Molecular mechanics analysis of drug-resistant mutants of HIV protease

Drug-resistant mutants of HIV-1 protease limit the long-termeffectiveness of current anti-viral therapy. In order to studydrug resistance, the wild-type HIV-1 protease and the mutantsR8Q, V32I, M46I, V82A, V82I, V82F, I84V, V32I/I84V and M46I/I84Vwere modeled with the inhibitors saquinavir and indinavir usingthe program AMMP. A new screen term was introduced to reproducemore correctly the electron distribution of atoms. The atomicpartial charge was represented as a delocalized charge distributioninstead of a point charge. The calculated protease–saquinavirinteraction energies showed the highly significant correlationof 0.79 with free energy differences derived from the measuredinhibition constants for all 10 models. Three different protonationstates of indinavir were evaluated. The best indinavir modelincluded a sulfate and gave a correlation coefficient of 0.68between the calculated interaction energies and free energiesfrom inhibition constants for nine models. The exception wasR8Q with indinavir, probably due to differences in the solvationenergy. No significant correlation was found using the standardmolecular mechanics terms. The incorporation of the new screencorrection resulted in better prediction of the effects of inhibitorson resistant protease variants and has potential for selectingmore effective inhibitors for resistant virus.     

Molecular dynamics simulation studies on Ca2+ -induced conformational changes of annexin I

Cryo-electron microscopy (EM) and X-ray studies proposed differentmechanisms for annexin-induced membrane aggregation. In thiswork, molecular dynamics (MD) simulation technique was utilizedto gain an insight into the calcium-induced conformational changeson annexin I and their implication in membrane aggregation mechanism.MD simulations were performed on the Ca²⁺-free annexin I withthe N-terminal domain buried inside the core (System 1), theCa²⁺-bound annexin I without N-terminal domain (System 2) andthe Ca²⁺-bound annexin I with the N-terminal domain exposed(System 3). Our results indicated that calcium binding increasesthe flexibility of annexin I core domain residues includingthe calcium coordinating residues. As a result, annexin I wasactivated to interact with the negatively charged membrane.The exposed N-terminal domain was very flexible and graduallylost the secondary structure during MD simulation, suggestingthat the N-terminal may adopt a favorable conformation to binda second membrane and also explaining the failure of attemptsto crystallize the full-length annexin I in the presence ofcalcium ions. The measured dimensions of the averaged simulationstructure of the Ca²⁺-bound annexin I with the N-terminal exposed(System 3) support the proposed membrane aggregation mechanismbased on X-ray studies.     

Generation and analysis of proline mutants in protein G

The pyrrolidine ring of the amino acid proline reduces the conformational freedom of the protein backbone in its unfolded form and thus enhances protein stability. The strategy of inserting proline into regions of the protein where it does not perturb the structure has been utilized to stabilize many different proteins including enzymes. However, most of these efforts have been based on trial and error, rather than rational design. Here, we try to understand proline's effect on protein stability by introducing proline mutations into various regions of the B1 domain of Streptococcal protein G. We also applied the Optimization of Rotamers By Iterative Techniques computational protein design program, using two different solvation models, to determine the extent to which it could predict the stabilizing and destabilizing effects of prolines. Use of a surface area dependent solvation model resulted in a modest correlation between the experimental free energy of folding and computed energies; on the other hand, use of a Gaussian solvent exclusion model led to significant positive correlation. Including a backbone conformational entropy term to the computational energies increases the statistical significance of the correlation between the experimental stabilities and both solvation models.     

Hinge-bending motions in annexins: molecular dynamics and essential dynamics of apo-annexin V and of calcium bound annexin V and I

Annexins are homologous proteins that bind to membranes in a calcium dependent manner, but for which precise physiological roles have yet to be defined. Most annexins are composed of a planar array of four homologous repeats, each containing five alpha-helices and associated into two modules. Annexin V forms a voltage-gated calcium channel in phospholipid bilayers. It has been proposed that the hydrophilic pore in the centre of the molecule may represent the ion conduction pathway and that a hinge movement in annexin V causes a variation of the inter- module angle and opens the calcium ion path. Here we present the results of molecular dynamics simulations of apo-annexin V and of calcium-bound annexin V and annexin I. The three simulations show significant differences in conformation and dynamics. The essential dynamics method was used to study the essential subspace of annexin V and showed that one of the essential motions corresponds to the postulated hinge motion. The hinge residues were located between repeats but belong to helices rather than to the links between helices. Calcium binding to annexin V led to a limitation of this hinge motion with more open conformations being favoured.      

Predicted structure for the calcium-dependent membrane-binding proteins p35, p36 and p32

A new family of proteins (annexins) that bind to membranes atmicromolar free Ca²⁺ has been recognized. Its members includean EGF-receptor kinase substrate (p35) a retroviral tyrosinekinase substrate (p36), the liver protein endonexin (p32) andan electric ray protein, calelectrin. Each protein containsfour sequence repeats with a further 2-fold Internal homology.Using the predicted secondary structure and pat tern of conservedhydrophobic residues in each repeat, we have built a three-dimensionalmodel that is largely isostruc tural with the known molecularconformation of bovine In testinal calcium-binding protein.The final (energy-refined) model had a core formed from theconserved hydrophobic residues. It differed from ICaBP principallyin the length of the two Ca²⁺ loops with only one loop beingable to bind. The model suuggest a mechanism for interactionof these new Ca²⁺ proteins with phospholipid bilayers.     

Grow to Fit Molecular Dynamics (G2FMD): an ab initio method for protein side-chain assignment and refinement

The rough energy landscapes and tight packing of protein interiors are two of the critical factors that have prevented the wide application of physics-based models in protein side-chain assignment and protein structure prediction in general. Complementing the rotamer-based methods, we propose an ab initio method that utilizes molecular mechanics simulations for protein side-chain assignment and refinement. By reducing the side-chain size, a smooth energy landscape was obtained owing to the increased distances between the side chains. The side chains then gradually grow back during molecular dynamics simulations while adjusting to their surrounding driven by the interaction energies. The method overcomes the barriers due to tight packing that limit conformational sampling of physics-based models. A key feature of this approach is that the resulting structures are free from steric collisions and allow the application of all-atom models in the subsequent refinement. Tests on a small set of proteins showed nearly 100% accuracy on both chi1 and chi2 of buried residues and 94% of them were within 20 degrees from the native conformation, 79% were within 10 degrees and 42% were within 5 degrees . However, the accuracy decreased when exposed side chains were involved. Further improvement and application of the method and the possible reasons that affect the accuracy on the exposed side chains are discussed.     

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

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

Theoretical studies of Rhizomucor miehei lipase activation

Computational methods have been used to study the extensiveconformational change of Rhizomucor miehei lipase upon activation.Hie present study considers the possible activation route, theenergies involved and molecular interactions during the conformationalchange of the lipase in a hydrophobic environment. The conformationalchange was studied by conventional molecular dynamics methodsand with a combined molecular dynamics and mechanics protocol,in which the conformational change was simulated by restrainingCor pseudotorsional angles in small steps between the two crystallographicallyobserved positions of the lid. In the closed conformer of theenzyme the active site is completely buried under a short helicalloop, ‘the lid’. The activation of the lipase consistsof a movement of the lid, which results in an open conformerwith an exposed active she. From the results of the simulationsin the present work we suggest that the lipase in a hydrophobicenvironment is stabilized in the open form by electrostaticinteractions.     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.     

Inhibitory mode of N-phenyl-4-pyrazolo[1,5-b] pyridazin-3-ylpyrimidin-2-amine series derivatives against GSK-3: molecular docking and 3D-QSAR analyses

Glycogen synthase kinase 3 (GSK-3) inhibition is an important research topic because of its wide range of associated health implications. The interaction mode of a series of N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amine compounds with human GSK-3 has been studied using molecular docking and 3D-QSAR approaches. In the 3D-QSAR studies, the molecular alignment and conformation determination are so important that they affect the success of a model. Flexible docking (AutoDock3.0.5) was used for the determination of 'active' conformation and molecular alignment. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used to develop 3D-QSAR models of 80 N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amine compounds. The r(2) values were 0.870 and 0.861 for CoMFA and CoMSIA models, respectively. The predictive ability of these models was validated by 10 compounds of the test set. Mapping these models back to the topology of the active site of GSK-3 led to a better understanding of the vital N-phenyl-4-pyrazolo[1,5-b]pyridazin-3-ylpyrimidin-2-amines-GSK-3 interactions. The results demonstrate that combination of ligand-based and receptor-based modeling is a powerful approach to build 3D-QSAR models. The interaction mode from this study may be helpful for the design of a novel inhibitor and guide the selection of candidate sites for further experimental studies on site-directed mutagenesis.     

Molecular modeling of coiled-coil {alpha}-tropomyosin: analysis of staggered and in register helix--helix interactions

In register and staggered models of tropomyosin coiled-coilwere built from X-ray C coordinates and refined via moleculardynamics. The two models show similar structural features withthe X-ray structure of GCN4 leucine zipper. Empirical energeticmethods used to compare the in register and staggered modelsindicate that both are equally probable. The two models havesimilar profiles of solvation free energy of folding for residuesat positions a and d of the repeating heptad, indicating thatresidues at these positions are as well buried in an in registerstructure as in a staggered one. Neither the in register northe 14 residues staggered structure can be ruled out based onhydrophobic or e–g' (g–e') electrostatic interactionswhich are not able to distinguish between the two models andare therefore not selective. However, the eg–b'c' electrostaticinteractions, although smaller in magnitude, are in favor ofthe in register model. Furthermore, analysis of hydrophobicand electrostatic interactions along the tropomyosin sequenceshows that bulky residues in positions a and d prevent the formationof inter-chain salt bridges.     

A novel mechanism of allosteric regulation of archaeal phosphoenolpyruvate carboxylase: a combined approach to structure-based alignment and model assessment

Phosphoenolpyruvate carboxylase (PEPC) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) and plays a crucial role in fixing atmospheric CO(2) in C(4) and CAM plants. The enzyme is widespread in plants and bacteria and mostly regulated allosterically by both positive and negative effectors. Archaeal PEPCs (A-PEPCs) have unique characteristics in allosteric regulation and molecular mass, distinct from their bacterial and eukaryote homologues, and their amino acid sequences have become available only recently. In this paper, we generated a structure-based alignment of archaeal, bacterial and eukaryote PEPCs and built comparative models using a combination of fold recognition, sequence and structural analysis tools. Our comparative modeling analysis identified A-PEPC-specific strong interactions between the two loops involved in both allostery and catalysis, which explained why A-PEPC is not influenced by any allosteric activators. We also found that the side-chain located three residues before the C-terminus appears to play a key role in determining the sensitivity to allosteric inhibitors. In addition to these unique features, we revealed how archaeal, bacterial and eukaryote PEPCs would share a common catalytic mechanism and adopt a similar mode of tetramer formation, despite their divergent sequences. Our novel observations will help design more efficient molecules for ecological and industrial use.     

Laboratory evolution of P450 BM3 for mediated electron transfer yielding an activity-improved and reductase-independent variant

One of the main obstacles in employing P450 monooxygenases for preparative chemical syntheses in cell-free systems is their requirement for cofactors such as NAD(P)H. In order to engineer P450 BM3 from Bacillus megaterium for cost-effective process conditions in vitro, a validated medium throughput screening system based on cheap Zn dust as an electron source and Cobalt(III)sepulchrate (Co(III)sep) as a mediator was reported. In the current study, the alternative cofactor system Zn/Co(III)sep was used in a directed evolution experiment to improve the Co(III)sep-mediated electron transfer to P450 BM3. A variant, carrying five mutations (R47F F87A V281G M354S D363H, Table I), P450 BM3 M5 was identified and characterized with respect to its kinetic parameters. P450 BM3 M5 achieved for mediated electron transfer a 2-fold higher k(cat) value and a 3-fold higher catalytic efficiency compared with the starting point mutant P450 BM3 F87A (k(cat): 62 min(-1) compared with 28 min(-1); k(cat)/K(m): 62 microM(-1)min(-1) compared to 19 microM(-1)min(-1)). For obtaining first insights on electron transfer contributions, three reductase-deficient variants were generated. The reductase-deficient mutant of P450 BMP M5 exhibited a catalytic efficiency of 69% and a k(cat) value of 89% of the values obtained for P450 BM3 M5.     

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

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

The Fourier-Green's function and the rapid evaluation of molecular potentials

Two tasks must be accomplished when calculating the bindingmodalities and binding energies of two molecules in solution:the calculation of the interaction energy and the calculationof the effects of solvation. It is the competition between theenergy of binding and the energy of remaining solvation whichdetermines the binding properties. It is necessary to calculate(or at least approximate in some manner) the partition functionin order to make a theoretical estimate of these effects. Anefficient algorithm for performing the energy evaluations necessaryfor this calculation is presented in this paper. The fast Fouriertransform (FFT) is used in combination with a polar factorizationof the potentials to calculate the interaction energy at allrelative translations between two molecules of fixed orientation.Thermodynamic quantities, including the partition function,internal and free energies can then be estimated from a setof these calculations covering the orientation space.     

Modelling the structure of latexin-carboxypeptidase A complex based on chemical cross-linking and molecular docking

We have determined the three-dimensional structure of the protein complex between latexin and carboxypeptidase A using a combination of chemical cross-linking, mass spectrometry and molecular docking. The locations of three intermolecular cross-links were identified using mass spectrometry and these constraints were used in combination with a speed-optimised docking algorithm allowing us to evaluate more than 3 x 10(11) possible conformations. While cross-links represent only limited structural constraints, the combination of only three experimental cross-links with very basic molecular docking was sufficient to determine the complex structure. The crystal structure of the complex between latexin and carboxypeptidase A4 determined recently allowed us to assess the success of this structure determination approach. Our structure was shown to be within 4 A r.m.s. deviation of Calpha atoms of the crystal structure. The study demonstrates that cross-linking in combination with mass spectrometry can lead to efficient and accurate structural modelling of protein complexes.     

Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides

Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.     

Secondary structure prediction: combination of three different methods

A combination of three complementary secondary structure predictionmethods is presented. The methods used are the GOR III method,the Homologue method and a new method, the bit pattern method,which is based on hydrophilic/hydrophobic residue patterns.For this purpose a hydropathy scale was developed and is presentedhere. The combination algorithm (Combine method) was designedto take the best results of each method and use their differencesin order to improve the prediction. The combination yields 65.5%correctly predicted residues in three states: -helix (H), ß-strand(E) and aperiodic structure (C) which is an improvement rangingfrom 2.5 to 6.5% compared with the individual methods when testedwith a 67-polypeptide chain database. Seventy-five per centof the regular secondary structure (H and E) runs are correctlylocated and ß-sheet runs are much better located bythe Combine method in comparison to the other methods.