Effect on inhibitory activity of mutation at reaction site P4 of the Streptomyces subtilisin inhibitor, SSI

The protein Streptomyces subtilisin inhibitor, SSI, efficientlyinhibits a bacterial serine protease, subtilisin BPN'. We recentlydemonstrated that functional change in SSI was possible simplyby replacing the amino acid residue at the reactive P1 site(methionine 73) of SSI. The present paper reports the additionaleffect of replacing methionine 70 at the P4 site of SSI(Lys73)on inhibitory activity toward two types of serine proteases,trypsin (or lysyl endopeptidase) and subtilisin BPN'. Conversionof methionine 70 at the P4 site of SSI(Lys73) to glycine oralanine resulted in increased inhibitory activity toward trypsinand lysyl endopeptidase, while replacement with phenylalanineweakened the inhibitory activity toward trypsin. This suggeststhat steric hindrance at the P4 site of SSI(Lys73) is an obstaclefor its binding with trypsin. In contrast, the same P4 replacementshad hardly any effect on inhibitory activity toward subtilisinBPN'. Thus the subsite structure of subtilisin BPN' is tolerantto these replacements. This contrast in the effect of P4 substitutionmight be due to the differences in the S4 subsite structuresbetween the trypsin-like and the subtilisin-like proteases.These findings demonstrate the importance of considering structuralcomplementarity, not only at the main reactive site but alsoat subsites of a protease, when designing stronger inhibitors.     

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].     

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.     

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Sequential rounds of error-prone PCR to introduce random mutationsand screenrng of the resultant mutant libraries have been usedto enhance the total catalytic activity of subtilisin E significantlyin a non-natural environment, aqueous dimethylformamide (DMF).Seven DNA substitutions coding for three new amino acid substitutionswere identified in a mutant isolated after two additional generationsof directed evolution carried out on 10M subtilisin E, previously‘evolved’ to increase its specific activity in DMF.A Bacillus subtilis-Escherichia coli shuttle vector was developedin order to increase the size of the mutant library that couldbe established in B.subtilis and the stringency of the screeningprocess was increased to reflect total as well as specific activity.This directed evolution approach has been extremely effectivefor improving enzyme activity in a non-natural environment:the resulting-evolved 13M subtilisin exhibits specific catalyticefficiency towards the hydrolysis of a peptide substrate succlnyl-Ala-Ala-Pro-Phe-p-nitroanilidein 60% DMF solution that is three times that of the parent 10Mand 471 times that of wild type subtilisin E. The total activityof the 13M culture supernatant is enhanced 16-fold over thatof the parent 10M.     

Geometric and energy parameters in lysine-retinal chromophores

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

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126–128.Substitution of Val102 by Trp results in a 12–fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(‘MAG’ mutant), Phe-Gln-Ser(‘FQS’ mutant) and Asn-Ser-Ala (‘NSA’mutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the ‘NSA’mutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein.     

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.     

Effects of engineered salt bridges on the stability of subtilisin BPN'

Variants designed using PROTEUS have been produced in an attemptto engineer stabilizing salt bridges into subtilisin BPN'. Allthe mutants constructed by site-directed mutagenesis were secretedby Bacillus subtillus, except L75K. Q19E, expressed as a singlevariant and also in a double variant, Q19E/Q271E, appears toform a stabilizing salt bridge based on X-ray crystal structuredetermination and differential scanning calorimeter measurements.Although the double mutant was found to be less thermodynamicallystable than the wild-type, it did exhibit an autolytic stabilityabout two fold greater under hydrophobic conditions. Four variants,A98K, S89E, V26R and L235R, were found to be nearly identicalto wild-type in thermal stability, indicative of stable structureswithout evidence of salt bridge formation. Variants Q271E, V51Kand T164R led to structures that resulted in varying degreesof thermodynamic and autolytic instability. A computer-modelinganalysis of the PROTEUS predictions reveals that the low percentageof salt bridge formation is probably due to an overly simplisticelectrostatic model, which does not account for the geometryof the pairwise interactions.     

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.     

Analysis of proteinprotein interactions by mutagenesis: direct versus indirect effects

Site-directed mutagenesis, including double-mutant cycles, isused routinely for studying protein–protein interactions.We now present a case analysis of chymotrypsin inhibitor 2 (CI2)and subtilisin BPN' using (i) a residue in CI2 that is knownto interact directly with subtilisin (Tyr42) and (ii) two CI2residues that do not have direct contacts with subtilisin (Arg46and Arg48). We find that there are similar changes in bindingenergy on mutation of these two sets of residues. It can thusbe difficult to interpret mutagenesis data in the absence ofstructural information.     

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

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

Proline versus charge concept for protein stabilization against proteolytic attack

The virtue of the so-called ‘proline concept’ andthe ‘charge concept’ for stabilizing protease-susceptibleregions of a protein structure was compared on bovine pancreaticribonuclease A. Alanine 20 and serine 21, both of whichare located in a loop that is susceptible to the unspecificproteases subtilisin Carlsberg, subtilisin BPN', proteinaseK and elastase, were replaced with proline or lysine by site-directedmutagenesis. The rate constant of proteolysis was decreasedby up to three orders of magnitude for the proline mutants dependingon the site of the mutation and the protease used. In contrast,substitution by lysine increased the proteolytic resistanceby only one order of magnitude characterizing the ‘prolineconcept’ as superior to the ‘charge concept’.Although the four applied proteases are considered to be unspecific,the degree of stabilization of the ribonuclease molecule variedconsiderably, indicating the impact of individual differencesin their substrate specificity on the proteolytic resistanceand degradation pathway of the target protein. Received May 12, 2003; revised October 23, 2003; accepted October 30, 2003     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisinE into an enzyme functionally equivalent to its thermophilichomolog thermitase from Thermoactinomyces vulgaris. Five generationsof random mutagenesis, recombination and screening created subtilisinE 5-3H5, whose half-life at 83°C (3.5 min) and temperatureoptimum for activity (T_opt, 76°C) are identical with thoseof thermitase. The T_opt of the evolved enzyme is 17°C higherand its half-life at 65°C is >200 times that of wild-typesubtilisin E. In addition, 5-3H5 is more active towards thehydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-typeat all temperatures from 10 to 90°C. Thermitase differsfrom subtilisin E at 157 amino acid positions. However, onlyeight amino acid substitutions were sufficient to convert subtilisinE into an enzyme equally thermostable. The eight substitutions,which include known stabilizing mutations (N218S, N76D) andalso several not previously reported, are distributed over thesurface of the enzyme. Only two (N218S, N181D) are found inthermitase. Directed evolution provides a powerful tool to unveilmechanisms of thermal adaptation and is an effective and efficientapproach to increasing thermostability without compromisingenzyme activity.     

An NMR study on the DNA-binding SPKK motif and a model for its interaction with DNA

The solution structure of one and two repeats of the ‘SPKK’DNA-binding motif is reported on the basis of NMR measurements.In dimethylsulphoxide (DMSO) the major population (approximately90%) of peptides, SPRKSPRK(S2) and GSPKKSPRK(S2b), adopts aconformation, which has two trans pralines. The two ‘SP(R/K)K’units hi these peptides are equivalent and each adopts a turnstructure exchanging with an extended structure. This is suggestedby an NOE connectivity of the /3-turn type, between the backboneamide protons of residues (j+2) and (j+3) and NOE connectivitiesof the Asx(a)-turn type, between protons of the tth Ser andthe backbone amide proton on residue (i+2). This suggests thateach SP(R/K)K unit has a structural intermediate between (ora combination of) a /3-turn and an Asx(a)-turn. In 90-10% DMSO/H2Oat 4C the two units of S2 are connected more tightly by foldinginto a short 3t0 helix, broken at the second proline. For anotherpeptide, Thr-Pro-Arg-Lys(Tl), the major population (75%) in100% DMSO comprises a /3-turn in rapid exchange with an extendedstructure. We did not observe an NOE connectivity of the Asx()type with the Tl peptide. A possible structure of the SPKK motifhi the complex with DNA is discussed.     

A structure-function relationship for the calcium affinities of regulatory proteins containing 'EF-hand' pairs

Using a series of homologous calcium-binding proteins, a quantitativestructure–activity relationship (QSAR), log(1/K_d) = –18.986– 1.6278(X₁) + 0.7981(X₂) + 0.2312(X₃), has been established,which relates the calcium-binding affinities (1/K_d) of the regulatoryproteins with (i) the net ligand charge (X₁) of the two calciumbinding loops, (ii) the hydrophobicity (X₂) of the ß-sheetsegment of the loops and (iii) the hydrophobicity (X₃) of thefour ‘EF-hand’ helices. It is found that the bindingaffinities are influenced by the ‘EF-hand’ pairrather than the individual ‘EF-hands’. The QSAR,in addition to explaining satisfactorily the large variationin the observed calcium affinities, can predict the affinitiesof the ‘EF-hand’ pairs in other proteins from theamino acid sequence and can also account for the changes inthe affinities caused by substitution in the hydrophobic and/ormetal-coordinating residues. Thus, this relationship can beemployed in protein design and engineering. The method is potentiallyuseful in the development of similar relationships for the bindingof other proteins to substrates, inhibitors, drugs and co-factors.     

Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone

We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.     

The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) is composed of structurallyautonomous domains. From the N-terminus of t-PA, a finger-likedomain (F), an epidermal growth factor-like domain (G), twokringle domains (Kl and K2) and a serine protease domain (P)can be discerned. The K2 domain of t-PA is known to be involvedin lysine binding, fibrin binding and fibrin-dependent plasminogenactivation. To study the functional autonomy of the K2 domainin t-PA we constructed, with the aid of a cassette t-PA gene[Rehberg et al. (1989) Protein Engng, 2,371–377], mutantt-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2Pand GK2K1P) in which the K2 domain was placed in two differentpositions in t-PA. The DNAs of wild-type t-PA and the t-PA variantswere expressed in Chinese hamster ovary cells and the recombinantproteins were purified by affinity chromatography.All moleculeswere expressed in their single-chain form and could be convertedto their two-chain form. With these molecules, lysine binding,fibrin binding and fibrin-dependent plasminogen activation werestudied. All variants showed affinity for lysyl-Sepharose andaminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versusFGK2K1P and GK1K2P versus GK2K1P) resulted in a 23–47%weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose.Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versusGK2K1P) resulted in a 20–70% improvement of the interactionslysyl-Sepharose and aminohexyl-Sepharose. All variants boundto a forming fibrin clot. Reversal of the K domains (FGK1K2Pversus FGK2K1P) reduced fibrin binding. In the presence of thelysine analogue -amino caproic acid, only FGK1K2P bound to fibrin.All variants activated plasminogen. In the absence of fibrinogenCNBr fragments (mimic of fibrin), the reversal of the K domain(FGK2K1P) resulted in a 2-fold improved plasminogen activation.In the presence of a fibrin mimic, the plasminogen activationsof the F domain deletion analogues GK1K2P and GK2K1P were foundto be decreased 2- to 4-fold. From these results we concludedthat the function of t-PA in lysine binding, fibrin bindingand fibrin-dependent plasminogen activation is dependent onthe correct spatial orientation of the K2 domain within thet-PA molecule     

Molecular mechanics analysis of inhibitor binding to HIV-1 protease

Crystallographic structures of HIV protease with three differentpeptide-mimetic inhibitors were subjected to energy minimizationusing molecular mechanics, the minimized structures analyzedand the inhibitor binding energies calculated. Partial chargeassignment for the hydrogen bonded catalytic aspartk acids,Asp25 and -25', was in good agreement with charge calculationsusing semi-empirical molecular orbital methods. Root mean squaredeviations on minimization were small and similar for both subunitsin the protease dimer. The surface loops, which had the largestB factors, changed most on minimization; the hydrophobic coreand the inhibitor binding site showed little change. The distance-dependentdielectric of D(r) = 4r was found to be preferable to D(r) =r. Distance restraints were applied for the intermolecular hydrogenbonds to maintain the conformation of the inhibitor bindingsite. Using the dielectric of D(r) = 4r, the calculated interactionenergy of the three inhibitors with the protease ranged from–53 to –56 kcal/mol. The groups of the inhibitorswere changed to add or remove a ‘transition state analogue’hydroxyl group, and the loss in energy on the removal of thisgroup was calculated to be 0.9–1.7 kcal/mol. This wouldrepresent 19–36% of the total measured difference in bindingenergy between the inhibitors JG365 and MVT-101.     

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

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