Salt effects on peptide conformers: a dielectric study of tuftsin.

Four 1-ns molecular dynamics computer simulations of tuftsin, Thr-Lys-Pro-Arg, are analyzed: (1) cis tuftsin in water, (2) trans tuftsin in water, (3) cis tuftsin in 1 M NaCl, and (4) trans tuftsin in 1 M NaCl. Independently of the salt concentration, the trans conformer has a higher dielectric constant than the cis conformer because the former exhibits a more widely distributed charge distribution in space. Independently of the peptide conformation, the presence of salt reduces the dielectric constants of both the peptide and the solvating water molecules because ions, on binding, restrict the motion of other atoms. In contrast to the dielectric constants, neither the peptide conformation nor the salt concentration shows a significant influence on the dielectric relaxation time of water molecules.     

Two TPX2-dependent switches control the activity of Aurora A

Aurora A is an important oncogenic kinase for mitotic spindle assembly and a potentially attractive target for human cancers. Its activation could be regulated by ATP cycle and its activator TPX2. To understand the activation mechanism of Aurora A, a series of 20 ns molecular dynamics (MD) simulations were performed on both the wild-type kinase and its mutants. Analyzing the three dynamic trajectories (Aurora A-ATP, Aurora A-ADP, and Aurora A-ADP-TPX2) at the residue level, for the first time we find two TPX2-dependent switches, i.e., switch-1 (Lys-143) and switch-2 (Arg-180), which are tightly associated with Aurora A activation. In the absence of TPX2, Lys-143 exhibits a "closed" state, and becomes hydrogen-bonded to ADP. Once TPX2 binding occurs, switch-1 is forced to "open" the binding site, thus pulling ADP away from Aurora A. Without facilitation of TPX2, switch-2 exits in an "open" conformation which accompanies the outward-flipping movement of P·Thr288 (in an inactive conformation), leading to the crucial phosphothreonine exposed and accessible for deactivation. However, with the binding of TPX2, switch-2 is forced to undergo a "closed" movement, thus capturing P·Thr288 into a buried position and locking its active conformation. Analysis of two Aurora A (K143A and R180A) mutants for the two switches further verifies their functionality and reliability in controlling Aurora activity. Our systems therefore suggest two switches determining Aurora A activation, which are important for the development of aurora kinase inhibitors.     

Crystal structures of the active site mutant (Arg-243-->Ala) in the T and R allosteric states of pig kidney fructose-1,6-bisphosphatase expressed in Escherichia coli.

The active site of pig kidney fructose-1,6-bisphosphatase (EC 3.1.3.11) is shared between subunits, Arg-243 of one chain interacting with fructose-1,6-bisphosphate or fructose-2,6-bisphosphate in the active site of an adjacent chain. In this study, we present the X-ray structures of the mutant version of the enzyme with Arg-243 replaced by alanine, crystallized in both T and R allosteric states. Kinetic characteristics of the altered enzyme showed the magnesium binding and inhibition by AMP differed slightly; affinity for the substrate fructose-1,6-bisphosphate was reduced 10-fold and affinity for the inhibitor fructose-2,6-bisphosphate was reduced 1,000-fold (Giroux E, Williams MK, Kantrowitz ER, 1994, J Biol Chem 269:31404-31409). The X-ray structures show no major changes in the organization of the active site compared with wild-type enzyme, and the structures confirm predictions of molecular dynamics simulations involving Lys-269 and Lys-274. Comparison of two independent models of the T form structures have revealed small but significant changes in the conformation of the bound AMP molecules and small reorganization of the active site correlated with the presence of the inhibitor. The differences in kinetic properties of the mutant enzyme indicate the key importance of Arg-243 in the function of fructose-1,6-bisphosphatase. Calculations using the X-ray structures of the Arg-243-->Ala enzyme suggest that the role of Arg-243 in the wild-type enzyme is predominantly electrostatic in nature.     

Molecular dynamics study of substance P peptides partitioned in a sodium dodecylsulfate micelle

Two neuropeptides, substance P (SP) and SP-tyrosine-8 (SP-Y8), have been studied by molecular dynamics (MD) simulation in an explicit sodium dodecylsulfate (SDS) micelle. Initially, distance restraints derived from NMR nuclear Overhauser enhancements (NOE) were incorporated in the restrained MD (RMD) during the equilibration stage of the simulation. It was shown that when SP-Y8 was initially placed in an insertion (perpendicular) configuration, the peptide equilibrated to a surface-bound (parallel) configuration in approximately 450 ps. After equilibration, the conformation and orientation of the peptides, the solvation of both the backbone and the side chain of the residues, hydrogen bonding, and the dynamics of the peptides were analyzed from trajectories obtained from the RMD or the subsequent free MD (where the NOE restraints were removed). These analyses showed that the peptide backbones of all residues are either solvated by water or are hydrogen-bonded. This is seen to be an important factor against the insertion mode of interaction. Most of the interactions come from the hydrophobic interaction between the side chains of Lys-3, Pro-4, Phe-7, Phe-8, Leu-10, and Met-11 for SP, from Lys-3, Phe-7, Leu-10, and Met-11 in SP-Y8, and the micellar interior. Significant interactions, electrostatic and hydrogen bonding, between the N-terminal residues, Arg-Pro-Lys, and the micellar headgroups were observed. These latter interactions served to affect both the structure and, especially, the flexibility, of the N-terminus. The results from simulation of the same peptides in a water/CCl4 biphasic cell were compared with the results of the present study, and the validity of using the biphasic system as an approximation for peptide-micelle or peptide-bilayer systems is discussed.     

The structure of residues 7-16 of the A alpha-chain of human fibrinogen bound to bovine thrombin at 2.3-A resolution.

The tetradecapeptide Ac-D-F-L-A-E-G-G-G-V-R-G-P-R-V-OMe, which mimics residues 7f-20f of the A alpha-chain of human fibrinogen, has been co-crystallized with bovine thrombin from ammonium sulfate solutions in space group P2(1) with unit cell dimensions of a = 83.0 A, b = 89.4 A, c = 99.3 A, and beta = 106.6 degrees. Three crystallographically independent complexes were located in the asymmetric unit by molecular replacement using the native bovine thrombin structure as a model. The standard crystallographic R-factor is 0.167 at 2.3-A resolution. Excellent electron density could be traced for the decapeptide, beginning with Asp-7f and ending with Arg-16f in the active site of thrombin; the remaining 4 residues, which have been cleaved from the tetradecapeptide at the Arg-16f/Gly-17f bond, are not seen. Residues 7f-11f at the NH2 terminus of the peptide form a single turn of alpha-helix that is connected by Gly-12f, which has a positive phi angle, to an extended chain containing residues 13f-16f. The major specific interactions between the peptide and thrombin are 1) a hydrophobic cage formed by residues Tyr-60A, Trp-60D, Leu-99, Ile-174, Trp-215, Leu-9f, Gly-13f, and Val-15f that surrounds Phe-8f; 2) a hydrogen bond linking Phe-8f NH to Lys-97 O;3) a salt link between Glu-11f and Arg-173; 4) two antiparallel beta-sheet hydrogen bonds between Gly-14f and Gly-216; and 5) the insertion of Arg-16f into the specificity pocket. Binding of the peptide is accompanied by a considerable shift in two of the loops near the active site relative to human D-phenyl-L-prolyl-L-arginyl chloromethyl ketone (PPACK)-thrombin.     

Predicted Folding of β-Structure in Myelin Basic Protein

Predictions of myelin basic protein secondary structure have not previously considered a major role for beta-structure in the organization of the native molecule because optical rotatory dispersion and circular dichroism studies have provided little, if any, evidence for beta-structure, and because a polycationic protein is generally considered to resist folding into a compact structure. However, the Chou-Fasman, Lim, and Robson algorithms identify a total of five beta-strands in the amino acid sequence. Four of these hydrophobic amino acid sequences (37-45, 87-95, 110-118, and 150-158) could form a hairpin intermediate that initiates folding of a Greek-key-type beta-structure. A second fold on the more hydrophobic side, with the addition of a strand from the N-terminus (residues 13-21), would complete the five-stranded antiparallel beta-sheet. A unique strand alignment can be predicted by phasing the hydrophobic residues. The unusual triproline sequence of myelin basic protein (100-102) is enclosed in the 14-residue hairpin loop. If these prolines are in the trans conformation, models show that a reverse turn could occur at residues 102-105 (Pro-Ser-Gln-Gly). Algorithms do not agree on the prediction of alpha-helices, but each of the two large loops could accommodate an alpha-helix. Myelin basic protein is known to be phosphorylated in vivo on as many as five Ser/Thr residues. Phosphorylation might alter the dynamics of folding if the nascent polypeptide were phosphorylated in the cytoplasm. In particular, phosphorylation of Thr-99 could neutralize cationic residues Lys-106 and Arg-108 within the hairpin loop. In addition, the methylation of Arg-108 might stabilize the hairpin loop structure through hydrophobic interaction with the side chain of Pro-97. The cationic side chains of arginine and lysine residues located on the faces of the beta-sheet (Arg-43, Arg-114, Lys-13, Lys-92, Lys-153, and Lys-156) could provide sites for interaction with phospholipids and other anionic structures on the surface of the myelin lipid bilayer.     

Structure of a fructose-1,6-bis(phosphate) aldolase liganded to its natural substrate in a cleavage-defective mutant at 2.3 A(,).

Class I fructose-1,6-bis(phosphate) aldolase is a glycolytic enzyme that catalyzes the cleavage of fructose 1,6-bis(phosphate) through a covalent Schiff base intermediate. Although the atomic structure of this enzyme is known, assigning catalytic roles to the various enzymic active-site residues has been hampered by the lack of a structure for the enzyme-substrate complex. A mutant aldolase, K146A, is unable to cleave the C3-C4 bond of the hexose while retaining the ability to form the covalent intermediate, although at a greatly diminished rate. The structure of rabbit muscle K146A-aldolase A, in complex with its native substrate, fructose 1,6-bis(phosphate), is determined to 2.3 A resolution by molecular replacement. The density at the hexose binding site differs between subunits of the tetramer, in that two sites show greater occupancy relative to the other two. The hexose is bound in its linear, open conformation, but not covalently linked to the Schiff base-forming Lys-229. Therefore, this structure most likely represents the bound complex of hexose just after hemiketal hydrolysis and prior to Schiff base formation. The C1-phosphate binding site involves the three backbone nitrogens of Ser-271, Gly-272, and Gly-302, and the epsilon-amino group of Lys-229. This is the same binding site previously found for the analogous phosphate of the product DHAP. The C6-phosphate binding site involves three basic side chains, Arg-303, Arg-42, and Lys-41. The residues closest to Lys-229 were relatively unchanged in position when compared to the unbound wild-type structure. The major differences between the bound and unbound enzyme structures were observed in the positions of Lys-107, Arg-303, and Arg-42, with the greatest difference in the change in conformation of Arg-303. Site-directed mutagenesis was performed on those residues with different conformations in bound versus unbound enzyme. The kinetic constants of these mutant enzymes with the substrates fructose 1, 6-bis(phosphate) and fructose 1-phosphate are consistent with their ligand interactions as revealed by the structure reported here, including differing effects on k(cat) and K(m) between the two substrates depending on whether the mutations affect C6-phosphate binding. In the unbound state, Arg-303 forms a salt bridge with Glu-34, and in the liganded structure it interacts closely with the substrate C6-phosphate. The position of the sugar in the binding site would require a large movement prior to achieving the proper position for covalent catalysis with the Schiff base-forming Lys-229. The movement most likely involves a change in the location of the more loosely bound C6-phosphate. This result suggests that the substrate has one position in the Michaelis complex and another in the covalent complex. Such movement could trigger conformational changes in the carboxyl-terminal region, which has been implicated in substrate specificity.     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

Detailed mechanism for AmtB conducting NH4+/NH3: molecular dynamics simulations

The mechanism by which the ammonium transporter, AmtB, conducts NH4+/NH3 into the cytoplasm was investigated using conventional molecular dynamics (MD) simulations. These simulations revealed that the neutral molecule, NH3, passes automatically through the channel upon its arrival at the Am2 site and that the function of the channel as a one-way valve for passage of NH3 is not determined by the cytoplasmic exit gate but, rather, by the periplasmic entrance gate of the channel. The NH3, produced by deprotonation of NH4+ at the entrance gate, is spontaneously conveyed to the central region of the channel via a hydrogen-bond network comprising His-168, His-318, Tyr-32, and the NH3 molecule. Finally, the NH3 molecule exits the channel through the exit gate formed by Phe-31, Ile-266, Val-314, and His-318. In addition, Ser-263 is shown to play a critical role in the final stages, acting as a pivoting arm to shunt the NH3 molecule from the cytoplasmic exit gate of the channel out into the cytoplasm. This finding is further supported by another simulation which shows that NH3 fails to be translocated through the channel formed by the Ser-263-Ala mutation. Thus, this study casts new insights on the mechanism of AmtB-mediated passage of NH3 across cellular membranes.     

Functional roles of charged residues in the putative voltage sensor of the HCN2 pacemaker channel

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels contribute to pacemaking activity in specialized neurons and cardiac myocytes. HCN channels have a structure similar to voltage-gated K(+) channels but have a much larger putative S4 transmembrane domain and open in response to membrane hyperpolarization instead of depolarization. As an initial attempt to define the structural basis of HCN channel gating, we have characterized the functional roles of the charged residues in the S2, S3, and S4 transmembrane domains. The nine basic residues and a single Ser in S4 were mutated individually to Gln, and the function of mutant channels was analyzed in Xenopus oocytes using two-microelectrode voltage clamp techniques. Surface membrane expression of hemagglutinin-epitope-tagged channel proteins was examined by chemiluminescence. Our results suggest that 1) Lys-291, Arg-294, Arg-297, and Arg-300 contribute to the voltage dependence of gating but not to channel folding or trafficking to the surface membrane; 2) Lys-303 and Ser-306 are essential for gating, but not for channel folding/trafficking; 3) Arg-312 is important for folding but not gating; and 4) Arg-309, Arg-315, and Arg-318 are crucial for normal protein folding/trafficking and may charge-pair with Asp residues located in the S2 and S3 domains.     

Isolation of four novel tachykinins from frog (Rana catesbeiana) brain and intestine

In a survey for unknown bioactive peptides in frog (Rana catesbeiana) brain and intestine, we isolated four novel peptides that exhibit potent stimulant effects on smooth muscle preparation of guinea pig ileum. By microsequencing and synthesis, these peptides were identified as Lys- Pro- Ser- Pro- Asp- Arg- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin A), Tyr- Lys- Ser- Asp- Ser- Phe- Tyr- Gly- Leu- Met- NH2 (ranatachykinin B), His- Asn- Pro- Ala- Ser- Phe- Ile- Gly- Leu- Met- NH2 (ranatachykinin C) and Lys- Pro- Ans- Pro- Glu- Arg- Phe- Tyr- Ala- Pro- Met- NH2 (ranatachykinin D). Ranatachykinin (RTK) A, B and C conserve the C- terminal sequence, Phe- X- Gly- Leu- Met- NH2, which is common to known members of the tachykinin family. On the other hand, RTK-D has a striking feature in its C-terminal sequence, Phe- Tyr- Ala- Pro- Met- NH2, which has never been found in other known tachykinins, and may constitute a new subclass in the tachykinin family.     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.     

Nuclear magnetic resonance studies on the structure of the tetrapeptide tuftsin, L-threonyl-L-lysyl-L-prolyl-L-arginine, and its pentapeptide analogue L-threonyl-L-lysyl-L-prolyl-L-prolyl-L-arginine.

Nuclear magnetic resonance spectroscopy has been used to investigate the solution conformation of tuftsin, threonyllysylprolylarginine, as well as a pentapeptide inhibitor of tuftsin, threonyllysylprolylprolylarginine. Both proton and carbon-13 studies were performed. In water, neither peptide gives evidence of a preferred conformation. In dimethyl-d6 sulfoxide, tuftsin appears to prefer a particular conformation, but the inhibitor does not. The conformation of tuftsin is one in which the amide NH proton of arginine is solvent shielded. The conformation does not, however, appear to be such that a normal 4 leads to 1 beta turn exists.     

Mapping multiple potential ATP binding sites on the matrix side of the bovine ADP/ATP carrier by the combined use of MD simulation and docking

The mitochondrial adenosine diphosphate/adenosine triphosphate (ADP/ATP) carrier-AAC-was crystallized in complex with its specific inhibitor carboxyatractyloside (CATR). The protein consists of a six-transmembrane helix bundle that defines the nucleotide translocation pathway, which is closed towards the matrix side due to sharp kinks in the odd-numbered helices. In this paper, we describe the interaction between the matrix side of the AAC transporter and the ATP(4-) molecule using carrier structures obtained through classical molecular dynamics simulation (MD) and a protein-ligand docking procedure. Fifteen structures were extracted from a previously published MD trajectory through clustering analysis, and 50 docking runs were carried out for each carrier conformation, for a total of 750 runs ("MD docking"). The results were compared to those from 750 docking runs performed on the X-ray structure ("X docking"). The docking procedure indicated the presence of a single interaction site in the X-ray structure that was conserved in the structures extracted from the MD trajectory. MD docking showed the presence of a second binding site that was not found in the X docking. The interaction strategy between the AAC transporter and the ATP(4-) molecule was analyzed by investigating the composition and 3D arrangement of the interaction pockets, together with the orientations of the substrate inside them. A relationship between sequence repeats and the ATP(4-) binding sites in the AAC carrier structure is proposed.     

Mass-weighted molecular dynamics simulation and conformational analysis of polypeptide.

Atomic motions in protein molecules have been studied by molecular dynamics (MD) simulations; dynamics simulation methods have also been employed in conformational studies of polypeptide molecules. It was found that when atomic masses are weighted, the molecular dynamics method can significantly increase the sampling of dihedral conformation space in such studies, compared to a conventional MD simulation of the same total simulation time length. Herein the theoretical study of molecular conformation sampling by the molecular dynamics-based simulation method in which atomic masses are weighted is reported in detail; moreover, a numerical scheme for analyzing the extensive conformational sampling in the simulation of a tetrapeptide amide molecule is presented. From numerical analyses of the mass-weighted molecular dynamics trajectories of backbone dihedral angles, low-resolution structures covering the entire backbone dihedral conformation space of the molecule were determined, and the distribution of rotationally stable conformations in this space were analyzed quantitatively. The theoretical analyses based on the computer simulation and numerical analytical methods suggest that distinctive regimes in the conformational space of the peptide molecule can be identified.     

New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E

Defective binding of apolipoprotein E (apoE) to heparan sulfate proteoglycans (HSPGs) is associated with increased risk of atherosclerosis due to inefficient clearance of lipoprotein remnants by the liver. The interaction of apoE with HSPGs has also been implicated in the pathogenesis of Alzheimer's disease and may play a role in neuronal repair. To identify which residues in the heparin-binding site of apoE and which structural elements of heparan sulfate interact, we used a variety of approaches, including glycosaminoglycan specificity assays, (13)C nuclear magnetic resonance, and heparin affinity chromatography. The formation of the high affinity complex required Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147 from apoE and N- and 6-O-sulfo groups of the glucosamine units from the heparin fragment. As shown by molecular modeling, using a high affinity binding octasaccharide fragment of heparin, these findings are consistent with a binding mode in which five saccharide residues of fully sulfated heparan sulfate lie in a shallow groove of the alpha-helix that contains the HSPG-binding site (helix 4 of the four-helix bundle of the 22-kDa fragment). This groove is lined with residues Arg-136, Ser-139, His-140, Arg-142, Lys-143, Arg-145, Lys-146, and Arg-147. In the model, all of these residues make direct contact with either the 2-O-sulfo groups of the iduronic acid monosaccharides or the N- and 6-O-sulfo groups of the glucosamine sulfate monosaccharides. This model indicates that apoE has an HSPG-binding site highly complementary to heparan sulfate rich in N- and O-sulfo groups such as that found in the liver and the brain.     

Computational simulations of interactions of scorpion toxins with the voltage-gated potassium ion channel

Based on a homology model of the Kv1.3 potassium channel, the recognitions of the six scorpion toxins, viz. agitoxin2, charybdotoxin, kaliotoxin, margatoxin, noxiustoxin, and Pandinus toxin, to the human Kv1.3 potassium channel have been investigated by using an approach of the Brownian dynamics (BD) simulation integrating molecular dynamics (MD) simulation. Reasonable three-dimensional structures of the toxin-channel complexes have been obtained employing BD simulations and triplet contact analyses. All of the available structures of the six scorpion toxins in the Research Collaboratory for Structural Bioinformatics Protein Data Bank determined by NMR were considered during the simulation, which indicated that the conformations of the toxin significantly affect both the molecular recognition and binding energy between the two proteins. BD simulations predicted that all the six scorpion toxins in this study use their beta-sheets to bind to the extracellular entryway of the Kv1.3 channel, which is in line with the primary clues from the electrostatic interaction calculations and mutagenesis results. Additionally, the electrostatic interaction energies between the toxins and Kv1.3 channel correlate well with the binding affinities (-logK(d)s), R(2) = 0.603, suggesting that the electrostatic interaction is a dominant component for toxin-channel binding specificity. Most importantly, recognition residues and interaction contacts for the binding were identified. Lys-27 or Lys-28, residues Arg-24 or Arg-25 in the separate six toxins, and residues Tyr-400, Asp-402, His-404, Asp-386, and Gly-380 in each subunit of the Kv1.3 potassium channel, are the key residues for the toxin-channel recognitions. This is in agreement with the mutation results. MD simulations lasting 5 ns for the individual proteins and the toxin-channel complexes in a solvated lipid bilayer environment confirmed that the toxins are flexible and the channel is not flexible in the binding. The consistency between the results of the simulations and the experimental data indicated that our three-dimensional models of the toxin-channel complex are reasonable and can be used as a guide for future biological studies, such as the rational design of the blocking agents of the Kv1.3 channel and mutagenesis in both toxins and the Kv1.3 channel. Moreover, the simulation result demonstrates that the electrostatic interaction energies combined with the distribution frequencies from BD simulations might be used as criteria in ranking the binding configuration of a scorpion toxin to the Kv1.3 channel.     

On the dependence of molecular conformation on the type of solvent environment: a molecular dynamics study of cyclosporin A

The dependence of the conformation of cyclosporin A (CPA), a cyclic undecapeptide with potent immunosuppressive activity, on the type of solvent environment is examined using the computer simulation method of molecular dynamics (MD). Conformational and dynamic properties of CPA in aqueous solution are obtained from MD simulations of a CPA molecule dissolved in a box with water molecules. Corresponding properties of CPA in apolar solution are obtained from MD simulations of CPA in a box with carbontetrachloride. The results of these simulations in H2O and in CCl4 are compared to each other and to those of previous simulations of crystalline CPA and of an isolated CPA molecule. The conformation of the backbone of the cyclic polypeptide is basically independent of the type of solvent. In aqueous solution the beta-pleated sheet is slightly weaker and the gamma-turn is a bit less pronounced than in apolar solution. Side chains may adopt different conformations in different solvents. In apolar solution the hydrophobic side chain of the MeBmt residue is in an extended conformation with its hydroxyl group hydrogen bonded to the backbone carbonyl group. In aqueous solution this hydrophobic side chain folds over the core of the molecule and the mentioned hydrogen bond is broken in favor of hydrogen bonding to water molecules. The conformation obtained from the MD simulation in CCl4 nicely agrees with experimental atom-atom distance data as obtained from nmr experiments in chloroform. In aqueous solution the relaxation of atomic motion tends to be slower than in apolar solution.     

The accessibilities of histones in nucleosome cores to an arginine-specific protease

Submaxillaris protease, reportedly specific for arginine residues, was used to probe accessible arginines of chicken erythrocyte nucleosome cores. The relative rates of digestion of histones in nucleosome cores by this protease were H3 greater than H2b greater than H4 much greater than H2a. At most, 8 of 52 total arginines among the four core histones were reasonably accessible to attack. Sites most rapidly cleaved were Arg-26 in H3, a site approximately 13 residues from the NH2 terminus of H2b, and Lys-12 or Arg-17 in H4. Five sites attacked more slowly were Arg-8, -128, or -129 and Arg-49, -52, or -53 in H3; Arg-3 and Arg-17 or -19 in H4; and a site near one terminus of H2b or H3. H2a and fragments resulting from the above cleavages were highly resistant to attack, even after prolonged incubation. Similar results were obtained upon digestion of histones in intact chromatin. H1 and H5 in whole chromatin were attacked at rates comparable to H3. Seven of the eight accessible sites lie outside of structure-forming histone sequences, i.e. sequences that are immobilized in histone complexes, indicating that these sequences are inaccessible in nucleosome cores. The single exceptional site noted, approximately 50 residues from the NH2 terminus of H3, is consistent with previous observations that Glu-51 and Glu-60 of H3 in nucleosomes are accessible to attack by S. aureus protease (Rill, R. L., and Oosterhof, D. K. (1981). J. Biol. Chem. 256, 12687-12691). The relationships of these protease accessibilities to NMR assignments of mobile histone tails in nucleosome cores are discussed.     

Substrate discrimination by formamidopyrimidine-DNA glycosylase: a mutational analysis

Formamidopyrimidine-DNA glycosylase (Fpg) is a primary participant in the repair of 8-oxoguanine, an abundant oxidative DNA lesion. Although the structure of Fpg has been established, amino acid residues that define damage recognition have not been identified. We have combined molecular dynamics and bioinformatics approaches to address this issue. Site-specific mutagenesis coupled with enzyme kinetics was used to test our predictions. On the basis of molecular dynamics simulations, Lys-217 was predicted to interact with the O8 of extrahelical 8-oxoguanine accommodated in the binding pocket. Consistent with our computational studies, mutation of Lys-217 selectively reduced the ability of Fpg to excise 8-oxoguanine from DNA. Dihydrouracil, also a substrate for Fpg, served as a nonspecific control. Other residues involved in damage recognition (His-89, Arg-108, and Arg-109) were identified by combined conservation/structure analysis. Arg-108, which forms two hydrogen bonds with cytosine in Fpg-DNA, is a major determinant of opposite-base specificity. Mutation of this residue reduced excision of 8-oxoguanine from thermally unstable mispairs with guanine or thymine, while excision from the stable cytosine and adenine base pairs was less affected. Mutation of His-89 selectively diminished the rate of excision of 8-oxoguanine, whereas mutation of Arg-109 nearly abolished binding of Fpg to damaged DNA. Taken together, these results suggest that His-89 and Arg-109 form part of a reading head, a structural feature used by the enzyme to scan DNA for damage. His-89 and Lys-217 help determine the specificity of Fpg in recognizing the oxidatively damaged base, while Arg-108 provides specificity for bases positioned opposite the lesion.