystallizewith one molecule in the asymmetric unit in space group P1,with a = 42.75 Å, b =43.87 Å, c = 58.17 Å, = 95.15° , ß = 86.85° and = 98.01°.The final structure has an R value of 0.188 at 1.85 Åresolution. Interactions between bound fluorescein, the complementarity-determiningregions (CDRs) of the Fab and the active-site mutants of the4-4-20 single-chain Fv will be discussed. Differences were foundbetween the structure reported here and the previously reported2.7 Å 4-4-20 Fab structure [Herron et al. (1989) Proteins:Struct. Fund., 5, 271–280]. Our structure determinationwas based on 26 328 unique reflections — four times theamount of data used in the previous report. Differences in thetwo structures could be explained by differences in interpretingthe electron density maps at the various resolutions. The r.m.s.deviations between the variable and constant domains of thetwo structures were 0.77 and 1.54 Å, respectively. Fourregions of the light chain and four regions of the heavy chainhad r.m.s. backbone deviations of >4 Å. The most significantof these was the conformation of the light chain CDR 1.     

Analysis of protein conformational characteristics related to thermostability

The thermal stability of proteins was studied, 195 single aminoacid residue replacements reported elsewhere being analysedfor several protein conformational characteristics: type ofresidue replacement; conservative versus nonconservative substitution;replacement being in a homologous stretch of amino acid residues;change in hydrogen bond, van der Waals and secondary structurepropensities; solvent-accessible versus inaccessible replacement;type of secondary structure involved in the substitution; thephysico-chemical characteristics to which the thermostabilityenhancement can be attributed; and the relationship of the replacementsite to the folding intermediates of the protein, when known.From the above analyses, some general rules arise which suggestwhere amino acid substitutions can be made to enhance proteinthermostability: substitutions are conservative according tothe Dayhoff matrix; mainly occur on conserved stretches of residues;preferentially occur on solvent-accessible residues; maintainor enhance the secondary structure propensity upon substitution;contribute to neutralize the dipole moment of the caps of helicesand strands; and tend to increase the number of potential hydrogenbonding or van der Waals contacts or improve hydrophobic packing.     

Monte Carlo docking of protein-DNA complexes: incorporation of DNA flexibility and experimental data

A Monte Carlo simulation program (MONTY) has been developedto dock proteins onto DNA. Protein and DNA interact via square-wellpotentials for hydrogen bond and van der Waals interactions.The effect of the inclusion of DNA flexibility and experimentallyderived restraints has been tested on members of the helix-turn-helixfamily of DNA binding proteins. Unwinding and bending the DNAdouble helix improves the number of correctly retrieved hydrogenbonds in simulations starting from the 434 cro protein monomercomplexed with a standard B-DNA O_Rl half-site. Agreement withphosphate ethylation interference and mutagenesis data is rewardedwith energy bonuses. This protocol was tested on protein-DNAcomplexes of 434 cro, lac headpiece and a mutant lac headpieceresembling the gal repressor headpiece with the recognitionhelices in correct and reversed orientations in the DNA majorgroove. The inclusion of experimental data gives an improvedconvergence of the correctly oriented structures and allowsfor an easier discrimination between correctly and incorrectlydocked complexes     

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.     

Solution conformations of human growth hormone releasing factor: comparison of the restrained molecular dynamics and distance geometry methods for a system without long-range distance data

A series of three-dimensional structures of the 1–29 fragmentof human growth hormone releasing factor in trifluoroethanolhave been determined by molecular dynamics and distance geometrymethods. The resulting structures satisfy information from nuclearOverhauser effect (NOE) distance data and an empirical potentialenergy function. Although the polypeptide was found to havean ordered structure in all simulations, the NOE data were notsufficient for global convergence to a unique three-dimensionalgeometry. Several satisfactory structures have been determined,all of which are extended conformations consisting of a shortß-strand and two -helices (residues 6–13 andresidues 16–29) connected by short segments of less welldefined secondary structure. Because of the lack of NOE dataconnecting the helix segments, their relative orientation isnot uniquely determined.     

Alternating charge clusters of side chains: new surface structural invariants observed in calf eye lens gamma-crystallins

A detailed stereochemical analysis of the oppositely chargedside chains of amino acid residues on the surface of calf eyelens protein gamma-crystallin B has been carried out. The refinedstructural data of very high quality obtained at 1.47 Åresolution have been used. Charge–charge interactionswere considered to be valuable for all the charged oxygen andnitrogen atoms situated at distances, d, between 2.4 and 7.0Å. This means we consider short contact ion pairs as thosewith interchange distances 2.4 < d 4.0 Å and distantcontact ion pairs as those with distances 4.0 < d 7.0 Å.Hydrogen bonding of the charged atomic groups with the structuralwater molecules also has been considered. We have not lookedat the side groups of histidines which are charged only partiallyat neutral pH. Five clusters of charged side chains which werelarge enough were observed. The clusters are comprised of fourto six residues which compose 543% of the total charged residuesin the protein. The clusters contain from eight to 12 chargedatoms and look like the bent chains of oppositely charged atoms.All clusters are of plane geometry and their maximal lineardimensions are from 11 to 18 Å. The root mean square deviationsof charged atoms from the cluster plane varied from 0.63 to0.86 Å for four clusters and was only 1.85 Å forthe largest cluster. All clusters include a number of watermolecules situated on the cluster boundary and grouped nearthe cluster plane. It was shown that the amino acid sequencepositions of charged residues are conservative for all the proteinsof the gamma-crystallin family of vertebrates including fish,frog, mouse, rat, calf and human. The cluster properties werediscussed both in their functional aspect for gamma-crystallinsand in other aspects common for globular proteins. As a result,the alternating charge clusters should be considered as newlyrecognized surface structural invariants. The importance ofthe charged side chain clusters is claimed for the updated conceptof the protein surface.     

Molecular dynamics simulation of winter flounder antifreeze protein variants in solution: correlation between side chain spacing and ice lattice

The solution structure of the 38 amino acid C-terminal regionof the precursor for the HPLC-6 antifreeze protein from winterflounder has been investigated with molecular dynamics usingthe AMBER software. The simulation for the peptide in aqueoussolution was carried out at a constant temperature of 0°Cand at atmospheric pressure. The simulation covered 120 ps andthe results were analyzed based on data sampled upon reachinga stable equilibrium phase. Information has been obtained onthe quality of constant temperature and pressure simulations,the solution structure and dynamics, the hydrogen bonding network,the helix-stabilizing role of terminal charges and the interactionwith the surrounding water molecules. The Lys18–Glu22interactions and the terminal charged residues are found tostabilize a helical structure with the side chains of Thr2,Thr13, Thr24 and Thr35 equally spaced on one side of the helix.The spacing between oxygen atoms in the hydroxyl group of thethreonine side chains exhibits fluctuations of the order of2–3 Å during the 120 ps of simulation, but valuessimultaneously close to the repeat distance of 16.6 Åbetween oxygen atoms along the [0112] direction in ice are observed.Furthermore, two engineered variants were studied using thesame simulation protocol.