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.     

Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding

Insulin-like growth factor-1 (IGF-1) is a serum protein whichunexpectedly folds to yield two stable tertiary structures withdifferent disulphide connectivities; native IGF-1 [18–61,6–48,47–52]and IGF-1 swap [18–61,6–47, 48–52]. Here we demonstratein detail the biological properties of recombinant human nativeIGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae.IGF-1 swap had a ~30 fold loss in affinity for the IGF-1 receptoroverexpressed on BHK cells compared with native IGF-1.The parallelincrease in dose required to induce negative cooperativity togetherwith the parallel loss in mitogenicity in NIH 3T3 cells impliesthat disruption of the IGF-1 receptor binding interaction ratherthan restriction of a post-binding conformational change isresponsible for the reduction in biological activity of IGF-1swap. Interestingly, the affinity of IGF-1 swap for the insulinreceptor was ~200 fold lower than that of native IGF-1 indicatingthat the binding surface complementary to the insulin receptor(or the ability to attain it) is disturbed to a greater extentthan that to the IGF-1 receptor. A 1.0 ns high-temperature moleculardynamics study of the local energy landscape of IGF-1 swap resultedin uncoiling of the first A-region -helix and a rearrangementin the relative orientation of the A- and B-regions. The modelof IGF-1 swap is structurally homologous to the NMR structureof insulin swap and CD spectra consistent with the model arepresented. However, in the model of IGF-1 swap the C-regionhas filled the space where the first A-region -helix has uncoiledand this may be hindering interaction of Val44 with the secondinsulin receptor binding pocket.     

Free energy perturbation studies on binding of A-74704 and its diester analog to HIV-1 protease

Free energy simulations have been employed to rationalize thebinding differences between A-74704, a pseudo C₂- symmetricinhibitor of HIV-1 protease and its diester analog. The diesteranalog inhibitor, which misses two hydrogen bonds with the enzymeactive site, is surprisingly only 10-fold weaker. The calculatedfree energy difference of 1.7 ± 0.6 kcal/mol is in agreementwith the experimental result. Further, the simulations showthat such a small difference in binding free energies is dueto (1) weaker hydrogen bond interactions between the two (P₁and P₁) NH groups of A-74704 with Gly27/Gly27' carbonyls ofthe enzyme and (2) the higher desolvation free energy of A-74704compared with its ester analog. The results of these calculationsand their implications for design of HIV-1 protease inhibitorsare discussed.     

Molecular modelling study of antigen binding to oxazolone-specific antibodies: the Ox1 idiotypic IgG and its mature variant with increased affinity to 2-phenyloxazolone

Structural models of the variable domains of the murine anti-2-phenyloxazoloneIgG (Oxl idiotype) and its somatic variant, which has higheraffinity to the hapten 2-phenyloxazolone, were constructed bycomputer-aided model building using known structures of highlyhomologous immunoglobulins as templates. Molecular dynamicssimulations were used to dock the hapten between the V_L andV_H domains. The hapten is predicted to bind to slightly differentsites in the two models. Hypotheses concerning the role of anumber of preferred mutations in anti-oxazolone variants arepresented. These can be tested by mutagenesis and crystallography.In particular, the higher binding affinities of the differentantibody variants are shown to correlate with better complementarityof electrostatics. The molecular dynamic simulations also suggestthat two mobile tryptophans at the mouth of the pocket may playan important role in the binding of hapten.     

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.     

An SS1-SS2 {beta}-barrel structure for the voltage-activated potassium channel

To examine the feasibility of a ß structure for thepore-lining region of the voltage-gated potassium channel, wehave characterized a family of 12 antiparallel ß-barrels.Each is comprised of four identical pairs of ß-strandsorganized with approximate 4-fold symmetry about a channel axis.The Cand N-termini of the ß-strand pairs are assumedto be at the extracellular end of the channel, and each pairis connected by a hairpin turn at the intracellular end of thechannel. The models differ in the residues located in the hairpinturn and in the orientation of the two strands of each pairin the barrel, i.e. whether the C-terminus of a pair is clockwise(CW) or counterclockwise (CCW) from the N-terminus when thechannel is viewed from outside the cell. Following known structureprecedents and potential energy predictions, the barrel is assumedto be right-twisting in all cases. All models have crowded layersof inward-projecting aromatic sidechains near the center ofthe channel which could regulate channel selectivity. The modelswith an odd number of amino acids in the hairpin turn have theadvantage of predicting that F433 points into the barrel, butthe disadvantage that V438 does not. Of these models, two ofthe models are most consistent with the external tetraethylanunonhim(TEA) block data, and of those, one (T439 CCW 3:5) is most consistentwith the internal TEA block data.     

Selection of single domain binding proteins by covalent DNA display

Selection technologies such as phage and ribosome display, which provide a physical linkage between genetic information and encoded polypeptide, are important tools for the engineering of proteins for diagnostic and therapeutic applications. We have recently described a selection strategy called covalent DNA display, in which individual proteins are covalently linked to the cognate encoding DNA template in separate droplets of a water-in-oil emulsion. We here report on the optimization of several experimental steps in covalent DNA display technology, such as the elution conditions and the PCR strategy used for the amplification of selected DNA templates. A PCR assembly strategy was developed, which allows the amplification of the DNA templates over repeated rounds of selection. In addition, we could demonstrate that approximately 50% of the DNA templates form a covalent adduct with the corresponding proteins in the compartments of a water-in-oil emulsion. In model selection experiments, differences in recovery efficiency <100 000 per round of selection could be observed when comparing a specific binding polypeptide with a binder of irrelevant specificity. Furthermore, the optimized protocol was successfully applied for the selection of single domain proteins, capable of specific binding to mouse serum albumin (MSA). A mutant derived from the SH3 domain of the Fyn kinase, with millimolar affinity to MSA, was affinity matured using covalent DNA display and yielded several MSA binding FynSH3 variants with dissociation constants in the 100 nM range.     

Characterization of the DNA binding domain of the mouse IRF-2 protein

The DNA binding domain of the interferon regulatory factor-2protein (IRF-2) has been produced and characterized, -chymotrypsindigestion of the purified IRF-2 protein bound to a syntheticbinding site yields a peptide fragment of 14 K in molecularweight. N-terminal analysis of this peptide fragment showedthat its sequence is the same as that of the intact IRF-2. Apeptide fragment of {small tilde} 14 K, IRF-2(113), which correspondsto the N-terminal 113 amino acids of the intact IRF-2 protein,has been expressed in a functional form in Escherichia coli.The first methionine was processed during the expression andthe purified IRF-2(113) thus contains 112 amino acids. DNaseI footprinting and gel retardation assaying showed that IRF-2(113)binds to a synthetic DNA having the consensus binding site andto the upstream regulatory sequence of the IFN-ß geneas intact IRF-2 does. These results showed that this peptidefragment, IRF-2(113), may be a good material for investigationof the DNA binding domain of IRF-2 and of the DNA–proteininteraction.     

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD

Protein engineering of the cholera toxin A1 subunit (CTA1) fusedto a dimer of the Ig-binding D-region of Staphylococcus aureusprotein A (DD) was employed to investigate the effect of specificamino acid changes on solubility, stability, enzymatic activityand capacity to act as an adjuvant in vivo. A series of CTA1-DDanalogues were selected by a rational modeling approach, inwhich surface-exposed hydrophobic amino acids of CTA1 were exchangedfor hydrophilic counterparts modeled for best structural fit.Of six different mutants initially produced, two analogues,CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated tohave 50 and 70% increased solubility, respectively, at neutralpH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at leastthreefold more soluble, demonstrating an additive effect ofthe two mutations. Only the Phe132Ser analogue retained fullbiological activity and stability compared with the native CTA1-DDfusion protein. Two mutants, Pro185Gln and Phe31His mutations,exhibited unaltered ADP-ribosyltransferase activity in vitro,but demonstrated markedly reduced adjuvant function. Since thePro185 and Phe31 amino acids are located in close vicinity onthe distal side of the molecule relative to the enzymaticallyactive cleft, it is conceivable that this region is involvedin mediating a biological function, separate from the enzymaticactivity but intrinsic to the adjuvant activity of CTA1.     

Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose binding surface

A total of 23 fungal cellulose-binding domain (CBD) sequenceswere aligned. Structural models of the cellulosebinding domainof an exoglucanase (CBHII) and of three endoglucanases (EGI,EGII and EGV) from Trichoderma reesei cellulases were homologymodelled based on the NMR structure of the fungal cellobiohydrolaseCBHI, from the same organism. The completed models and the knownstructure of the CBHI cellulose-binding domain were refinedby molecular dynamics simulations in water. All four modelswere found to be very similar to the structure of the CBHI cellulose-bindingdomain and sequence comparison indicated that in general thethree-dimensional structures of fungal cellulose-binding domainsare very similar. In all the CBDs studied, two disulphide bridgesapparently stabilize the polypeptide fold. From the models,an additional disulphide bridge was predicted in EGI and CBHII,and in eight further CBDs from other organisms. Three highlyconserved aromatic residues on the hydrophilic side of the wedgemake this surface flat This surface is expected to make contactwith the substrate. Three invariant amino acids, Gln7, Asn29and Gln34, on this flat face are in suitable positions for hydrogenbonding with the cellulose surface. Analysis of the differencesin the protein surface properties indicated that the endoglucanasestend to be more hydrophilic than the exoglucanases. The largeststructural variation was found around positions 12-16. The fungalCBD sequences are discussed in relation to variations in functionand pH dependence. Comparison of the modelled structures withexperimental binding data for the CBHI and EGI allowed the formulationof a qualitative relationship to cellulose affinity. This relationshipwas used to predict the cellulose affinities for 21 CBDs.     

Molecular dynamics of the 5-HT1a receptor and ligands

A 3-D model of the human 5-HT_1a receptor was constructed fromits amino acid sequence by computer graphics techniques, molecularmechanics calculations and molecular dynamics simulations. Themodel has seven -helical membrane spanning segments, which forma central core containing a putative ligand binding site. Electrostaticpotentials 1.4 Å outside the water accessible surfacewere mainly negative on the synaptic side of the receptor modeland at the postulated ligand binding site, and positive in thecytoplasmic domains. The negative electrostatic potentials aroundthe synaptic domains indicate that positively charged ligandsare attracted to the receptor by electrostatic forces. Moleculardynamics simulations of the receptor model with serotonin, ipsapirone,R(–)-methiothepin or S(+)- methiothepin in the centralcore suggested that up to 22 different amino acid residues mayform a ligand binding pocket, and contribute to the specificityof ligand recognition and binding.     

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.     

Comparative molecular modeling of Amphioxus calcium vector protein with calmodulin and troponin C

Calcium vector protein (CaVP), a new protein isolated from Amphioxusmuscle, binds in a Ca²⁺ -rgulated manner to a 27 kd target protein,named CaVPT, whose function has not been elucidated yet. CaVPbears significant sequence homology to both calmodulin and skeletalmuscle troponin C, especially in the C-tenninal half of themolecule, which presumably contains the two functional Ca²⁺sites. The N-terminal half contains two abortive EF-hands andis intramolecularly crosslinked with a disulfide bond. Usingthe crystallographic structures of calmodulin and striated muscletroponin C as a framework, we constructed two different three-dimensionalmodels of CaVP and modeled the intramolecular disulfide bridge.The modeling based upon the coordinates of calmodulin yieldsa Ca²⁺ -filled sites configuration in the N-terminal half ofthe molecule, even though no Ca²⁺ is bound in this half, whereasthe troponin C-derived model generates a Ca²⁺ -empty sites configuration.The models predict that neither in the Ca²⁺ nor in the Ca²⁺-empty sites conformation is there any steric and/or energeticobstacle for the formation of the disulfide bridge and thatthe disulfide bond is poorly accessible to reducing reagents.The optical properties of the Trp and Tyr residues of CaVP indicatethat the calmodulin-derived model represents the most plausibleprediction.     

A mutational analysis of receptor binding sites of interleukin-1{beta}:differences in binding of human interleukin-1{beta} muteins to human and mouse receptors

The 3-D crystal structure of interleukin-1ß(IL-1ß)has been used to define its receptor binding surface by mutationalanalysis. The surface of IL-1ß was probed by site-directedmutagenesis. A total of 27 different IL-1ß muteinswere constructed, purified and analyzed. Receptor binding measurementson mouse and human cell lines were performed to identify receptoraffinities. IL-1ß muteins with modified receptor affinitywere evaluated for structural integrity by CD spectroscopy orX-ray crystallography. Changes in six surface loops, as wellas in the C- and N-termini, yielded muteins with lower bindingaffinities. Two muteins with intact binding affinities showed10- to 100-fold reduced biological activity. The surface regioninvolved in receptor binding constitutes a discontinuous areaof 1000 Å2 formed by discontinuous polypeptide chain stretches.Based on these results, a subdivision into two distinct localareas is proposed. Differences in receptor binding affinitiesfor human and mouse receptors have been observed for some muteins,but not for wild-type IL-1ß. This is the first timea difference in binding affinity of IL-1ß muteinsto human and mouse receptors has been demonstrated     

The binding mode of an E-64 analog to the active site of cathepsin B

Two binding modes of the isobutyl-NH-Eps-Leu-Pro inhibitor tocathepsin B have been proposed. Molecular docking using an empiricalforce field was carried out to distinguish between the two modes.The search began with manual docking, followed by random perturbationsof the docking conformation and cycles of Monte Carlo minimization.Finally, molecular dynamics was carried out for the most favorabledocking conformations. The present calculations predict thatthe isobutyl-NH-Eps-Leu-Pro inhibitor preferentially binds tothe S' rather than the S subsites of cathepsin B. The S' bindingmode prediction is supported by the X-ray crystal structureof cathepsin B bound to a closely related ethyl-O-Eps-Ile-Proinhibitor, which was found to bind in the S' subsite with theC-terminal epoxy ring carbon making a covalent bond to the sulfuratom of Cys29. This agreement, in turn, validates our dockingstrategy. Furthermore, the calculations provide evidence thatthe dominant contribution to the total stabilization energyof the enzyme–inhibitor complex stems from the strongelectrostatic interaction between the negatively charged C-terminalcarboxylate group of the ligand and the positively charged imidazoliumrings of His110 and His111. The latter are stabilized and heldin an optimal orientation for interactions with the C-terminalend of the ligand through a salt bridge between the side chainsof His110 and Asp22. By comparison with the crystal structure,some insight into the specificity of the epoxyldipeptide familytowards cathepsin B inhibition has been extracted. Both thecharacteristics of the enzyme (e.g. subsite size and hydrophobicity)as well as the nature of the inhibitor influence the selectivityof an inhibitor towards an enzyme.     

Cloning of rabbit interleukin-1{beta}: differential evolution of IL-1{alpha} and IL-1{beta} proteins

We have cloned the rabbit IL-1ß cDNA, which encodesa 268 amino acid precursor similar in length to other sequencedIL-1 precursors. Comparison of all published IL-1 and IL-1ßsequences respectively indicates that the IL-1 gene family isevolving faster than the IL-1ß family, and that thetwo genes diverged –270 million years ago. Surprisingly,there are differences in the regions preferentially conservedwithin the two families. The IL-1 family is most conserved atthe amino terminus whereas the IL-1ß family is mostconserved in the carboxy-terminal half. This is despite thefact that the carboxy-terminal half encodes the active portionof both molecules and would be expected to adopt a similar ß-sheetstructure in IL-1 as in the published X-ray structure of matureIL-1ß. These findings suggest that differences inthe function and properties of the IL-1 and IL-1ßprecursor molecules may have been conserved. These differencesmay therefore provide an explanation for the existence of twoIL-1 molecules.