Network pattern of residue packing in helical membrane proteins and its application in membrane protein structure prediction

De novo protein structure prediction plays an important role in studies of helical membrane proteins as well as structure-based drug design efforts. Developing an accurate scoring function for protein structure discrimination and validation remains a current challenge. Network approaches based on overall network patterns of residue packing have proven useful in soluble protein structure discrimination. It is thus of interest to apply similar approaches to the studies of residue packing in membrane proteins. In this work, we first carried out such analysis on a set of diverse, non-redundant and high-resolution membrane protein structures. Next, we applied the same approach to three test sets. The first set includes nine structures of membrane proteins with the resolution worse than 2.5 A; the other two sets include a total of 101 G-protein coupled receptor models, constructed using either de novo or homology modeling techniques. Results of analyses indicate the two criteria derived from studying high-resolution membrane protein structures are good indicators of a high-quality native fold and the approach is very effective for discriminating native membrane protein folds from less-native ones. These findings should be of help for the investigation of the fundamental problem of membrane protein structure prediction.     

Use of a quantitative structure-property relationship to design larger model proteins that fold rapidly

A quantitative structure–property relationship (QSPR)was used to design model protein sequences that fold repeatedlyand relatively rapidly to stable target structures. The specificmodel was a 125-residue heteropolymer chain subject to MonteCarlo dynamics on a simple cubic lattice. The QSPR was derivedfrom an analysis of a database of 200 sequences by a statisticalmethod that uses a genetic algorithm to select the sequenceattributes that are most important for folding and a neuralnetwork to determine the corresponding functional dependenceof folding ability on the chosen attributes. The QSPR dependson the number of anti-parallel sheet contacts, the energy gapbetween the native state and quasi-continuous part of the spectrumand the total energy of the contacts between surface residues.Two Monte Carlo procedures were used in series to optimize boththe target structures and the sequences. We generated 20 fullyoptimized sequences and 60 partially optimized control sequencesand tested each for its ability to fold in dynamic MC simulations.Although sequences in which either the number of anti-parallelsheet contacts or the energy of the surface residues is non-optimalare capable of folding almost as well as fully optimized ones,sequences in which only the energy gap is optimized fold markedlymore slowly. Implications of the results for the design of proteinsare discussed.     

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.     

The Trp-cage: optimizing the stability of a globular miniprotein

The Trp-cage, as the smallest miniprotein, remains the subject of numerous computational and experimental studies of protein folding dynamics and pathways. The original Trp-cage (NLYIQWLKDGGPSSGRPPPS, Tm = 42 degrees C) can be significantly stabilized by mutations; melting points as high as 64 degrees C are reported. In helical portions of the structure, each allowed replacement of Leu, Ile, Lys or Ser residues by Ala results in a 1.5 (+/-0.35) kJ/mol fold stabilization. No changes in structure or fluxionality of the core results upon stabilization. Contrary to the initial hypothesis, specific Pro/Trp interactions are not essential for core formation. The entropic advantage of Pro versus Ala (DeltaDeltaS(U) = 11 +/- 2 J/mol K) was measured at the solvent-exposed P17 site. Pro-Ala mutations at two of the three prolines (P12 and P18) that encage the indole ring result in less fold destabilization (2.3-3.4 kJ/mol). However, a P19A mutation reduces fold stability by 16 kJ/mol reflecting a favorable Y3/P19 interaction as well as Trp burial. The Y3/P19 hydrophobic staple interaction defines the folding motif as an 18-residue unit. Other stabilizing features that have been identified include a solvent-exposed Arg/Asp salt bridge (3.4-6 kJ/mol) and a buried H-bonded Ser side chain ( approximately 10 kJ/mol).     

Structural analysis of the CD2 T lymphocyte antigen by site-directed mutagenesis to introduce a disulphide bond into domain 1

Many proteins have been predicted to contain domains with immunoglobulin-Iikefolds and hence to be members of the immunoglobulin superfamily(IgSF). However, several members lack the Cys residues capableof forming the disulphide bond that forms a characteristic bridgebetween the ß sheets in the Ig fold, e.g. domain 1of the lymphocyte antigen CD2. The assignment of ßstrands in CD2 by sequence analysis was tested by attemptingto introduce a disulphide bridge between the ß sheetsby mutating two residues in the relevant positions to Cys. Mutant,soluble forms of CD2 were expressed in Chinese hamster ovarycell lines and amino add sequencing showed that a disulphidebond was formed as predicted, but not in the control where oneCys residue was misplaced by four residues. Evidence that bothmutated molecules folded correctly is given by the indistinguishablebinding of three monoclonal antibodies recognising differentepftopes on CD2. The 3-D structure of rat CD2 domain 1 has beendetermined by NMR spectroscopy and X-ray crystallography, confirmingthe predictions from the sequence. Applications of this methodof insertion of disulphide residues for probing protein structuresare discussed, together with other structures of IgSF domainslacking the typical inter-sheet disulphide bond.     

Design of four-helix bundle protein as a candidate for HIV vaccine

To be efficient, a synthetic vaccine should contain differentT and B cell epitopes of human immunodeficiency virus (HIV)antigens, and the B epitope regions in the vaccine and in theHIV should be conformationally similar. We have suggested previouslythe construction of vaccines in the form of a protein with apredetermined tertiary structure, namely a four--helix bundle.Antigenic determinants of cellular and humoral immunity areblocks for the vaccine design. From experimentally studied HIV-1T and B cell epitopes, we constructed a sequence of a four-helixprotein TBI (T and B cell epitopes containing immunogen). Thegene of the protein was synthesized and the protein was producedin C600 Escherichia coli cells under recA promoter from Proteusmirabelis. CD spectroscopy of the protein demonstrated that30% of amino acid residues adopt an -helical conformation. Miceimmunized with TBI have shown both humoral and cellular immuneresponses to HIV-1. The obtained data show that the design ofTBI was successful. The synthesized gene structure makes possiblefurther reconstruction and improvement of the protein vaccinestructure.     

Normal-mode analysis suggests important flexibility between the two N-terminal domains of CD4 and supports the hypothesis of a conformational change in CD4 upon HIV binding

Human CD4 is the receptor for human immunodeficiency virus (HTV).It is well established that the first domain of CD4 binds withhigh affinity to gp120, an envelope protein of HIV, but it hasalso been demonstrated that amino acids located in its seconddomain, within or close to residues 120–127 or 163–166(lying 15 Å away from the binding site), play a role invirus infectivity. We show here that these two stretches ofamino acids happen to be important for the largest amplitudemotion obtained with the normal-mode theory for the two N-terminaldomains of human CD4: an overall rigid-body displacement ofone domain with respect to the other. Such a ‘hinge-bending’motion is unexpected since these two domains were found by crystallographersto be tightly abutting. On the other hand, since for severalproteins the hinge-bending motion experimentally observed uponligand binding was found to be similar to the largest amplitudemotion obtained with the normal-mode theory for these proteins,our results suggest that CD4 may undergo such a kind of conformationalchange upon HTV binding.     

Monovalent antibody scFv fragments selected to modulate T-cell activation by inhibition of CD86-CD28 interaction

Beside the interaction of the antigen-presenting major histocompatibility complex with the T-cell receptor, a co-stimulatory signal is required for T-cell activation in an immune response. To reduce immune-mediated graft rejection in corneal transplantation, where topical application of drugs in ointments or eye-drops may be possible, we selected single-chain antibody fragments (scFv) with binding affinity to rat CD86 (B7.2) that inhibit the co-stimulatory signal. We produced the IgV-like domain of rat CD86 as a fusion protein in Escherichia coli by refolding from inclusion bodies. This protein was used as a target for phage display selection of scFv from HuCAL-1, a fully artificial human antibody library. Selected binding molecules were shown to specifically bind to rat CD86 and inhibit the interaction of CD86 with CD28 and CTLA4 (CD152) in flow cytometry experiments. In an assay for CD86-dependent co-stimulation, the selected scFv fragment successfully inhibited the proliferation of T-cells induced by CD86-expressing P815 cells.     

Designing amino acid sequences to fold with good hydrophobic cores

We present two methods for designing amino acid sequences ofproteins that will fold to have good hydrophobic cores. Giventhe coordinates of the desired target protein or polymer structure,the methods generate sequences of hydrophobic (H) and polar(P) monomers that are intended to fold to these structures.One method designs hydrophobic inside, polar outside; the otherminimizes an energy function in a sequence evolution process.The sequences generated by these methods agree at the levelof 60–80% of the sequence positions in 20 proteins inthe Protein Data Bank. A major challenge in protein design isto create sequences that can fold uniquely, i.e. to a singleconformation rather than to many. While an earlier lattice-basedsequence evolution method was shown not to design unique folders,our method generates unique folders in lattice model tests.These methods may also be useful in designing other types offoldable polymer not based on amino acids     

Stabilization of TRAIL, an all-beta-sheet multimeric protein, using computational redesign

Protein thermal stability is important for therapeutic proteins, both influencing the pharmacokinetic and pharmacodynamic properties and for stability during production and shelf-life of the final product. In this paper we show the redesign of a therapeutically interesting trimeric all-beta-sheet protein, the cytokine TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), yielding variants with improved thermal stability. A combination of tumor necrosis factor (TNF) ligand family alignment information and the computational design algorithm PERLA was used to propose several mutants with improved thermal stability. The design was focused on non-conserved residues only, thus reducing the use of computational resources. Several of the proposed mutants showed a significant increase in thermal stability as experimentally monitored by far-UV circular dichroism thermal denaturation. Stabilization of the biologically active trimer was achieved by monomer subunit or monomer-monomer interface modifications. A double mutant showed an increase in apparent T(m) of 8 degrees C in comparison with wild-type TRAIL and remained biologically active after incubation at 73 degrees C for 1 h. To our knowledge, this is the first study that improves the stability of a large multimeric beta-sheet protein structure by computational redesign. A similar approach can be used to alter the characteristics of other multimeric proteins, including other TNF ligand family members.     

Two-dimensional surface display of functional groups on a beta-helical antifreeze protein scaffold

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a beta-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of beta-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded beta-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.     

Identification of important functional environs in protein tertiary structures from the analysis of residue variation in 3-D: application to cytochromes c and carboxypeptidases A and B

A simple methodology is described to apply to aligned proteinsequence sets for which at least one representative 3-D C structureis known. The evolutionary variation observed at each residueposition in the sequence alignment is qualified by taking intoaccount the residue variation that has occurred at other positionslocated within 7 A (according to the probable chain fold). Thisexpresses the evolutionary behaviour of any residue positionin the more appropriate context of its immediate surroundingsand distinguishes between invariant residues on the basis ofthe variation of their environment. The highest mechanisticsignificance is attached to conserved residues in conservedsurroundings, but the quantitative nature of the analysis meansthat all residue vicinities can be ranked and merged accordingto the degree of conservation that they exhibit and the residuepositions that comprise them. Therefore, with the aid of thechain fold, contour maps can be constructed that show gradedfoci of evolutionary conservation in the underlying superstructureof the protein type, and the irregular shapes and extents oflarge conserved areas. To test the methodology, it was appliedto cytochromes c and the carboxypeptidases A and B.     

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.     

Finding a new vaccine in the ricin protein fold

Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivating protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly, RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aerosolized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.     

Potential of genetic algorithms in protein folding and protein engineering simulations

Genetic algorithms are very efficient search mechanisms whichmutate, recombine and select amongst tentative solutions toa problem until a near optimal one is achieved. We introducethem as a new tool to study proteins. The identification andmotivation for different fitness functions is discussed. Theevolution of the zinc finger sequence motif from a random startis modelled. User specified changes of the repressor structurewere simulated and critical sites and exchanges for mutagenesisidentified. Vast conformational spaces are efficiently searchedas illustrated by the ab initio folding of a model protein ofa four ß strand bundle. The genetic algorithm simulationwhich mimicked important folding constraints as overall hydrophobicpackaging and a propensity of the betaphilic residues for transpositions achieved a unique fold. Cooperativity in the ßstrand regions and a length of 3–5 for the interconnectingloops was critical. Specific interaction sites were considerablyless effective in driving the fold.     

Global folding of proteins using a limited number of distance constraints

A Monte Carlo method is presented which can obtain the correcttertiary fold of a protein given the secondary structure andas few as three interactions between each secondary structureunit. This method was used to fold hemerythrin, Qavodoxin, bovinepancreatic trypsin inhibitor and a variable light domain froman immunoglobulin using the known secondary structures of theseproteins. Each of the proteins was successfully folded to obtaina structure resembling the initial X-ray structure. Reasonablesuccess was also achieved when using a secondary structure predictionalgorithm to assign secondary structure. The r.m.s. deviationsbetween the folded proteins and the crystal structures are inthe order of 3–5 A for the backbone coordinates. Evaluationof the r.m.s. deviations between members of the globin familyindicates that two equivalent overall folds may have r.m.s.deviations of this or even larger magnitude. The limiting numberof constraints necesssary to achieve the correct fold is discussed.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminalextracellular domains of human CD4 antigen, a cell surface glycoprotein,is reported. This region of CD4, particularly the first domain,has been identified as containing the binding region for theenvelope gp120 protein of the human immuno-deficiency virus.The model was predicted based on the sequence homology of eachdomain with the variable light chain of immunoglobulins. Theframework ß-sheet regions were taken from the crystalcoordinates of REI. For one region in the first domain of CD4there was an ambiguity in the alignment with REI and two alternatemodels are presented. Loops connecting the framework were modeledfrom fragments selected from a database of main chain coordinatesfrom all known protein structures. Residues identified as involvedin binding gp120 have been located in several other studieswithin the first domain of CD4. Epitopes from eight monoclonalantibodies have been mapped onto residues in both domains. Competitionof these antibodies with each other and with gp120 can be interpretedfrom the structural model.     

Design, synthesis and structure of an amphipathic peptide with pH-inducible haemolytic activity

A synthetic, 26–residue peptide having a strong helixforming potential in the protonated state was designed to interactwith lipid bilayers in a pH–dependent way. On the basisof this concept a cluster of four glutamk acid residues wasinserted in the central region of the amphipathic peptide topromote helix destabilization by mutual charge repulsion atneutral pH. Protonation of these residues might then bring aboutboth a pH-mediated change in hydrophobteity and conformationforming a membrane–active amphiphilk helix. The sequenceGLGTLLTLLEFLLEELLEFLKRKRQQamide produced by the design strategyinduced pH–triggered lysis of human erythrocytes. A molecularmodel correlating the lytic activity to the formation of transmembranepores which were detected by electron microscopy in erythrocytemembranes is discussed. Circular dichroism studies indicateda selfassociation of the monomeric random coil form with increasingpeptide concentration leading to the apparent induction of stronga-helix formation ({small tilde} 100% helkity) in the fullyaggregated state. However, no pH–dependent helixrandomcoil transition was observed, implying that interhelical hydrophobkand ionic interactions not only govern the self–associationbut also decisively influence the conformational stability ofthe peptide.     

Easy adaptation of protein structure to sequence

An investigation into the conservation of coarse, medium andfine grain structural properties has been performed over a dataset of 175 protein tertiary structures in 34 different families,each characterized by a common core fold and a library of conservedsites formed for each family. It is shown that, while the conservationof coarse and medium grain properties correlates to the structuraldeviation between the proteins, fine grain properties are poorlyconserved except in functional sites. This flexibility in finegrain properties suggests that folding can be viewed as an optimizationprocess whereby side chains have freedom to position themselvesas best as possible given environmental conformationa] constraintsand that given a basic framework, the local structure is ableto adapt easily to sequence variation. The conserved cores ofthe 34 families are used to estimate a minimal core size of35% of the fold, consistent with buried residue considerations.Finally, conservation in side chain l torsion angles is combinedwith structural deviation, sequence deviation and resolutionto suggest a set of example structure pairs suitable for testingautomatic homology modelling programs     

Nuclear magnetic resonance chemical shift: comparison of estimated secondary structures in peptides by nuclear magnetic resonance and circular dichroism

Traditionally, CD has been used extensively for peptides insecondary structure analysis. In recent years, NMR chemicalshifts and nuclear Overhauser enhancements have been widelyused in conjunction with CD to assess the secondary structuresof peptides and proteins; however, there are many instanceswhere the estimation of secondary structure contents differssignificantly between the two methods. In order to elucidatethe perceived differences between the two methods, secondarystructure estimations by CD and ¹H NMR chemical shifts werecompared for over 50 peptides. The linear peptides investigatedwere largely unstructured, {small tilde}15–50 residuesin size, and lacked stable tertiary conformation. These peptideswere studied in different solvent systems including water, alcohol—water,micelles and urea. A strong correlation exists for secondarystructure assessment by CD and NMR chemical shifts; however,an interesting trend of higher estimation of helical contentsby NMR was observed for peptide fragments from globular proteinsstudied in water. This may be a result of associative propertiesof these peptides in water. Addition-ally, a new method of quantitatingsecondary structure contents based on ¹H NMR chemical shiftsis reported.