Effects of dimerization of Serratia marcescens endonuclease on water dynamics

The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.     

The role of flexibility and hydration on the sequence-specific DNA recognition by the Tn916 integrase protein: a molecular dynamics analysis

The N-terminal domain of the Tn916 integrase protein (INT-DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site-specific association is facilitated by numerous protein-DNA contacts from the face of a three-stranded beta-sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein-DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non-polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein-DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT-DBD-DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site-specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA-bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein-DNA contacts. The protein-DNA hydrogen-bonding network is highly dynamic. The simulation identifies protein-DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the 'wet' protein-DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT-DBD alone and in complex with DNA.     

Molecular dynamics simulations of aptamer-binding reveal generalized allostery in thrombin

Thrombin is an attractive target for antithrombotic therapy due to its central role in thrombosis and hemostasis as well as its role in inducing tumor growth, metastasis, and tumor invasion. The thrombin-binding DNA aptamer (TBA), is under investigation for anticoagulant drugs. Although aptamer binding experiments have been revealed various effects on thrombin’s enzymatic activities, the detailed picture of the thrombin’s allostery from TBA binding is still unclear. To investigate thrombin’s response to the aptamer-binding at the molecular level, we compare the mechanical properties and free energy landscapes of the free and aptamer-bound thrombin using microsecond-scale all-atom GPU-based molecular dynamics simulations. Our calculations on residue fluctuations and coupling illustrate the allosteric effects of aptamer-binding at the atomic level, highlighting the exosite II, 60s, γ and the sodium loops, and the alpha helix region in the light chains involved in the allosteric changes. This level of details clarifies the mechanisms of previous experimentally demonstrated phenomena, and provides a prediction of the reduced autolysis rate after aptamer-binding. The shifts in thrombin’s ensemble of conformations and free energy surfaces after aptamer-binding demonstrate that the presence of bound-aptamer restricts the conformational freedom of thrombin suggesting that conformational selection, i.e. generalized allostery, is the dominant mechanism of thrombin-aptamer binding. The profound perturbation on thrombin’s mechanical and thermodynamic properties due to the aptamer-binding, which was revealed comprehensively as a generalized allostery in this work, may be exploited in further drug discovery and development.     

HLA-DP2 binding prediction by molecular dynamics simulations

Major histocompatibility complex (MHC) II proteins bind peptide fragments derived from pathogen antigens and present them at the cell surface for recognition by T cells. MHC proteins are divided into Class I and Class II. Human MHC Class II alleles are grouped into three loci: HLA-DP, HLA-DQ, and HLA-DR. They are involved in many autoimmune diseases. In contrast to HLA-DR and HLA-DQ proteins, the X-ray structure of the HLA-DP2 protein has been solved quite recently. In this study, we have used structure-based molecular dynamics simulation to derive a tool for rapid and accurate virtual screening for the prediction of HLA-DP2-peptide binding. A combinatorial library of 247 peptides was built using the "single amino acid substitution" approach and docked into the HLA-DP2 binding site. The complexes were simulated for 1 ns and the short range interaction energies (Lennard-Jones and Coulumb) were used as binding scores after normalization. The normalized values were collected into quantitative matrices (QMs) and their predictive abilities were validated on a large external test set. The validation shows that the best performing QM consisted of Lennard-Jones energies normalized over all positions for anchor residues only plus cross terms between anchor-residues.     

Specific DNA recognition by the Antp homeodomain: MD simulations of specific and nonspecific complexes

Four molecular dynamics simulation trajectories of complexes between the wild-type or a mutant Antennapedia homeodomain and 2 DNA sequences were generated in order to probe the mechanisms governing the specificity of DNA recognition. The starting point was published affinity measurements showing that a single protein mutation combined with a replacement of 2 base pairs yields a new high-affinity complex, whereas the other combinations, with changes on only 1 macromolecule, exhibited lower affinity. The simulations of the 4 complexes yielded fluctuating networks of interaction. On average, these networks differ significantly, explaining the switch of affinity caused by the alterations in the macromolecules. The network of mostly hydrogen-bonding interactions involving several water molecules, which was suggested both by X-ray and NMR structures of the wild-type homeodomain and its DNA operator sequence, could be reproduced in the trajectory. More interestingly, the high-affinity complex with alterations in both the protein and the DNA yielded again a dynamic but very tight network of intermolecular interactions, however, attributing a significantly stronger role to direct hydrophobic interactions at the expense of water bridges. The other 2 homeodomain-DNA complexes, with only 1 molecule altered, show on average over the trajectories a clearly reduced number of protein-DNA interactions. The observations from these simulations suggest specific experiments and thus close the circle formed by biochemical, structural, and computational studies. The shift from a water-dominated to a more "dry" interface may prove important in the design of proteins binding DNA in a specific manner.     

Molecular dynamics simulations to understand LRKK2 mutations in Parkinson

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of Parkinson's disease (PD). LRRK2 contains a Ras of complex proteins (ROC) domain that may acts as a GTPase to regulate its protein kinase activity. Here, we performed 10 ns molecular dynamics simulations on LRRK2 Apo, complex with GDP and mutations (R1441C, R1441G and R1441H). Our results strongly suggest that the formations of helix in L1 and its pliable plays a major role in the LRRK2 functions.     

登革病毒2型E蛋白基因真核表达和DNA免疫的研究

将编码登革病毒2型(DV2)氨基末端80％的E蛋白的DNA片段克隆到真核表达载体pCXN2 AG强启动子下游,构建成DV2E重组真核表达质粒pCXN-E。间接免疫荧光显示其可在COS-7细胞中表达。ELISA法检测pCXN2-E DNA免疫BALB/c鼠血清中的E抗体变化和维持规律,结果显示三次免疫后2周已有抗体产生,15周时仍维持较高的水平;血清空斑减数中和实验显示其中和滴度高于1:640;流式细胞计数仪(FACS)检测DNA免疫鼠CD4~+、CD8~+T淋巴细胞变化情况,与注射空载体pCXN2的阴性鼠相比,CD4~+淋巴细胞水平略有上升。CD8~+细胞水平有较大升高(p<0.01);动物保护性实验结果显示,当用致死剂量登革病毒攻击免疫鼠时,其保护率为60％。以上结果表明:pCXN2-E在实验动物内表达出的DV2E蛋白可以诱导免疫动物的体液免疫和细胞免疫应答,尤其是MHC-I限制性杀伤性CD8~+T淋巴细胞水平的提高对清除病毒是十分有利的。因此,DV2 E DNA免疫为登革病毒DNA疫苗的发展进行了有益的探索。     

Effect of DNA on the conformational dynamics of the endonucleases I‐DmoI as provided by molecular dynamics simulations

The conformational behavior of the wild‐type endonucleases I‐DmoI and two of its mutants has been studied in the presence and in the absence of DNA target sequences by means of extended molecular dynamics simulations. Our results show that in the absence of DNA, the three protein forms explore a similar essential conformational space, whereas when bound to the same DNA target sequence of 25 base pairs, they diversify and restrain the subspace explored. In addition, the differences in the essential subspaces explored by the residues near the catalytic site for both the bound and unbound forms are discussed in background of the experimental protein activity.     

登革病毒2型E蛋白基因真核表达和DNA免疫的研究

将编码登革病毒2型(DV2)氨基末端80%的E蛋白的DNA片段克隆到真核表达载体pCXN2 AG强启动子下游,构建成DV2E重组真核表达质粒pCXN-E.间接免疫荧光显示其可在COS-7细胞中表达.ELISA法检测pCXN2-E DNA免疫BALB/c鼠血清中的E抗体变化和维持规律,结果显示三次免疫后2周已有抗体产生,15周时仍维持较高的水平;血清空斑减数中和实验显示其中和滴度高于1640;流式细胞计数仪(FACS)检测DNA免疫鼠CD4+、CD8+T淋巴细胞变化情况,与注射空载体pCXN2的阴性鼠相比,CD4+淋巴细胞水平略有上升,CD8+细胞水平有较大升高(p＜0.01);动物保护性实验结果显示,当用致死剂量登革病毒攻击免疫鼠时,其保护率为60%.以上结果表明pCXN2-E在实验动物内表达出的DV2E蛋白可以诱导免疫动物的体液免疫和细胞免疫应答,尤其是MHC-Ⅰ限制性杀伤性CD8+T淋巴细胞水平的提高对清除病毒是十分有利的.因此,DV2 E DNA免疫为登革病毒DNA疫苗的发展进行了有益的探索.     

Molecular dynamics study of the membrane interaction of a membranotropic dengue virus C protein-derived peptide

Dengue virus C protein, essential in the dengue virus life cycle, possesses a segment, peptide PepC, known to bind membranes composed of negatively charged phospholipids. To characterize its interaction with the membrane, we have used the molecular dynamics HMMM membrane model system. This approach is capable of achieving a stable system and sampling the peptide/lipid interactions which determine the orientation and insertion of the peptide upon membrane binding. We have been able to demonstrate spontaneous binding of PepC to the 1,2-divaleryl-sn-glycero-3-phosphate/1,2-divaleryl-sn-glycero-3-phosphocholine membrane model system, whereas no binding was observed at all for the 1,2-divaleryl-sn-glycero-3-phosphocholine one. PepC, adopting an α-helix profile, did not insert into the membrane but did bind to its surface through a charge anchor formed by its three positively charged residues. PepC, maintaining its three-dimensional structure along the whole simulation, presented a nearly parallel orientation with respect to the membrane when bound to it. The positively charged amino acid residues Arg-2, Lys-6, and Arg-16 are mainly responsible for the peptide binding to the membrane stabilizing the structure of the bound peptide. The segment of dengue virus C protein where PepC resides is a fundamental protein–membrane interface which might control protein/membrane interaction, and its positive amino acids are responsible for membrane binding defining its specific location in the bound state. These data should help in our understanding of the molecular mechanism of DENV life cycle as well as making possible the future development of potent inhibitor molecules, which target dengue virus C protein structures involved in membrane binding.     

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and functionality in human Group IIA phospholipase A2

Human Group IIA phospholipase A₂ (hGIIA) promotes inflammation in immune‐mediated pathologies by regulating the arachidonic acid pathway through both catalysis‐dependent and ‐independent mechanisms. The hGIIA crystal structure, both alone and inhibitor‐bound, together with structures of closely related snake‐venom‐derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis‐independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A₂ of Daboia russelli pulchella (svPLA₂). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827–842. © 2016 Wiley Periodicals, Inc.     

Molecular beacons for detecting DNA binding proteins: mechanism of action

New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.     

Molecular dynamics simulations of excimer forming (+)-anti-BPDE-DNA adducts in aqueous solution

The chemical carcinogen (+)-anti BPDE preferentially binds covalently to the guanine base in the minor groove of DNA. Fluorescence spectroscopic studies have shown that the BPDE molecules bound to DNA can interact in their photo-excited state giving strong excimer fluorescence when bound to poly(dGdC) · poly(dGdC). It was suggested that the formation of such excited state complexes is most probable when the two (+)-anti-BPDE bind to guanines of adjacent base pairs on the two different strands of the DNA. In the present work a model for such an excimer forming DNA-BPDE double adduct system has been constructed and shown to be stable over a 300 ps molecular dynamics simulation in a water box. The model is a d(CG)₃ · d(CG)₃ molecule with two BPDE molecules bound to the guanines at the 4th position on each strand, located in the minor groove and each oriented towards the 5 end of the modified strand, respectively. The results of 300 ps MD simulation show that the two BPDE chromophores exhibited on the average a relative geometry favourable for excimer formation. The local structure at the adduct position was considerably distorted and the helix axis was bent. The modified bases were found to be paired through a stable single non-Watson Crick type of hydrogen bond. Correspondence to: A. Gräslund     

An NMR-based molecular dynamics simulation of the interaction of the lac repressor headpiece and its operator in aqueous solution

The results of a 125 psec molecular dynamics simulation of a lac headpiece-operator complex in aqueous solution are reported. The complex satisfies essentially all experimental distance information derived from two-dimensional nuclear magnetic resonance (2-D-NMR) studies. The interaction between lac repressor headpiece and its operator is based on many direct- and water-mediated hydrogen bonds and nonpolar contacts which allow the formation of a tight complex. No stable hydrogen bonds between side chains and bases are found, while specific contacts occur between both nonpolar groups and, to a lesser extent, through water-mediated hydrogen bonds. The simulated complex structure in water is intrinsically stable without application of nuclear Overhauser effect (NOE) distance restraints, while being compatible with most of the available biochemical, genetic, and chemically induced dynamic nuclear polarization (CIDNP) data.     

Molecular dynamics simulations of ethanol binding to the transmembrane domain of the glycine receptor: implications for the channel potentiation mechanism

The glycine receptor (GlyR) is potentiated by ethanol and other anesthetics. The potentiation mechanism at the molecular level is unknown and remains elusive, but mutagenic studies have shown that ethanol and other volatile anesthetics bind to a pocket between TM1, TM2, and TM3. The present study extends previous studies (Cheng et al., Proteins 2007;68:581-593) wherein we conducted homology modeling and molecular dynamics (MD) simulations to construct models of the homopentameric alpha1 subunits of the GlyR transmembrane domain in open and closed states. To understand the potentiation of GlyR by ethanol we compare the binding of ethanol molecules to the channel in these different states. We observe that ethanol stably resides inside solvent-accessible cavities found in the open state of GlyR that are formed by I229 (of TM1) in one subunit and S267 and A288 (of TM2 and TM3, respectively) in the adjacent subunit. The volume of these putative binding pockets is state-dependent. Selective binding to the open states of receptors has been proposed to explain the potentiating actions of this class of anesthetics. In accordance with this model, our MD simulations suggest that the potentiation of ethanol on GlyR may be effected through preferential binding of ethanol molecules to an inter-subunit binding pocket in the open state.     

HPV-2E2蛋白(A338V)突变体对E2蛋白DNA结合能力增强的研究

HPV-2是引起皮肤寻常疣的常见HPV型别,病毒E2蛋白可抑制病毒早期启动子的活性。我们曾经报道来自一例巨大寻常疣患者的HPV-2突变E2蛋白对病毒早期启动子活性的抑制作用明显减弱,该E2蛋白在其C末端的DNA结合区域带有A338V的点突变。本研究利用原核表达系统表达纯化了突变E2(A338V)和HPV-2原毒株的羧基端和全长蛋白。电泳迁移率实验结果显示,E2蛋白可与带有E2蛋白特异性结合位点的寡核苷酸探针形成复合物,突变E2蛋白比原毒株E2蛋白的DNA结合能力强。这提示DNA结合能力的增强可能为E2蛋白对病毒启动子活性影响的分子基础,与患者出现罕见巨大寻常疣这一临床表型关联。