Ji-Long Zhang

Structural and Dynamic Basis of Human Cytochrome P450 7B1: A Survey of Substrate Selectivity and Major Active Site Access Channels

Cytochrome P450 (CYP) 7B1 is a steroid cytochrome P450 7α-hydroxylase that has been linked directly with bile salt synthesis and hereditary spastic paraplegia type 5 (SPG5). The enzyme provides the primary metabolic route for neurosteroids dehydroepiandrosterone (DHEA), cholesterol derivatives 25-hydroxycholesterol (25-HOChol), and other steroids such as 5α-androstane-3β,17β-diol (anediol), and 5α-androstene-3β,17β-diol (enediol). A series of investigations including homology modeling, molecular dynamics (MD), and automatic docking, combined with the results of previous experimental site-directed mutagenesis studies and access channels analysis, have identified the structural features relevant to the substrate selectivity of CYP7B1. The results clearly identify the dominant access channels and critical residues responsible for ligand binding. Both binding free energy analysis and total interaction energy analysis are consistent with the experimental conclusion that 25-HOChol is the best substrate. According to 20 ns MD simulations, the Phe cluster residues that lie above the active site, particularly Phe489, are proposed to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. The investigation of CYP7B1-substrate binding modes provides detailed insights into the poorly understood structural features of human CYP7B1 at the atomic level, and will be valuable information for drug development and protein engineering.

Molecular Dynamics Simulations Suggest Ligand’s Binding to Nicotinamidase/Pyrazinamidase

The research on the binding process of ligand to pyrazinamidase (PncA) is crucial for elucidating the inherent relationship between resistance of Mycobacterium tuberculosis and PncA’s activity. In the present study, molecular dynamics (MD) simulation methods were performed to investigate the unbinding process of nicotinamide (NAM) from two PncA enzymes, which is the reverse of the corresponding binding process. The calculated potential of mean force (PMF) based on the steered molecular dynamics (SMD) simulations sheds light on an optimal binding/unbinding pathway of the ligand. The comparative analyses between two PncAs clearly exhibit the consistency of the binding/unbinding pathway in the two enzymes, implying the universality of the pathway in all kinds of PncAs. Several important residues dominating the pathway were also determined by the calculation of interaction energies. The structural change of the proteins induced by NAM’s unbinding or binding shows the great extent interior motion in some homologous region adjacent to the active sites of the two PncAs. The structure comparison substantiates that this region should be very important for the ligand’s binding in all PncAs. Additionally, MD simulations also show that the coordination position of the ligand is displaced by one water molecule in the unliganded enzymes. These results could provide the more penetrating understanding of drug resistance of M. tuberculosis and be helpful for the development of new antituberculosis drugs.

Exploring the molecular basis of dsRNA recognition by Mss116p using molecular dynamics simulations and free-energy calculations.

DEAD-box proteins are the largest family of helicase that are important in nearly all aspects of RNA metabolism. However, it is unclear how these proteins recognize and bind RNA. Here, we present a detailed analysis of the related DEAD-box protein Mss116p-RNA interaction, using molecular dynamics simulations with MM-GBSA calculations. The energetic analysis indicates that the two strands of double strands RNA (dsRNA) are recognized asymmetrically by Mss116p. The strand 1 of dsRNA provides the main binding affinity. Meanwhile, the nonpolar interaction provides the main driving force for the binding process. Although the contribution of polar interaction is small, it is vital in stabilizing the protein-RNA interaction. Compared with the wild type Mss116p, two studied mutants Q412A and D441A have obviously reduced binding free energies with dsRNA because of the decreasing of polar interaction. Three important residues Lys409, Arg415 and Arg438 lose their binding affinity significantly in mutants. In conclusion, these results complement previous experiments to advance comprehensive understanding of Mss116p-dsRNA interaction. The results also would provide support for the application of similar approaches to the understanding of other DEAD-box protein-RNA complexes.

Influence of Hyperthermophilic Protein Cren7 on the Stability and Conformation of DNA: Insights from Molecular Dynamics Simulation and Free Energy Analysis

Cren7, a novel chromatin protein highly conserved among crenarchaea, plays an important role in genome packaging and gene regulation. However, the detail dynamical structural characteristic of the Cren7-DNA complex and the detail study of the DNA in the complex have not been done. Focused on two specific Cren7-DNA complexes (PDB codes 3LWH and 3LWI ), we applied molecular dynamics (MD) simulations at four different temperatures (300, 350, 400, and 450 K) and the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculation at 300 and 350 K to examine the role of Cren7 protein in enhancing the stability of DNA duplexes via protein-DNA interactions, and to study the structural transition in DNA. The simulation results indicate that Cren7 stabilizes DNA duplex in a certain temperature range in the binary complex compared with the unbound DNA molecules. At the same time, DNA molecules were found to undergo B-like to A-like form transitions with increased temperature. The results of statistical analyses of the H-bond and hydrophobic contacts show that some residues have significant influence on the structure of DNA molecules. Our work can give important information to understand the interactions of proteins with nucleic acids and other ligands.

Molecular basis of the recognition of arachidonic acid by cytochrome P450 2E1 along major access tunnel

Cytochrome P450 2E1 is widely known for its ability to oxidize both low molecular weight xenobiotics and endogenous fatty acids (e.g., arachidonic acid (AA)). In this study, we investigated the structural features of the AA‐bound CYP2E1 complex utilizing molecular dynamics (MD) and found that the distinct binding modes for both AA and fatty acid analog are conserved. Moreover, multiple random acceleration MD simulations and steered MD simulations uncovered the most possible tunnel for fatty acids. The main attractions are derived from three key residues, His107, Ala108, and His109, whose side chains reorient to keep ligands bound via hydrogen bonds during the initial unbinding process. More importantly, based on the calculated binding free energy results, we hypothesize that the hydrogen bonds between the receptor and the ligand are the most important contributors involved in the binding affinity. Thus, it is inferred that the hydrogen bonds between these three residues and the ligand may help offer insights into the structural basis of the different ligand egress mechanisms for fatty acids and small weight compounds. Our investigation provides detailed atomistic insights into the structural features of human CYP2E1–fatty acid complex structures. Furthermore, the ligand‐binding characteristics obtained in the present study are helpful for both experimental and computational studies of CYPs and may allow future researchers to achieve desirable changes in enzymatic activities.

Analysis of clinically relevant substrates of CYP2B6 enzyme by computational methods

Mounting evidence thus far indicates that human cytochrome P450 2B6 (CYP2B6), an enzyme expressed at a relatively low level functionally, is primarily responsible for the metabolism of several clinically relevant drugs, including propofol, efavirenz, bupropion, mephobarbital, and the propofol analog 2,6-di-sec-butyl phenol. We used molecular dynamics and molecular docking methods to predict such interactions and to compare with experimentally measured metabolisms. Insight II and Discover Studio 2.5 were used to carry out the docking of these substrates into CYP2B6 to explore the critical residues and interaction energies of the complexes. Phe297, Glu301, Thr302 and Val367 were identified as major drug-binding residues, which is consistent with previous data on site-directed mutagenesis, crystallography structure, and from modeling and docking studies. In addition, our docking results suggest that nonpolar amino acid clusters and heme also participate in binding to mediate drug oxidative metabolism. The binding modes of the five clinically relevant substrates mentioned above for metabolism on CYP2B6 are presented.

Insight into the dynamic interaction of different carbohydrates with human surfactant protein D: molecular dynamics simulations.

The unbinding process of three monosaccharides--galactose, glucose, and mannose--from human surfactant protein D (hSP-D) was investigated by the molecular docking and molecular dynamics methods to explore the cause of different dynamic interaction between these monosaccharides and the protein. The results show that the low affinity of galactose for hSP-D is attributed to the different binding conformation from the other two monosaccharides. The sugar coordinates to the calcium ion by the hydroxyl groups in the C2 and C3 atoms, so it cannot form the effective interaction with hSP-D. Glucose and mannose have similar binding conformations with hSP-D. Their difference in the affinity is induced by the interaction between the hydroxyl group in the C2 atom and the residue Asp325. The direction of the hydroxyl group in mannose results in the formation of the hydrogen bond with Asp325 and further makes mannose hydrogen-bond to the residues Glu329 and Arg343 by the hydroxyl groups in the C3, C4, and C6 atoms. As glucose only forms three hydrogen bonds with the residues Glu321, Asn323, and Glu329 by the hydroxyl groups in the C3 and C4 atoms, its interaction with hSP-D is weaker than that of mannose. Thus glucose has a lower energy barrier of dissociation. This work could provide the more penetrating understanding of hSP-D physiological functions.

How Does (E)-2-(Acetamidomethylene)succinate Bind to Its Hydrolase? From the Binding Process to the Final Result

The binding of (E)-2-(acetamidomethylene)succinate (E-2AMS) to E-2AMS hydrolase is crucial for biological function of the enzyme and the last step reaction of vitamin B6 biological degradation. In the present study, several molecular simulation methods, including molecular docking, conventional molecular dynamics (MD), steered MD (SMD), and free energy calculation methods, were properly integrated to investigate the detailed binding process of E-2AMS to its hydrolase and to assign the optimal enzyme-substrate complex conformation. It was demonstrated that the substrate binding conformation with trans-form amide bond is energetically preferred conformation, in which E-2AMSs pose not only ensures hydrogen bond formation of its amide oxygen atom with the vicinal oxyanion hole but also provides probability of the hydrophobic interaction between its methyl moiety and the related enzymes hydrophobic cavity. Several key residues, Arg146, Arg167, Tyr168, Arg179, and Tyr259, orientate the E-2AMSs pose and stabilize its conformation in the active site via the hydrogen bond interaction with E-2AMS. Sequentially, the binding process of E-2AMS to E-2AMS hydrolase was studied by SMD simulation, which shows the surprising conformational reversal of E-2AMS. Several important intermediate structures and some significant residues were identified in the simulation. It is stressed that Arg146 and Arg167 are two pivotal residues responsible for the conformational reversal of E-2AMS in the binding or unbinding. Our research has shed light onto the full binding process of the substrate to E-2AMS hydrolase, which could provide more penetrating insight into the interaction of E-2AMS with the enzyme and would help in the further exploration on the catalysis mechanism.

Exploring the mechanism how Marburg virus VP35 recognizes and binds dsRNA by molecular dynamics simulations and free energy calculations.

Filoviruses often cause terrible infectious disease which has not been successfully dealt with pharmacologically. All filoviruses encode a unique protein termed VP35 which can mask doubled‐stranded RNA to deactivate interferon. The interface of VP35–dsRNA would be a feasible target for structure‐based antiviral agent design. To explore the essence of VP35–dsRNA interaction, molecular dynamics simulation combined with MM‐GBSA calculations were performed on Marburg virus VP35–dsRNA complex and several mutational complexes. The energetic analysis indicates that nonpolar interactions provide the main driving force for the binding process. Although the intermolecular electrostatic interactions play important roles in VP35–dsRNA interaction, the whole polar interactions are unfavorable for binding which result in a low binding affinity. Compared with wild type VP35, the studied mutants F228A, R271A, and K298A have obviously reduced binding free energies with dsRNA reflecting in the reduction of polar or nonpolar interactions. The results also indicate that the loss of binding affinity for one dsRNA strand would abolish the total binding affinity. Three important residues Arg271, Arg294, and Lys298 which makes the largest contribution for binding in VP35 lose their binding affinity significantly in mutants. The uncovering of VP35–dsRNA recognition mechanism will provide some insights for development of antiviral drug.

Constant pH molecular dynamics (CpHMD) and molecular docking studies of CquiOBP1 pH-induced ligand releasing mechanism

The odorant binding protein of Culex quinquefasciatus (CquiOBP1), expressed on the insect antenna, is crucial for the investigation of trapping baited with oviposition semi-chemicals and controlling mosquito populations. The acidic titratable residues pKa prediction and the ligand binding poses investigation in two systems (pH 7 and pH 5) are studied by constant pH molecular dynamics (CpHMD) and molecular docking methods. Research results reveal that the change of the protonation states would disrupt some important H-bonds, such as Asp 66-Asp 70, Glu 105-Asn 102, etc. The cleavage of these H-bonds leads to the movement of the relative position of hydrophobic tunnel, N- and C- termini loops and pH-sensing triad (His23-Tyr54-Val125) in acid solution. Ligand MOP has lower affinity and shows different binding poses to protein CquiOBP1 at pH 5. This ligand may be released from another tunnel between helices α3 and α4 in acidic environment. However, it would bind to the protein with high affinity in neutral environment. This work could provide more penetrating understanding of the pH-induced ligand-releasing mechanism.