Introducing folding stability into the score function for computational design of RNA‐binding peptides boosts the probability of success

A computational strategy that integrates our peptide search algorithm with atomistic molecular dynamics simulation was used to design rational peptide drugs that recognize and bind to the anticodon stem and loop domain (ASL^Lys3) of human for the purpose of interrupting HIV replication. The score function of the search algorithm was improved by adding a peptide stability term weighted by an adjustable factor λ to the peptide binding free energy. The five best peptide sequences associated with five different values of λ were determined using the search algorithm and then input in atomistic simulations to examine the stability of the peptides' folded conformations and their ability to bind to ASL^Lys3. Simulation results demonstrated that setting an intermediate value of λ achieves a good balance between optimizing the peptide's binding ability and stabilizing its folded conformation during the sequence evolution process, and hence leads to optimal binding to the target ASL^Lys3. Thus, addition of a peptide stability term significantly improves the success rate for our peptide design search. Proteins 2016; 84:700–711. © 2016 Wiley Periodicals, Inc.     

HCV NS3/4A蛋白酶与Faldaprevir类似物的分子识别机制及其复合物运动模式

Faldaprevir类似物(Faldaprevir analogue molecule,FAM)能有效抑制HCV NS3/4A蛋白酶的催化活性,是一种潜在抗HCV先导化合物。通过生物信息学统计分析了已报道的HCV NS3/4A蛋白酶晶体结构,得到了FAM-HCV NS3/4A蛋白酶晶体结构。对FAM-HCV NS3/4A蛋白酶复合物进行了20.4 ns的分子动力学模拟,重点从氢键和结合自由能两个角度分析了二者分子识别中的关键残基及结合驱动力。氢键和范德华力是促使FAM特异性结合到蛋白V132?S139、F154?D168、D79?D81和V55的活性口袋中的主要驱动力,这与实验数据较为吻合。耐药性突变实验分析了R155K、D168E/V和V170T定点突变对FAM分子识别的影响,为可能存在的FAM耐药性提供了分子依据。最后,用自由能曲面和构象聚类两个方法探讨了体系的构象变化,给出体系的4种优势构象,为后续的基于HCV NS3/4A蛋白酶结构的Faldaprevir类似物抑制剂分子设计提供一定的理论帮助。     

3D-QSAR and molecular recognition of Klebsiella pneumoniae NDM-1 inhibitors

New Delhi metallo-β-lactamase-1 (NDM-1) as a target for the development of anti-superbug agents, plays an important role in the resistance of β-lactam antibiotics and has received worldwide attention. Sulfhydryl propionic acid derivatives can effectively inhibit the catalytic activity of NDM-1, but the quantitative structure–activity relationship (QSAR) and inhibitor-target recognition mechanism both remain unclear. In this work, CoMFA and CoMSIA models of sulfhydryl propionic acid inhibitors with high predictive ability were obtained, from which the effect of different substituents on the inhibitory activity against NDM-1 were revealed at the molecular level. Then, two 120-nanosecond comparative molecular dynamics (MD) simulations for NDM-1 enzyme and NDM-1-inhibitor complex systems were performed to study the recognition and inhibition mechanism of sulfhydryl propionic acid derivatives. The binding of inhibitors alters the entire H-bond network of the NDM-1 system accompanied by the formation of strong interactions with I35, W93, H120, H122, D124, H189 and H250, further weakens the recognition of NDM-1 by its endogenic substrates. Finally, the results of free energy landscape and conformation cluster analyses show that NDM-1 underwent a significant conformational change after the association with sulfhydryl propionic acid inhibitors. Our findings can provide theoretical support and help for anti-superbug agents design based on the structures of NDM-1 and sulfhydryl propionic acid derivatives.     

Molecular mechanism study of several inhibitors binding to BRD9 bromodomain based on molecular simulations

Bromodomain-containing protein 9 (BRD9) has been employed as a potential target for anticancer drugs in recent years. In this work, molecular docking, molecular dynamics (MD) simulations, binding free energy calculations, and per residue energy decomposition approaches were performed to elucidate the different binding modes between four pyridinone-like scaffold inhibitors and BRD9 bromodomain. Analysis results indicate that non-polar contribution mainly deriving from van der Waals energy is a critical impact on binding affinity of inhibitors against BRD9. Some key residues Phe44, Phe47, Val49, and Ile53 (at ZA loop) enhance the binding energy of inhibitors in BRD9 by means of providing hydrophobic interactions. Moreover, it is observed that BRD9 is anchored by the formation of a stable hydrogen bond between the carbonyl of the inhibitors and the residue Asn100 (at BC loop), and a strong π–π stacking interaction formed between the residue Tyr106 (at BC loop) and the inhibitors. The existence of dimethoxyphenyl structure and the aromatic ring merged to pyridinone scaffold are useful to enhance the BRD9 binding affinity. These findings should guide the rational design of more prospective inhibitors targeting BRD9.

Communicated by Ramaswamy H. Sarma     

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2‐3)Gal: a study by in silico mutations and molecular dynamics simulations

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM‐PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.     

Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.     

Functional roles of magnesium binding to extracellular signal-regulated kinase 2 explored by molecular dynamics simulations and principal component analysis

Molecular dynamics (MD) simulations coupled with principal component (PC) analysis were carried out to study functional roles of Mg²⁺ binding to extracellular signal-regulated kinase 2 (ERK2). The results suggest that Mg²⁺ binding heavily decreases eigenvalue of the first principal component and totally inhibits motion strength of ERK2, which favors stabilization of ERK2 structure. Binding free energy predictions indicate that Mg²⁺ binding produces an important effect on binding ability of adenosine triphosphate (ATP) to ERK2 and strengthens the ATP binding. The calculations of residue-based free energy decomposition show that lack of Mg²⁺ weakens interactions between the hydrophobic rings of ATP and five residues I29, V37, A50, L105, and L154. Hydrogen bond analyses also prove that Mg²⁺ binding increases occupancies of hydrogen bonds formed between ATP and residues K52, Q103, D104, and M106. We expect that this study can provide a significant theoretical hint for designs of anticancer drugs targeting ERK2.     

Molecular insights into the binding selectivity of a synthetic ligand DAAG to Fc fragment of IgG

Affinity chromatography with synthetic ligands has been focused as the potential alternative to protein A‐based chromatography for antibody capture because of its comparable selectivity and efficiency. Better understanding on the molecular interactions between synthetic ligand and antibody is crucial for improving and designing novel ligands. In this work, the molecular interaction mechanism between Fc fragment of IgG and a synthetic ligand (DAAG) was studied with molecular docking and dynamics simulation. The docking results on the consensus binding site (CBS) indicated that DAAG could bind to the CBS with the favorable orientation like a tripod for the top‐ranked binding complexes. The ligand‐Fc fragment complexes were then tested by molecular dynamics simulation at neutral condition (pH 7.0) for 10 ns. The results indicated that the binding of DAAG on the CBS of Fc fragment was achieved by the multimodal interactions, combining the hydrophobic interaction, electrostatic interaction, hydrogen bond, and so on. It was also found that multiple secondary interactions endowed DAAG with an excellent selectivity to Fc fragment. In addition, molecular dynamics simulation conducted at acidic condition (pH 3.0) showed that the departure of DAAG ligand from the surface of Fc fragment was the result of reduced interaction energies. The binding modes between DAAG and CBS not only shed light on the molecular mechanisms of DAAG for antibody purification but also provide useful information for the improvement of ligand design. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular dynamics study of segment peptides of Bax,Bim, and Mcl-1 BH3 domain of the apoptosis-regulating proteins bound to the anti-apoptotic Mcl-1 protein

Mcl-1 has emerged as a potential therapeutic target in the treatment of several malignancies. Peptides representing BH3 region of pro-apoptotic proteins have been shown to bind the hydrophobic cleft of anti-apoptotic Mcl-1 and this segment is responsible for modulating the apoptotic pathways in living cells. Understanding the molecular basis of protein–peptide interaction is required to develop potent inhibitors specific for Mcl-1. Molecular dynamics simulations were performed for Mcl-1 in complex with three different BH3 peptides derived from Mcl-1, Bax, and Bim. Accordingly, the calculated binding free energies using MM-PBSA method are obtained and comparison with the experimentally determined binding free energies is made. The interactions involving two conserved charged residues (Aspi, and Arg/Lysi-4) and three upstream conserved hydrophobic residues (Leui-5, Ile/Vali-2, and Glyi-1, respectively) of BH3 peptides play the important roles in the structural stability of the complexes. The calculated results exhibit that the interactions of Bim BH3 peptides to Mcl-1 is stronger than the complex with Bax 19BH3 peptides. The hydrophobic residues (position i???9, i???8 and i?+?2) of BH3 peptides can be involved in their inhibitory specificity. The calculated results can be used for designing more effective MCL-1 inhibitors.     

A critical role of water in the specific cleavage of the anticodon loop of some eukaryotic methionine initiator tRNAs

We have noticed that during a long storage and handling, the plant methionine initiator tRNA is spontaneously hydrolyzed within the anticodon loop at the C34-A35 phosphodiester bond. A literature search indicated that there is also the case for human initiator tRNA^Met but not for yeast tRNA^Met _i or E. coli tRNA^Met _f. All these tRNAs have an identical nucleotide sequence of the anticodon stems and loops with only one difference at position 33 within the loop. It means that cytosine 33 (C33) makes the anticodon loop of plant and human tRNA^Met _i susceptible to the specific cleavage reaction. Using crystallographic data of tRNA^Met _f of E. coli with U33, we modeled the anticodon loop of this tRNA with C33. We found that C33 within the anticodon loop creates a pocket that can accomodate a hydrogen bonded water molecule that acts as a general base and catalyzes a hydrolysis of C-A bond. We conclude that a single nucleotide change in the primary structure of tRNA^Met _i made changes in hydration pattern and readjustment in hydrogen bonding which lead to a cleavage of the phosphodiester bond.     

Hierarchy of simulation models in predicting molecular recognition mechanisms from the binding energy landscapes: structural analysis of the peptide complexes with SH2 domains.

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature-dependent binding free energy landscapes. Once some native peptide-SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel-like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide-SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C-terminal to the pTyr group. The topological features of the peptide-protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino-terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy-terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide-SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region.     

Molecular determinants of the binding specificity of BH3 ligands to BclXL apoptotic repressor

B‐cell lymphoma extra‐large protein (BclXL) serves as an apoptotic repressor by virtue of its ability to recognize and bind to BH3 domains found within a diverse array of proapoptotic regulators. Herein, we investigate the molecular basis of the specificity of the binding of proapoptotic BH3 ligands to BclXL. Our data reveal that while the BH3 ligands harboring the LXXX[A/S]D and [R/Q]XLXXXGD motif bind to BclXL with high affinity in the submicromolar range, those with the LXXXGD motif afford weak interactions. This suggests that the presence of a glycine at the fourth position (G+4)—relative to the N‐terminal leucine (L0) within the LXXXGD motif—mitigates binding, unless the LXXXGD motif also contains arginine/glutamine at the ?2 position. Of particular note is the observation that the residues at the +4 and ?2 positions within the LXXX[A/S]D and [R/Q]XLXXXGD motifs appear to be energetically coupled—replacement of either [A/S]+4 or [R/Q]‐2 with other residues has little bearing on the binding affinity of BH3 ligands harboring one of these motifs. Collectively, our study lends new molecular insights into understanding the binding specificity of BH3 ligands to BclXL with important consequences on the design of novel anticancer drugs. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 573–582, 2014.     

Reducing CDK4/6-p16(INK4a) interface. Computational alanine scanning of a peptide bound to CDK6 protein

The tumor suppressor gene p16INK4a is commonly found altered in numerous and different types of cancer. The encoded protein arrests cell cycle in G1 phase by binding to CDK4 and CDK6, inhibiting their kinase function. In 1995, a 20-residue peptide, extracted from p16INK4a protein sequence, was discovered that retains the cell cycle inhibition properties of the endogenous tumor suppressor. However, its structure has not been determined yet. In this article, the features of a theoretical structure of the peptide bound to CDK6 are reported. The complex was modeled from CDK6-p16INK4a X-ray crystal structure and through molecular dynamics. Final structure was assessed by comparing computed binding free energy changes, when single-alanine substitutions were brought about on the peptide, to experimental data. Better concordance was obtained when including a high level of solvation effects. Solute-solvent vdW energy and electrostatic energy between solute and first shells of water, computed through a force field and considering explicit waters, were also to be included to achieve reasonably good concordance between theoretical and experimental data.     

NaV1.2 EFL domain allosterically enhances Ca2+ binding to sites I and II of WT and pathogenic calmodulin mutants bound to the channel CTD

1. Download : Download high-res image (287KB)
2. Download : Download full-size image