抑制剂BD2对PKA与PKC βII的抑制选择性研究

蛋白激酶A (PKA)和蛋白激酶C (PKC)的过度表达导致细胞生长分化异常, 是治疗肿瘤的潜在靶点. 抑制剂BD2对PKA和PKC抑制作用存在高选择性. 为了探讨BD2高选择性机制, 本工作以PKA与BD2复合物的晶体结构为模板, 通过同源模建结合分子对接的方法构建PKC βII与BD2复合物的结构, 并对PKA-BD2复合物和PKC-BD2复合物进行了2.5 ns的分子动力学模拟, 运用MM-GBSA方法计算了结合自由能, 通过能量分解的方法考察PKA和PKC的主要残基与BD2之间的相互作用和识别机制. 结合能分析结果很好地描述了BD2对PKA抑制活性比其对PKC抑制活性高这一实验现象. 氢键分析和能量分解结果共同说明了BD2的B环和酰胺链部分与PKA和PKC中相应位点的残基之间的相互作用存在差异, 这是BD2存在选择性的内在因素. BD2高选择性作用机制的阐明为进一步基于结构的balanol类抑制剂的结构设计和优化提供了合理的指导.     

抑制剂BD2对PKA与PKCβⅡ的抑制选择性研究

蛋白激酶A(PKA)和蛋白激酶C(PKC)的过度表达导致细胞生长分化异常,是治疗肿瘤的潜在靶点.抑制剂BD2对PKA和PKC抑制作用存在高选择性.为了探讨BD2高选择性机制,本工作以PKA与BD2复合物的晶体结构为模板,通过同源模建结合分子对接的方法构建PKC βⅡ与BD2复合物的结构,并对PKA-BD2复合物和PKC-BD2复合物进行了2.5 ns的分子动力学模拟,运用MM-GBSA方法计算了结合自由能,通过能量分解的方法考察PKA和PKC的主要残基与BD2之间的相互作用和识别机制.结合能分析结果很好地描述了BD2对PKA抑制活性比其对PKC抑制活性高这一实验现象.氢键分析和能量分解结果共同说明了BD2的B环和酰胺链部分与PKA和PKC中相应位点的残基之间的相互作用存在差异,这是BD2存在选择性的内在因素.BD2高选择性作用机制的阐明为进一步基于结构的balanol类抑制剂的结构设计和优化提供了合理的指导.     

人类丝氨酸消旋酶的同源模建及其与多肽类抑制剂的分子对接

利用同源模建和分子动力学模拟方法构建了人类丝氨酸消旋酶(hSR)的三维结构, 并利用profile-3D和procheck方法评估了模型的可靠性. 在此基础上用分子对接程序(affinity)将多肽类抑制剂A和B分别与hSR进行对接, 获得了其复合物结构的理论模型. 通过配体与受体之间相互作用能和结构分析给出了此类抑制剂与hSR的具体结合方式, 明确了hSR与此类抑制剂结合时起重要作用的氨基酸残基, 为基于人类丝氨酸消旋酶三维结构的药物设计提供重要的参考信息.     

2,3,3'-三氯联苯与人血清白蛋白之间相互作用的计算模拟及光谱研究

利用分子对接、分子动力学模拟、荧光光谱、紫外光谱及同步荧光光谱法研究了2,3,3'-三氯联苯(PCB-20)与人血清白蛋白(HSA)的相互作用。分子对接结果表明,PCB-20与HSA通过疏水作用力稳定结合于HSA的疏水空腔内。光谱法实验结果表明,PCB-20通过与HSA形成HSA-PCB20复合物从而对HSA具有荧光猝灭作用,猝灭原因是静态猝灭和非辐射能量转移,热力学参数也表明两者结合的主要驱动力为疏水作用力,计算模拟与实验结果吻合度较高。分子动力学模拟结果表明,PCB-20能够与HSA稳定结合,且与同步荧光光谱实验共同证明其对HSA的构象变化产生了一定影响。     

双金属存在下整合酶和抑制剂5CITEP的分子对接研究

在HIV-1整合酶(IN)和5CITEP复合物晶体结构的基础上, 用分子对接程序(Affinity)将含有单Mg2+和双Mg2+ 的HIV-1 IN核心区与抑制剂5CITEP进行对接, 获得了能形成复合物结构的理论模型. 通过配体与受体之间的相互作用能和结构分析给出此种抑制剂的结合模式, 并与晶体结构进行比较, 揭示出引入的第二个Mg2+原子在整合过程中所起的重要作用. 前后相互作用能的变化趋势很明显, 配体和受体的作用模式比单Mg2+体系更加清晰. 由单Mg2+体系的4种作用方式改变到双Mg2+体系的两种作用方式, 相互作用能提高了将近40 kJ/mol. 为基于整合酶结构的药物设计提供了参考信息.     

抗原肽与MHC分子相互作用QM/MM分子动力学模拟研究

在MHC I类(major histocompatibility complex class I)分子抗原加工提呈过程中抗原蛋白在抗原提呈细胞(antigen-presenting cells, APC)的胞浆中被蛋白酶体(proteasome)裂解成短肽peptide, 由转运相关蛋白(transporter associated with antigen processing, TAP)将蛋白酶体裂解产生的短肽片段从胞浆转运至内质网腔. 短肽peptide在内质网中与新生成的MHC I类分子结合, 形成peptide-MHC复合体被提呈到APC细胞表面, 与T细胞表面抗原受体(T cell receptor, TCR)特异性识别结合, 使得CTL细胞开始活化、增殖、分化, 进而对肿瘤细胞进行特异性杀伤. 目前对CTL细胞如何识别抗原肽-MHC复合物分子及抗原短肽peptide如何与主要组织相容性复合体MHC分子的相互作用识别结合的机理还不是很清楚. 传统的预测CTL细胞表位的方法没有考虑受体与配体结合过程中电子结构的变化, 电子结构的变化需要用量子力学方法来处理. 本文采用QM/MM多尺度生物大分子的分子动力学模拟方法, 以天然抗原肽TAX (LLFGYPVYVYU)与HLA-A*0201分子结合的晶体结构为模板, 替换抗原肽“锚点”氨基酸, 将口袋氨基酸残基的原子极化电荷在空间形成的静电势用电多极矩分量表示. 用箱线图分析每个口袋氨基酸分子静电势变化和功能, 确定Pocket B的Glu63和Lys66的功能是精细识别氨基酸和一级结合氨基酸, Pocket F的Asp77, Tyr84的功能是精细识别氨基酸, 而Asp77, Lys146是一级结合氨基酸, 表明QM/MM方法在提取抗原肽与MHC I类分子识别结合特异性信息是可行的, 这对了解免疫识别机理和指导肿瘤疫苗的开发都具有指导意义.     

L1 β-Lactamase催化反应机理研究

用混合量子力学和分子力学(QM/MM)方法和密度泛函理论讨论了L1 β-Lactamase催化Nitrocefin水解的过程, 研究结果表明, 反应为多步反应: 第一步亲核进攻反应为反应的决速步骤, 并且伴随着酰胺键的断裂, 第二步反应为质子迁移反应. 同时讨论了金属锌在反应中的作用.     

2,3,3′-三氯联苯与人血清白蛋白之间相互作用的计算模拟及光谱研究

利用分子对接、分子动力学模拟、荧光光谱、紫外光谱及同步荧光光谱法研究了2,3,3′-三氯联苯(PCB-20)与人血清白蛋白(HSA)的相互作用。分子对接结果表明,PCB-20与HSA通过疏水作用力稳定结合于HSA的疏水空腔内。光谱法实验结果表明,PCB-20通过与HSA形成HSA-PCB20复合物从而对HSA具有荧光猝灭作用,猝灭原因是静态猝灭和非辐射能量转移,热力学参数也表明两者结合的主要驱动力为疏水作用力,计算模拟与实验结果吻合度较高。分子动力学模拟结果表明,PCB-20能够与HSA稳定结合,且与同步荧光光谱实验共同证明其对HSA的构象变化产生了一定影响。     

HIV-1整合酶链转移抑制剂的3D-QSAR、分子对接和分子动力学模拟研究

为了获得高活性、结构新颖的整合酶链转移(INST)抑制剂,本文采用Co MFA和Co MSIA两种方法对32个萘啶类INST抑制剂进行了三维定量构效关系研究,并建立了相关模型,其交叉验证系数分别为q~2=0. 809和q~2=0. 816,拟合验证系数分别为r~2=0. 998和r~2=0. 981,表明所建立的模型是可靠的且具有一定的预测能力。利用分子对接探讨小分子化合物与INST蛋白的相互作用模式,结果表明,萘啶类化合物主要通过疏水作用和氢键作用与INSTIs蛋白结合。最后通过分子动力学模拟进一步验证对接结果发现,对接的结合模式与分子动力学模拟得到的结果是一致的。本研究获得的综合模型和推论可以为开发有效的HIV INSTIs提供重要的理论信息。     

酶催化过程的全程模拟

酶催化包括底物到活性区的输运、选择催化化学反应及产物释放等复杂过程,由于复杂的蛋白质环境效应,任一化学和非化学过程都有可能是决定酶活性的关键步骤。为了全面认识酶催化活性,我们对几类酶催化过程进行了广泛的组合量子/分子力学(QM/MM)和经典分子力学(MM)动力学模拟(MD)研究,详细地讨论了整个酶催化过程的分子机制、关键残基的作用和蛋白质环境效应,丰富了对酶催化活性的认识。随着多尺度模型和计算模拟方法的进一步完善与发展,有望实现超大复杂生物酶催化过程的全程模拟研究,为酶工程领域的相关研究提供支持。     

molUP: A VMD plugin to handle QM and ONIOM calculations using the gaussian software

The notable advances obtained by computational (bio)chemistry provided its widespread use in many areas of science, in particular, in the study of reaction mechanisms. These studies involve a huge number of complex calculations, which are often carried out using the Gaussian suite of programs. The preparation of input files and the analysis of the output files are not easy tasks and often involve laborious and complex steps. Taking this into account, we developed molUP: a VMD plugin that offers a complete set of tools that enhance the preparation of QM and ONIOM (QM/MM, QM/QM, and QM/QM/MM) calculations. The starting structures for these calculations can be imported from different chemical formats. A set of tools is available to help the user to examine or modify any geometry parameter. This includes the definition of layers in ONIOM calculations, choosing fixed atoms during geometry optimizations, the recalculation or adjustment of the atomic charges, performing SCANs or IRC calculations, etc. molUP also extracts the geometries from the output files as well as the energies of each of them. All of these tasks are performed in an interactive GUI that is extremely helpful for the user. MolUP was developed to be easy to handle by inexperienced users, but simultaneously to be a fast and flexible graphical interface to allow the advanced users to take full advantage of this plugin. The program is available, free of charges, for macOS, Linux, and Windows at the PortoBioComp page https://www.fc.up.pt/PortoBioComp/database/doku.php?id=molup . © 2018 Wiley Periodicals, Inc.     

锌酶的计算模拟:挑战与最新进展

锌酶在人体中分布非常广泛,种类繁多,是当前最受关注的金属酶之一。由于在锌配位结构上的多样性以及Zn²⁺饱和的d轨道带来的“光谱寂静”性,导致许多实验研究手段受限。计算模拟在锌酶的研究中发挥着越来越重要的作用,已经成为不可或缺的研究工具。现代量子化学计算模拟方法,特别是被视为研究生物大分子体系非常有效的QM/MM组合方法,目前已经被广泛应用于探讨复杂多变的锌配位结构以及锌酶催化反应机理。通过在QM/MM水平下开展的分子动力学模拟,可以揭示锌酶体系中结构与功能间的相互关系。此外,分子力场方法在锌酶研究中同样发挥了不可替代的作用,由于传统力场普遍无法正确描述锌配位结构,因此,锌酶分子力场的开发具有迫切的现实意义。本文总结了近年来锌酶计算模拟领域的最新进展,提出了锌酶计算研究中还有待解决的一些问题。     

Structure and dynamics of La(III) in aqueous solution – An ab initio QM/MM MD approach

Ab initio QM/MM MD simulations have allowed to clarify some of the ambiguities arising from various studies on the hydrated La(III) ion. Both nine- and ten-coordinated hydrates co-exist and interchange in a dissociative process on the nano- or even subnanosecond scale, and thus much faster than any other trivalent main group or transition metal ions. The weak ion–ligand bond (53 N/m) supplies a reasonable explanation for it. The simulation results for La(III) are also compared to those for the isoelectronic ions Cs(I) and Ba(II) obtained by the same ab initio MD procedure, leading to conclusions on the influence of central ion charge on structural and dynamic properties of hydrate complexes.     

Protonation Equilibrium in the Active Site of the Photoactive Yellow Protein

The role and existence of low-barrier hydrogen bonds (LBHBs) in enzymatic and protein activity has been largely debated. An interesting case is that of the photoactive yellow protein (PYP). In this protein, two short HBs adjacent to the chromophore, p-coumaric acid (pCA), have been identified by X-ray and neutron diffraction experiments. However, there is a lack of agreement on the chemical nature of these H-bond interactions. Additionally, no consensus has been reached on the presence of LBHBs in the active site of the protein, despite various experimental and theoretical studies having been carried out to investigate this issue. In this work, we perform a computational study that combines classical and density functional theory (DFT)-based quantum mechanical/molecular mechanical (QM/MM) simulations to shed light onto this controversy. Furthermore, we aim to deepen our understanding of the chemical nature and dynamics of the protons involved in the two short hydrogen bonds that, in the dark state of PYP, connect pCA with the two binding pocket residues (E46 and Y42). Our results support the existence of a strong LBHB between pCA and E46, with the H fully delocalized and shared between both the carboxylic oxygen of E46 and the phenolic oxygen of pCA. Additionally, our findings suggest that the pCA interaction with Y42 can be suitably described as a typical short ionic H-bond of moderate strength that is fully localized on the phenolic oxygen of Y42.     

QuanPol: A full spectrum and seamless QM/MM program

The quantum chemistry polarizable force field program (QuanPol) is implemented to perform combined quantum mechanical and molecular mechanical (QM/MM) calculations with induced dipole polarizable force fields and induced surface charge continuum solvation models. The QM methods include Hartree–Fock method, density functional theory method (DFT), generalized valence bond theory method, multiconfiguration self‐consistent field method, Møller–Plesset perturbation theory method, and time‐dependent DFT method. The induced dipoles of the MM atoms and the induced surface charges of the continuum solvation model are self‐consistently and variationally determined together with the QM wavefunction. The MM force field methods can be user specified, or a standard force field such as MMFF94, Chemistry at Harvard Molecular Mechanics (CHARMM), Assisted Model Building with Energy Refinement (AMBER), and Optimized Potentials for Liquid Simulations‐All Atom (OPLS‐AA). Analytic gradients for all of these methods are implemented so geometry optimization and molecular dynamics (MD) simulation can be performed. MD free energy perturbation and umbrella sampling methods are also implemented. © 2013 Wiley Periodicals, Inc.     

Molecular dynamics simulations of the detoxification of paraoxon catalyzed by phosphotriesterase

Combined QM(PM3)/MM molecular dynamics simulations together with QM(DFT)/MM optimizations for key configurations have been performed to elucidate the enzymatic catalysis mechanism on the detoxification of paraoxon by phosphotriesterase (PTE). In the simulations, the PM3 parameters for the phosphorous atom were reoptimized. The equilibrated configuration of the enzyme/substrate complex showed that paraoxon can strongly bind to the more solvent‐exposed metal ion Zn_β, but the free energy profile along the binding path demonstrated that the binding is thermodynamically unfavorable. This explains why the crystal structures of PTE with substrate analogues often exhibit long distances between the phosphoral oxygen and Zn_β. The subsequent S_N2 reaction plays the key role in the whole process, but controversies exist over the identity of the nucleophilic species, which could be either a hydroxide ion terminally coordinated to Zn_α or the μ‐hydroxo bridge between the α‐ and β‐metals. Our simulations supported the latter and showed that the rate‐limiting step is the distortion of the bound paraoxon to approach the bridging hydroxide. After this preparation step, the bridging hydroxide ion attacks the phosphorous center and replaces the diethyl phosphate with a low barrier. Thus, a plausible way to engineer PTE with enhanced catalytic activity is to stabilize the deformed paraoxon. Conformational analyses indicate that Trp131 is the closest residue to the phosphoryl oxygen, and mutations to Arg or Gln or even Lys, which can shorten the hydrogen bond distance with the phosphoryl oxygen, could potentially lead to a mutant with enhanced activity for the detoxification of organophosphates. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

In Silico Identification of Possible Inhibitors for Protein Kinase B (PknB) of Mycobacterium tuberculosis

With tuberculosis still being one of leading causes of death in the world and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), researchers have been seeking to find further therapeutic strategies or more specific molecular targets. PknB is one of the 11 Ser/Thr protein kinases of Mtb and is responsible for phosphorylation-mediated signaling, mainly involved in cell wall synthesis, cell division and metabolism. With the amount of structural information available and the great interest in protein kinases, PknB has become an attractive target for drug development. This work describes the optimization and application of an in silico computational protocol to find new PknB inhibitors. This multi-level computational approach combines protein–ligand docking, structure-based virtual screening, molecular dynamics simulations and free energy calculations. The optimized protocol was applied to screen a large dataset containing 129,650 molecules, obtained from the ZINC/FDA-Approved database, Mu.Ta.Lig Virtual Chemotheca and Chimiothèque Nationale. It was observed that the most promising compounds selected occupy the adenine-binding pocket in PknB, and the main interacting residues are Leu17, Val26, Tyr94 and Met155. Only one of the compounds was able to move the active site residues into an open conformation. It was also observed that the P-loop and magnesium position loops change according to the characteristics of the ligand. This protocol led to the identification of six compounds for further experimental testing while also providing additional structural information for the design of more specific and more effective derivatives.     

Dynamic structures of phosphodiesterase-5 active site by combined molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations

Various quantum mechanical/molecular mechanical (QM/MM) geometry optimizations starting from an x-ray crystal structure and from the snapshot structures of constrained molecular dynamics (MD) simulations have been performed to characterize two dynamically stable active site structures of phosphodiesterase-5 (PDE5) in solution. The only difference between the two PDE5 structures exists in the catalytic, second bridging ligand (BL2) which is HO- or H2O. It has been shown that, whereas BL2 (i.e. HO-) in the PDE5(BL2 = HO-) structure can really bridge the two positively charged metal ions (Zn2+ and Mg2+), BL2 (i.e. H2O) in the PDE5(BL2 = H2O) structure can only coordinate Mg2+. It has been demonstrated that the results of the QM/MM geometry optimizations are remarkably affected by the solvent water molecules, the dynamics of the protein environment, and the electronic embedding charges of the MM region in the QM part of the QMM/MM calculation. The PDE5(BL2 = H2O) geometries optimized by using the QM/MM method in different ways show strong couplings between these important factors. It is interesting to note that the PDE5(BL2 = HO-) and PDE5(BL2 = H2O) geometries determined by the QM/MM calculations neglecting these three factors are all consistent with the corresponding geometries determined by the QM/MM calculations that account for all of these three factors. These results suggest the overall effects of these three important factors on the optimized geometries can roughly cancel out. However, the QM/MM calculations that only account for some of these factors could lead to considerably different geometries. These results might be useful also in guiding future QM/MM geometry optimizations on other enzymes.