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.     

Structure prediction of subtilisin BPN' mutants using molecular dynamics methods

In this paper we describe the achievements and pitfalls encounteredin doing structure predictions of protein mutants using moleculardynamics simulation techniques in which properties of atomsare slowly changed as a function of time. Basically the methodconsists of a thermodynamic integration (slow growth) calculationused for free energy determination, but aimed at structure prediction;this allows for a fast determination of the mutant structure.We compared the calculated structure of the mutants Met222Ala,Met222Phe and Met222Gln of subtilisin BPN' with the respectiveX-ray structures and found good agreement between predictedand X-ray structure. The conformation of the residue subjectto the mutation is relatively easy to predict and is mainlydetermined by packing criteria. When the side chain has polargroups its exact orientation may pose problems; long-range Coulombinteractions may generate a polarization feedback involvingsystem relaxation times beyond the simulation time. Changesinduced in the environment are harder to predict using thismethod. In particular, rearrangement of the hydration structurewas difficult to predict correctly, probably because of thelong relaxation times. In all conversions made the changes observedin the environment were found to be history-dependent and inparticular the hydrogen bonding patterns provided evidence formetastable substates. In all cases the structure predicted wascompared with available kinetic data and the reduced activitycould be explained in terms of changes in the configurationof the active site.     

Stabilized variant of Streptomyces subtilisin inhibitor and its use in stabilizing subtilisin BPN'

Protein protease inhibitors could potentially be used to stabilize proteases in commercial products such as liquid laundry detergents. However, many protein protease inhibitors are susceptible to hydrolysis inflicted by the protease. We have engineered Streptomyces subtilisin inhibitor (SSI) to resist proteolysis by adding an interchain disulfide bond and removing a subtilisin cleavage site at leucine 63. When these stabilizing changes were combined with changes to optimize the affinity for subtilisin, the resulting inhibitor provided complete protease stability for at least 5 months at 31 degrees C in a subtilisin-containing liquid laundry detergent and allowed full recovery of the subtilisin activity upon the dilution that occurs in a North American washing machine.     

Contribution of a salt bridge to binding affinity and dUMP orientation to catalytic rate: mutation of a substrate-binding arginine in thymidylate synthase

Invariant arginine 179, one of four arginines that are conservedin all thymidylate synthases (TS) and that bind the phosphatemoiety of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP),can be altered even to a negatively charged glutainic acid withlittle effect on k_cat. In the mutant structures, ordered wateror the other phosphate binding arginines compensate for thehydrogen bonds made by Arg179 in the wild-type enzyme and thereis almost no change in the conformation or binding site of dUMP.Correlation of dUMP K_ds for TS R179A and TS R179K with the structuresof their binary complexes shows that the positive charge onArg179 contributes significantly to dUMP binding affinity. k_cat/Kmfor dUMP measures the rate of dUMP binding to TS during theordered bi-substrate reaction, and in the ternary complex dUMPprovides a binding surface for the cofactor. k_cat/Km reflectsthe ability of the enzyme to accept a properly oriented dUMPfor catalysis and is less sensitive than is K_d to the changesin electrostatics at the phosphate binding site.     

Comparative modeling of the three CP modules of the {beta}-chain of C4BP and evaluation of potential sites of interaction with protein S

A computer model of the ß-chain of C4b-binding protein(C4BP) was constructed, using the backbone fold of the NMR structuresof the sixteenth CP module of factor H (H16) and of a pair ofmodules consisting of the fifteenth and sixteenth CPs of factorH (H15-16). The characteristic hydrophobic core responsiblefor dictating the three-dimensional structure of the CP familyis conserved in the amino acid sequence of C4BP ßl, ß2and ß3. The distribution of the electrostatic potentialshows that the model is mainly covered by a negative contour.Interestingly, a positive area is observed in the C-terminalregion of the first CP module, enclosing peptide 31-45, knownto be a binding site for protein S. This observation suggeststhat electrostatic interactions can be of importance for theinteraction of C4BP to protein S. A solvent-accessible hydrophobicpatch, located nearby and involving the peptide 31-45, was alsofound in the model, further confirming that this area is involvedin the interaction with protein S. The contribution of ß-chainresidues 31-45 to the affinity for protein S was studied furtherby means of synthetic mutant peptides. The results suggest thatboth electrostatic and hydrophobic interactions are importantfor the binding to protein S.     

Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme

To elucidate correlative relationships between structural changeand thermodynamic stability in proteins, a series of mutanthuman lysozymes modified at two buried positions (Ile56 andIle59) were examined. Their thermodynamic parameters of denaturationand crystal structures were studied by calorimetry and X-raycrystallography. The mutants at positions 56 and 59 exhibiteddifferent responses to a series of amino acid substitutions.The changes in stability due to substitutions showed a linearcorrelation with changes in hydrophobicity of substituted residues,having different slopes at each mutation site. However, thestability of each mutant was found to be represented by a uniqueequation involving physical properties calculated from mutantstructures. By fitting present and previous stability data formutant human lysozymes substituted at various positions to theequation, the magnitudes of the hydrophobicity of a carbon atomand the hydrophobicity of nitrogen and neutral oxygen atomswere found to be 0.178 and –0.013 kJ/mol.Ų, respectively.It was also found that the contribution of a hydrogen bond witha length of 3.0 Å to protein stability was 5.1 kJ/moland the entropy loss of newly introduction of a water moleculeswas 7.8 kJ/mol.     

Relationship between thermal stability and 3-D structure in a homology model of 3-isopropylmalate dehydrogenase from Escherichia coli

To reveal the structural basis of the increased thermal stabilityof 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus,an extreme thermophile, the homology-based structural modelof one mesophilic (Escherichia coli) counterpart, was constructed.Both IPMDHs are homodimeric proteins. We built a model of onesubunit using the 3-D structures of the Th.thermophilus IPMDHand the homologous E.coli isocitrate dehydrogenase. Energy minimizationand molecular dynamics simulated annealing were performed onthe dimer, including a surrounding solvation shell. No seriouserrors were detected in the refined model using the 3-D profilemethod. The resulting structure was scrutinized and comparedwith the structure of the Th.thermophilus IPMDH. Significantdifferences were found in the non-specific interactions includingthe hydrophobic effect. The model predicts a higher number ofion pairs in the Th.thermophilus than hi the E.coli enzyme.An increase was observed in the stabilities of -helical regionshi the thermophilic protein. The preliminary X-ray coordinatesof the E.coli IPMDH were received after the completion of thiswork, allowing an assessment of the model in terms of the X-raystructure. The comparison proved that most of the structuralfeatures underlying the stability differences between the twoenzymes were predicted correctly.     

Increasing thermal stability of subtilisin from mutations suggested by strongly interacting side-chain clusters

In this paper we present for seven subtilisin structures a systematiccomparison of densely packed side-group clusters (defined asan ensemble of side chains with extensive internal atomic contactsas compared with those made with the surrounding protein environmentand measured relative to the maximum possible for each residuetype). Spatially consistent clusters are observed at structurallyequivalent positions in the proteins, as revealed by carefulmultiple superpositioning of the respective backbone atoms.The clusters are positioned at strategic loop-connecting sitesnear the protein surfaces. The residues within consistent clustersdisplaying extensive association show varying conservation atstructurally equivalent alignment sites. Suggestions for residuesubstitutions, as observed over the seven tertiary structures,were taken from the cluster positions and were shown to be consistentwith a number of point mutations in one of the seven structures(savinase) that result in increased thermal stability.     

Study of cysteine residues in the {alpha} subunit of Escherichia coli tryptophan synthase. 1. Role in conformational stability

To eludicate the role in conformational stability of Cys residuesburied in the interior of a protein, the thermodynamic propertiesof denaturation of mutant subunit of Escherichia coli tryptophansynthase, in which Ser, Ala, Val or Gly was substituted foreach of the three Cys residues, were analyzed using calorimetry.The mutants were less stable than the wild type, indicatingthat Cys residues contribute greatly to the stability of the subunit. In most cases, a large decrease in denaturation enthalpywas observed, compensated for by the denaturation entropy toa major extent. The extent of changes in the denaturation Gibbsenergy and denaturation enthalpy varied greatly depending onboth substituting residues and positions. Of all the mutantproteins, the Cys154Ser mutant showed the greatest decreasein denaturation enthalpy; its denaturation enthalpy was halfthat of the wild type, and was considerably repaired by addinga ligand of the subunit. Because the enthalpy of ligand bindingto Cys154Ser in the native state did not change. it seems thatthe decrease in the denaturation enthalpy of Cys154Ser and itsrecovery by ligand binding are caused by conformational changesin the denatured state due to the mutation.     

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replicaplate screen facilitated the isolation of mutant subtilisinsfrom Bacillus amyloliquefaciens that had altered autolytic stabilityunder alkaline conditions. Out of about 4000 clones screened,approximately 70 produced subtilisins with reduced stability(negatives). Two dones produced a more stable subtilisin (positives)and were identified as having a single mutation, either IIe107Valor Lys2l3Arg (the wild-type amino acid is followed by the codonposition and the mutant amino acid). One of the negative mutants,Met50Val, was at a site where other homologous subtilisins containeda Phe. When the Met50Phe mutation was introduced into the B.amyloliquefaciens gene, the mutant subtilisin was more alkalinestable. The double mutant IIe107Val/Lys2l3Arg) was more stablethan the isolated single mutant parents. The triple mutant (Met50Phe/IIel07Val/Lys2l3Arg)was even more stable than IIe107Val/Lys2l3Arg (up to two timesthe autolytic half-time of wild-type at pH 12). These studiesdemonstrate the feasibility for improving the alkaline stabilityof proteins by random mutagenesis and identifying potentialsites where substitutions from homologous proteins can improvealkaline stability.     

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.     

Conformational modeling of substrate binding to endocellulase E2 from Thermomonospora fusca

Molecular mechanics calculations have been used to place a cellotetraosesubstrate into the active site of the crystallographlcally determinedstructure of endocellulase E2 from Thermomonospora fusca. Inthe lowest energy model structure, the second residue of thesubstrate oligosaccharide is tilted away from the planar ribbongeometry of cellulose as it is in the X–ray structureof the E2_cd–cellobiose cocrystal. This tilt is the resultof the topology of the binding site, and results in severalstrong carbohydrate–protein hydrogen bonds. The tiltingproduces a twisting of the glycosidic linkage of the cleavagesite between residues two and three. In the predicted enzyme–substratecomplex both of the Asp residues believed to function in generalacid and base roles in the previously proposed model for themechanism are distant from the bond being cleaved. Moleculardynamics simulations of the complex were conducted, and whilethe putative catalytic Asp residues remained distant from thecleavage site, the proton of Tyr73 briefly came within van derWaals contact of the linkage oxygen.     

Humanization of mouse anti-human IL-2 receptor antibody B-B10

Mouse monoclonal anti–human IL–2 receptor antibody(BB10) inhibits EL–2–dependent human T–cellproliferation. It has been used in clinical trials in the transplantationfield and promising results are being accumulated. Mouse B–B10antibody was humanized by grafting all CDRs and some frameworkamino add residues onto human antibodies, KAS for V_H and PAYfor V_x. Nine humanized B–BlOs with differently graftedframework residues were constructed and assessed for their biologicalactivities. One of these humanized B–B10, M5, showed nearlythe same activity as the mouse B–B10. The 49th residueof V_x was demonstrated to play a crucial role in the antigen–antibodyinteraction by 3–D structure analysis with a computermodeling system.     

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.     

Computational modeling of type I collagen fibers to determine the extracellular matrix structure of connective tissues

A method is presented for generating computer models of biological tissues. The method uses properties of extracellular matrix proteins to predict the structure and physical chemistry of the elements that make up the tissue. The method begins with Protein Data Bank coordinate positions of amino acids as input into TissueLab software. From the amino acid sequence, a type I collagen-like triple helix backbone was computationally constructed and boundary spheres were added based on known chemical and physical properties of the amino acids. Boundary spheres determined the contact surface characteristics of the collagen molecules and intermolecular interactions were then determined by considering the relationships of the contact surfaces and by resolving the energy-minimum state using feasible sequential quadratic programming. From this, the software created fibrils that corresponded exactly to known collagen parameters and were further confirmed by finite element modeling. Computationally derived fibrils were then used to create collagen fibers and three-dimensional collagen matrices. By resolving the energy-minimum state, large complex components of the extracellular space as well as other structures can be determined to provide three-dimensional structure of molecules, molecular interactions and the tissues that they form.     

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceousprotease inhibitor which inhibits serine proteases by forminga stable Michaelis complex. The flexible loop region (Thr64–Val69)is a very flexible region in an SSI molecule and its importancein interactions with proteases has been suggested, since conformationalchange of this loop was found to occur for the smooth bindingof SSI with various proteases. In this study, mutated SSIs lackingone or two residues in this region were generated and the effectsof deletions on the interaction with proteases were investigated.Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73)both known to be trypsin inhibitors, to examine the effectsof deletion on interactions with subtilisin BPN' or trypsin.The deletion of one residue (Gly66) caused increased inhibitoryactivity toward trypsin, indicating the protruding flexibleloop hinders binding with trypsin. Reduction of such hindranceby one-residue shortening in this loop is shown to be effectivefor the interaction of SSI(Lys73) with trypsin. In contrast,one-residue shortening had virtually no effect on inhibitiontoward subtilisin BPN'. Differences in the subsite structuresof these proteases may have been the reason for this contrast.The deletion of two residues (Thr64 and Gly66) in this regionconverted SSI into a temporary inhibitor. Structural analysisof the degradation intermediate showed that the peptide bondat the reactive site of doubly deleted SSI was cleaved by subtilisinBPN' after its binding with protease. Thus, the irreversibilityof the cleaved peptide bond at the reactive site of mutatedSSI in the complex with protease may possibly be the cause forits temporary inhibition. Irregular conformation around thereactive site caused by the deletion of two residues in theflexible loop would convert SSI into a temporary inhibitor.Thus, moderate flexibility in the flexible loop region may possiblybe a structural requirement for SSI to function.     

A structure-based model for the halphilic adaptiation of dihydrofolate reductase from Halobacterium volcanii

‘Halophilic adaptation’ of proteins, i.e. the requirementfor high concentrations of monovalent ions for thermodynamicstability of proteins from halophilic organisms, is not fullyunderstood. In this work, an explanation for the halophilicbehavior of dihydrofolate reductase (h-DHFR) from Halobacteriumvolcanii is attempted, based on a model structure derived fromcomparative modeling to dihydrofolate reductase from Escherichiacoli. The model structure of h-DHFR shows an unique asymmetricalcharge distribution over the protein surface, with positivelycharged amino acids centered around the active site and negativecharges on the opposite side of the enzyme. This particularcharge distribution and the correlated molecular dipole arefunctionally relevant. The negative charges on the surface formclusters which are shielded at high salt concentrations; atlow salt, they repulse each other, thus destabilizing the protein.Results are in accordance with denaturation data and, thus,provide an explanation for the exceptional stability propertiesof h-DHFR.     

The refined crystal structure of subtilisin Carlsberg at 2.5 ? resolution

We report here the X-ray crystal structure of native subtilisinCarlsberg, solved at 2.5 ? resolution by molecular replacementand refined by restrained least squares to a crystal-lographicresidual of 0.206. we compare this structure to the crystal structure of subtilisin BPN'. We find that, despite82 amino acid substitutions and one deletion in subtilisin Carlsbergrelative to subtilisin BPN', the structures of these enzymesare remarkably similar. We calculate an r.m.s. difference betweenequivalent a-carbon positions in subtilisin Carlsberg and subtilisinBPN' of only 0.55 ?. This confirms previous reports of extensivestructural bomology between these two subtilisins based on X-raycrystal structures of the complex of eglin-c with subtilisinCarlsberg [McPhalen,C.A., Schnebli.H.P. and James,M.N.G. (1985)FEBS Lett., 188, 55; Bode,W., Papamokos,E. and Musil,D. (1987)Eur. J. Biochem., 166, 673-692]. In addition, we find that thenative active sites of subtilisins Carlsberg and BPN' are virtuallyidentical. While conservative substitutions at residues 217and 156 may have subtle effects on the environments of substrate-bindingsites SI' and SI respectively, we find no obvious structuralcorrelate for reports that subtilisins Carlsberg and BPN' differin their recognition of model substrates. In particular, wefind no evidence that the hydrophobic binding pocket SI in subtilisinCarlsberg is ‘deeper’, ‘narrower’ or'less polar' than the corresponding binding site hi subtilisinBPN' [Karasaki and Ohno (1978) J. Biochem., Tokyo, 84, 531–538].     

Homology modeling study of the human interleukin-7 receptor complex

Following a recent model of human interleukin-7 (IL-7), we presenthere a modeling study of the extracellular part of the humanIL-7 receptor complex, including the IL-7 specific (IL-7R) andthe common gamma (_c) chains. The investigation is based on structuralhomology to the complex of human growth hormone (hGH) boundto its receptor (hGHR). For domain 1 of IL-7R two differentmodels are presented which differ in the alignment to hGHR inthree regions. However, these differences affect binding toIL-7 in only one region, at the interface between loop EF ofdomain 1 of IL-7R and helix C of IL-7. The disulfide patternin domain 1 of IL-7R is predicted to deviate from that observedin hGHR in that the C'–E disulfide (hGHR) is replacedby a C-C' cross-link. The prediction for the _c chain is comparedwith two previous studies. The models of the complex provideinsight into the binding of IL-7 to its receptor and have implicationsfor the suggestion of mutagenesis experiments and the designof (ant)agonists.     

Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme

The titration behavior of the ionizable residues of the HyHEL-5–henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pK_a values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pK_a values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 1–2 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the Arg–Glu salt bridges that have beenproposed to be important in antibody-antigen binding, had pK_avalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed