Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA

Cellobiohydrolase CbhA from Clostridium thermocellum cellulosome is a multi-modular protein composed starting from the N-terminus of a carbohydrate-binding module (CBM) of family 4, an immunoglobulin(Ig)-like module, a catalytic module of family 9 glycoside hydrolases (GH9), X1(1) and X1(2) modules, a CBM of family 3 and a dockerin module. Deletion of the Ig-like module from the Ig-GH9 construct results in complete inactivation of the GH9 module. The crystal structure of the Ig-GH9 module pair reveals the existence of an extensive module interface composed of over 40 amino acid residues of both modules and maintained through a large number of hydrophilic and hydrophobic interactions. To investigate the importance of these interactions between the two modules, we compared the secondary and tertiary structures and thermostabilities of the individual Ig-like and GH9 modules and the Ig-GH9 module pair using both circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). Thr230, Asp262 and Asp264 of the Ig-like module are located in the module interface of the Ig-GH9 module pair and are suggested to be important in 'communication' between the modules. These residues were mutated to alanyl residues. The structure, stability and catalytic properties of the native Ig-GH9 and its D264A and T230A/D262A mutants were compared. The results indicate that despite being able to fold relatively independently, the Ig-like and GH9 modules interact and these interactions affect the final fold and stability of each module. Mutations of one or two amino acid residues lead to destabilization and change of the mechanism of thermal unfolding of the polypeptides. The enzymatic properties of native Ig-GH9, D264A and T230A/D262A mutants are similar. The results indicate that inactivation of the GH9 module occurs as a result of multiple structural disturbances finally affecting the topology of the catalytic center.     

The role of extra-membranous inter-helical loops in helix-helix interactions

The effect of a short loop connecting two transmembrane alpha-helices was studied using molecular dynamics simulations. Helices F and G from bacteriorhodopsin and two corresponding polyalanine helices were embedded in octane and POPC membranes in a transmembrane configuration both with and without the inter-helical loop. The results indicate that the membrane environment and the sequence of the loop are more influential on the dynamics and structure of the motif than the presence of a loop as such, at least for the time-scales investigated. The four residues in the FG loop are stabilized by four hydrogen bonds. These hydrogen bonds are not present in the polyalanine loop, causing it to be more flexible than the FG loop. This effect was observed independently of the protein environment, stressing the importance of the sequence. The structural analysis indicates that the loop has weak stabilizing properties in all environments. The stabilization due to the presence of the loop was strongest in a simulation of the FG fragment in a membrane-mimetic octane slab. In the simulations of the helix-loop-helix motif embedded in an explicit lipid bilayer model, the lipid bilayer interface compensates to a large extent for the absence of the loop.     

A simple algorithm for the calculation of multiple site titration curves

A simple algorithm for the calculation of multiple site titrationcurves is proposed. It is based on a hybridization of two computationaltechniques: (i) a modified Tanford-Roxby iterative procedure[Tanford and Roxby (1972) Biochemistry, 11, 2193–2198]and (ii) the Boltzmann statistics. The sites characterized bystrong electrostatic coupling were selected for statisticalmechanical treatment, whereas all other sites were treated bymeans of the modified Tanford-Roxby procedure. The selectionof the two sets was made on the basis of a criterion relatedto the interaction energy between the titratable sites in theprotein molecule. The algorithm was tested for bovine pancreatictrypsin inhibitor and the pK values calculated were discussedin the light of experimental data and theoretical results obtainedby other authors. The algorithm can easily be coded and incorporatedinto any program package for the calculation of electrostaticinteractions in proteins.     

Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity

A mutant of bovine pancreatic DNase I containing two additionalresidues in a loop next to C173 has been expressed in Escherichiacoli, purified and characterized biochemically. Modelling studiessuggest that the inserted arginine and glutamate side chainsof the modified loop sequence C173-R-E-G-T-V176 could contactthe bases 3' to the cleaved bond in the major groove of a boundDNA, and that up to 10 bp could interact with the enzyme andpotentially influence its cutting rate. The loop insertion mutanthas an 800-fold lower specific activity than wild-type and showsoverall cleavage characteristics similar to bovine pancreaticDNase I. Compared with the wild-type enzyme, the mutant showsa strongly enhanced preference for cutting the inverted repeat:5'-GACTT A AAGTC-3' CTGAA T TTCAG or close variants thereof.Unexpectedly for a minor groove binding protein, the preferredcutting sites in opposite strands are staggered by 1 bp in the5' direction, causing the cleavage of a TA and a TT step, respectively.This finding demonstrates that the sequence context is relativelymore important for the cutting frequency than the nature ofthe dinucleotide step of the cleaved bond, and clearly showsthat base recognition is involved in determining the sequenceselectivity of the mutant. The importance of the sequence 5'to the cleaved bond for the cutting rate suggests that the additionalmajor groove contacts may require a distortion of the DNA associatedwith a higher energy barrier, resulting in an increased selectivityfor flexible DNA sequences and a lower overall activity of themutant enzyme.     

Application of scaled particle theory to model the hydrophobic effect: implications for molecular association and protein stability

The energetics of alkane dissolution and partition between waterand organic solvent are described in terms of the energy ofcavity formation and solute-solvent interaction using scaledparticle theory. Thermodynamic arguments are proposed that allowcomparison of experimental measurements of the surface areawith values calculated from an all-atom representation of thesolute. While the surface tension relating to the accessiblesurface is shape dependent, it is found that for the molecularsurface it is not. This model rationalizes the change in surfacetension between the microscopic (20–30 cal/mol/A2) andmacroscopic (70–75 cal/mol/A2) regimes without the needto invoke Flory-Huggins theory or to apply other corrections.The difference in the values arises (i) to a small extent asa result of the curvature dependence of surface tension and(ii) to a large extent due to the difference in the molecularsurface derived from the experiment and that calculated froman extended all-atom model. The model suggests that the primarydriving force for alkane association in water is due to thetendency of water to reduce the solute cavity surface. It isargued that to model the energetics of alkane association, thesurface tension should be related to the molecular surface (ratherthan the accessible surface) with a surface tension near themacroscopic limit for water. This model is compared with resultsfrom theoretical simulations of the hydrophobic effect for twowell-studied systems. The implications for antibody– antigeninteractions and the effect of hydrophobic amino acid deletionon protein stability are discussed. The approach can be usedto model the solute cavity formation energy in solution as afirst step in the continuum modelling of biomolecular interactions     

Characterization of His-X3-His sites in {alpha}-helices of synthetic metal-binding bovine somatotropin

Variants of bovine somatotropin have been engineered to containsynthetic metal-binding sites consisting of two solvent-exposedhistidines separated by a single turn of an -helix (His-X₃-Hisvariants). The affinities of these proteins for Cu(II) werecharacterized by measuring their partitioning coefficients inan aqueous two-phase polymer system. The partition coefficientswere used to generate binding constants for formation of a complexbetween the engineered metal-binding site and Cu(II) chelatedto an iminodiacetic acid derivative of polyethylene glycol.For three His-X₃-His variants described here, these constantsrange from 2x104 to 1.6x106 M-1. The metal affinity of a His-X₃-Hissite depends on the rigidity of the helix into which the siteis engineered. The affinities of the His-X₃-His sites for Cu(II)are large enough to dramatically increase not only the partitioningof these proteins in aqueous two-phase systems, but also theirretention times on a metal-affinity chromatography column. Boththese features can greatly facilitate the purification of engineeredproteins. Criteria for choosing positions for incorporatingmetal-binding sites are discussed.     

Analysis of RNA phage fr coat protein assembly by insertion, deletion and substitution mutagenesis

A structure-function analysis of the icosahedral RNA bacteriophagefr coat protein (CP) assembly was undertaken using linker-insertion,deletion and substitution mutagenesis. Mutations were specificallyintroduced into either pre-existing or artificially createdrestriction enzyme sites within fr CP gene expressed in Escherichiacoli from a recombinant plasmid. This directs synthesis of wildtype protein that undergoes self-assembly and forms capsid-likeparticles indistinguishable morphologically and immunologicallyfrom native phage particles. A series of fr CP variants containingsequence alterations in the regions which are (i) exposed onthe external surface of capsid or (ii) located on the contactingareas between CP subunits were obtained and their assembly propertiesinvestigated. The majority of mutants demonstrated reductionof assembly ability and formed either CP dimers (mutations atresidues 2, 10, 63 or 129) or both dimer and capsid structures(residue 2 or 69). The exceptions were variants demonstratingnormal assembly and containing insertions at residues 2, 50or 129 of thefr CP. A third type of assembled structure wasformed by a variant with a single amino acid substitution I104T.The aA-helix region (residues 97-111) is particularly sensitiveto mutation and any alteration in this region decreases accumulationof mutant protein in E.coli. The relative contributions of particularfr CP domains in maintenance of capsid structural integrityas well as the possible capsid assembly mechanism are discussed.     

Electrostatic screening in molecular dynamics simulations

The screened Coulombic potential has been shown to describesatisfactorily equilibrium properties like pK shifts, the effectsof charged groups on redox potentials and binding constantsof metal ions. To test how well the screening of the electrostaticpotential describes the dynamical trajectory of a macromolecularsystem, a series of comparative simulations have been carriedout on a protein system which explicitly included water moleculesand a system in vacuo. For the system without solvent the resultsof using (i) the standard potential form were compared withresults of (ii) the potential where the Coulomb term was modifiedby the inclusion of a distance dependent dielectric, (r), tomodel the screening effect of bulk water, and (iii) standardpotential modified by reducing the charge on ionized residueside chains. All molecular dynamics simulations have been carriedout on bovine pancreatic trypsin inhibitor. Comparisons betweenthe resulting trajectories, averaged structures, hydrogen bondingpatterns and properties such as solvent accessible surface areaand radius of gyration are described. The results show thatthe dynamical behaviour of the protein calculated with a screenedelectrostatic term compares more favourably with the time-dependentstructural changes of the full system with explicitly includedwater than the standard vacuum simulation.     

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis

The Escherichia coli aspartate receptor is a dimer with twotransmembrane sequences per monomer that connect a periplasmicligand binding domain to a cytoplasmic signaling domain. Themethod of 'hydrophobic-biased' random mutagenesis, that we describehere, was used to construct mutant aspartate receptors in whicheither the entire transmembrane sequence or seven residues nearthe center of the transmembrane sequence were replaced withhydrophobic and polar random residues. Some of these receptorsresponded to aspartate in an in vivo chemotaxis assay, whileothers did not. The acceptable substitutions included hydrophobicto polar residues, small to larger residues, and large to smallerresidues. However, one mutant receptor that had only a few hydrophobicsubstitutions did not respond to aspartate. These results addto our understanding of sequence specificity in the transmembraneregions of proteins with more than one transmembrane sequence.This work also demonstrates a method of constructing familiesof mutant proteins containing random residues with chosen characteristics.     

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.     

Native-like {beta}-hairpin structure in an isolated fragment from ferredoxin: NMR and CD studies of solvent effects on the N-terminal 20 residues

The conformational properties of protein fragments have beenwidely studied as models of the earliest initiation events inprotein folding. While native-like -helices and ß-turnshave been identified, less is known about the factors that underlyß-sheet formation, in particular ß-hairpins,where considerably greater long-range order is required. TheN-terminal 20 residue sequence of native ferredoxin I (fromthe blue-green alga Aphanothece sacrum ) forms a ß-hairpinin the native structure and has been studied in isolation byNMR and CD spectroscopy. Local native-like interactions aloneare unable to stabilize significantly a folded conformationof the 20-residue fragment in purely aqueous solution. However,we show that the addition of low levels of organic co-solventspromotes formation of native-like ß-hairpin structure.The results suggest an intrinsic propensity of the peptide toform a native-like ß-hairpin structure, and that theorganic co-solvent acts in lieu of the stabilizing influenceof tertiary interactions (probably hydrophobic contacts) whichoccur in the folding of the complete ferredoxin sequence. Thestructure of the isolated hairpin, including the native-likeregister of interstrand hydrogen bonding interactions, appearsto be determined entirely by the amino acid sequence. The solventconditions employed have enabled this intrinsic property tobe established.     

Effect of Lys->Arg mutation on the thermal stability of Cu,Zn superoxide dismutase: influence on the monomer-dimer equilibrium

The thermal stability of two single (K3R, K67R) and one double(K3R-K67R) mutants of Xenopus laevis B Cu,Zn superoxide dismutasehas been studied to test LysArg substitution as an ‘electrostaticallyconservative’ strategy to increase protein stability.The K3R mutant displays an increased thermostability with respectto the wild-type enzyme, whilst a decreased stability was observedin the case of the K67R and K3R-K67R mutants. Concentrationdependence of the apparent inactivation constant (k_app) of thelatter mutants, as compared to that of the wild type enzymeand K3R mutant, indicates that their higher sensitivity to heatinactivation is due to a perturbation of the dimer association.These results are confirmed also by fluorescence anisotropymeasurements of the internal probe Tyr149. The possible roleof Arg67 in perturbing the dimer dissociation equilibrium towardthe monomeric form is discussed.     

The structure of interfaces between subunits of dimeric and tetrameric proteins

The structures of the interfaces of nine dimeric and nine tetramericproteins have been analyzed and have been seen to follow generalprinciples. These interfaces are combinations of four structuralmotifs, which resemble features of monomeric proteins. Theseare: (i) extended beta sheet; (ii) helix–helix packing;(iii) sheet–sheet packing; and (iv) loop interactions.Other common structural features in the interfaces studied aretwo-fold symmetry, charged hydrogen bonds and channel formation(found only in tetramers). Monomer–monomer interfacesare intermediate in hydrophobicity and charge between the interfacesbetween secondary structures of monomeric proteins and the exteriorsof monomeric proteins. A typical interface has one of the firstthree of the structural motifs at its centre and loop interactionsaround the outside, where most of the charge resides.     

EcoRV-T94V: a mutant restriction endonuclease with an altered substrate specificity towards modified oligodexynucleotides

Synthetic oligodeoxynucleotides with single methyl phosphonate(mp) substitutions were used for an analysis of the contributionof phosphate contacts to the recognition of the cleavage siteby the restriction endonuclease EcoRV. Only in the last positionwithin the recognition sequence, is the methyl phosphonate substitutiontolerated by the enzyme. The wild-type enzyme cleaves the S_Pdiastereomer of the oligodeoxynucleotide GACGATAT_mpCGTC andthe unmodified sequence with equal rates, whereas the R_P diastereomeris cleaved much more slowly. Inspection of the crystal structureof an EcoRV–DNA complex revealed that the non-bridgingoxygen atoms of the phosphodiester bond between the T and Cbases are in hydrogen bonding distance of the hydroxyl groupof the amino acid Thr94. We therefore tried to engineer a variantof EcoRV that would prefer a methyl phosphonate linkage overa normal phosphodiester bond and produced mutants with aminoacid exchanges at position 94. One of them, Thr94Val, showsa dramatically reduced activity towards the unmodified DNA anddoes not accept the R_p diastereomer, but cleaves the S_P diastereomerwith the same rate as wild-type EcoRV. Its selectivity, i.e.the ratio of cleavage rates determined for the unmodified andmodified substrates, differs by three orders of magnitude fromthat of the wild-type enzyme.     

The net energetic contribution of interhelical electrostatic attractions to coiled-coil stability

The net energetic contribution of interhelical electrostaticattractions to coiled-coil stability has been quantitated usingde novo designed synthetic coiled-coils. The synthesized modelcoiled-coil (EK), denoted by amino acid res-idues in positionse and g, which contains only interhelical ionic interactionswithout any possible (i, i + 3) and (i, i + 4) intrahelicalionic interaction, consists of two identical 35 residue polypeptidechains with a heptad repeat KgLaG-bAcLdEeKf. Three mutant coiled-coilswere prepared where five Glu residues at e positions in EK weremutated to Gin residues (QK); five Lys residues at g positionswere altered to Gin residues (EQ) or these mutations were effectedat both positions e and g (QQ). The stabilities of the fourcoiled-coils were determined by measuring the ellipticitiesat 220 nm as a function of urea concentration at 20C. By usinga double-mutant cycle analysis it was possible to isolate theenergetic contribution of interhelical ionic attractions tocoiled-coil stability from the other contributions such as helicalpreference and hydro-phobicity. The 0.37 0.01 kcal/mol ofenergetic contribution of one interhelical ion pair to the coiled-coilstability was obtained from three independent comparisons. Thisfinding suggests that a large number of weak interhelical electrostaticinteractions on the surface of a protein can make a substantialcontribution to protein stability. In addition, the energeticcontributions of a single mutation E^– Q, K⁺Q, Q E andE^– Ewere also determined (G = 0.22, 0.26, 0.46 and 0.65kcal/mol for the single mutations, respectively). The greatercontribution of a protonated Glu residue to coiled-coil stabilitycompared with an ionized Glu residue (0.65 kcal/mol) can outweighthe relatively smaller contribution of an interhelical ion pair(0.37 kcal/mol), which clearly explains why most coiled-coilsare more stable at acidic pH compared with neutral pH even wheninterhelical salt bridges contribute to the coiled-coil stabilityat neutral pH.     

Empirical free energy calculations of ligand-protein crystallographic complexes. I. Knowledge-based ligand-protein interaction potentials applied to the prediction of human immunodeficiency virus 1 protease binding affinity

The steadily increasing number of high-resolution human immunodeficiencyvirus (HIV) 1 protease complexes has been the impetus for theelaboration of knowledge-based mean field ligand-protein interactionpotentials. These potentials have been linked with the hydrophobicityand conformational entropy scales developed originally to explainprotein folding and stability. Empirical free energy calculationsof a diverse set of HIV-1 protease crystallographic complexeshave enabled a detailed analysis of binding thermodynamics.The thermodynamic consequences of conformational changes thatHIV-1 protease undergoes upon binding to all inhibitors, anda substantial concomitant loss of conformational entropy bythe part of HIV-1 protease that forms the ligand-protein interface,have been examined. The quantitative breakdown of the entropy-drivenchanges occurring during ligand-protein association, such asthe hydrophobic contribution, the conformational entropy termand the entropy loss due to a reduction of rotational and translationsaldegrees of freedom, of a system composed of ligand, proteinand crystallographic water molecules at the ligand-protein interfacehas been carried out The proposed approach provides reasonableestimates of distinctions in binding affinity and gives an insightinto the nature of enthalpy-entropy compensation factors detectedin the binding process.     

Light-induced denaturation of bacteriorhodopsin solubilized by octyl-{beta}-glucoside

The structural stability of bacteriorhodopsin (bR) solubilizedby octyl-ß-glucoside was studied by measuring the denaturationkinetics under visible light irradiation and in the dark. Thedenaturation of bR solubilized by 50 mM octyl-ß-glucosidewas very slow at room temperature when it was left in the dark.However, its spontaneous denaturation was accelerated when thesolubilized bR was irradiated by visible light. The denaturationkinetics under visible light irradiation and in the dark couldbe well described by a single decay constant. The activationenergy for the denaturation of bR was estimated from the temperaturedependence of decay time constants. The activation energy undervisible light irradiation was 12.5 kcal/mol, which was muchsmaller than the corresponding value in the dark, 26.2 kcal/mol.These results strongly suggest that some of the photointermediatestates are less stable than the ground state of bR. The criticaltemperature and the activation energy for denaturation of bRin the solubilized state were much lower than those in the 2Dcrystalline state. Comparing the denaturation behavior in the2D crystalline state and that in the octyl-ß-glucoside-solubilizedstate, our findings suggest that protein–protein interactioncontributes to the stability of this protein.     

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.     

A proposal for the catalytic mechanism in phospholipase C based on interaction energy and distance geometry calculations

The non-specific phospholipase C from Bacillus cereus preferentiallyhydrolyses phosphatidylcholine but is also active against phosphatidylserine,phosphatidylethanolaniine and at a much lower level, sphingomyelin.A minimal substrate model containing all required structuraland configurational elements of a high affinity substrate wasdocked into the active site. The enzyme–substrate attachmentpoints were from molecular interaction energy calculations usingthe program GRID and from a previous phosphate inhibitor complexstructure. Available conformational space for the substratewas sampled by distance geometry calculations using the programDGEOM. This investigation clearly identifies the attacking nucleophile,a catalytically favourable orientation of the phosphate groupin its tetra-, as well as its penta-, coordinated state anda crucial stabilizing environment for the alkoxide intermediate.Based on this information a complete catalytic cycle is proposed.