Structural Features And Dynamic Basis Of Several Important Cytochrome P450s: A Survey Of Substrate Selectivity And Tunnel Gating Mechanism

Many organisms have evolved to use oxygen to paticipate in the life sustaining process and to lower its toxic effects since oxygen has become part of the biochemical processes. Cytochrome P450s (CYPs) are ubiquitous heme-containing monooxygenases that catalyze the hydroxylation of nonactivated hydrocarbon reactions, dealkylation, epoxidation, and dehydrogenation reactions involved in the oxidative metabolism. They play an important role in the metabolism of endogenous and exogenous substrates like drugs and environmental chemicals, whereby mediate the drug-drug interactions and profoundly affect the safety and efficacy of drug therapies. Knowing how CYPs recognize and bind drugs is crucial for drug design. To date, the investigation in CYPs has gone deep into molecular level. Molecular dynamics simulation has been applied to provide important insights into the structure-function relationships of CYPs.In this dissertation, we investigated several interesting CYPs (CYP7B1, CYP2E1and CYP17A1) by means of molecular dynamics simulation method, mainly included in six parts. In Chapter I, we made an brief introduction of the enzymes and cytochrome P450s. In Chapter II, we summarize the theoretical methods used in this dissertation. Then, we carried on the theoretically investigations on the structural features of three interesting cytochromes P450s in Chapter III to Chapter VI. 1. Molecular dynamics studies of the substrate selectivity of CYP7B1and major access tunnels analysisP4507B1(CYP7B1) is one of the CYP7family members, which is widely expressed in brain, particularly in the hippocampus, also in liver and kidney with a much lower level. Hepatic CYP7B1activity is crucial for the inactivation of steroids, loss of the enzyme activity is associated with liver failure in children. Recent studies show that mutations in CYP7B1gene are directly responsible for spastic paraplegia type5(SPG5), which is known as a progressive neuropathy. The active site volume of CYP7B1was calculated to be826A3, which is inferred to accommodate multiple conformations of medium chain steroids within its active site.In the absence of an X-ray crystal structure, the structural features relevant to the substrate selectivity of CYP7B1remain unknown at the atomic level. In the present study, a series of methods including homology modeling, automatic docking and MD simulations are used to investigate the metabolic activation of four representative substrates:25-HOChol, DHEA, anediol, and enediol. The results clearly identify two binding modes. The calculated dynamic properties indicate that those residues with hydrophobic side chains in the active site, particularly Trp291and Phe489, are important for CYP7B1selectivity and have a significant impact on the active-site architecture and substrate binding. Phe489is proposed mainly to merge the active site with the adjacent channel to the surface and accommodate substrate binding in a reasonable orientation. Moreover, two major access channels are presented for the entry of substrates with dissimilar chain lengths. The Phe111-His139and Phe223-Phe489pairs are suggested to mediate the sufficient opening of the tunnels as gate keepers. Our work provides detailed insight into the poorly understood structural features of human CYP7B1at the atomic level, and could help identify candidate analogues for further drug leads. 2. Molecular basis of the recognition of arachidonic acid by CYP2E1along major access tunnelAmong the various isoforms of CYPs, CYP2E1is widely known for its ability to oxidize both low molecular weight xenobiotics and endogenous fatty acids with its smallest active site size (182A3). For example, the analgesic acetaminophen can be oxidized by CYP2E1and the created metabolite is responsible for hepatotoxicity and death in acetaminophen overdose cases. In addition to the xenobiotic compounds, CYP2E1can also oxidize endogenous fatty acids, such as arachidonic acid (AA). The metabolism of AA via induction of CYP enzymes, particularly CYP2E1, could promote oxidative damage and generation of free radical species. There is a wealth of biochemical information about the CYP2E1-xenobiotic complexes, but scarce binding information of CYP2E1with fatty acids has been available to date. It is suggested that long length fatty acids and small weight compounds, which are either more or less hydrophobic, may follow different access/egress routes.In the present study, we identified potential openings via a combination of MD, RAMD, and SMD simulations to investigate the new structural basis of the pathway gating mechanism. Results identified tunnel2c as the most possible tunnel for fatty acids rather than tunnel2a. We also compared the structural features of the CYP2E1-AA complex with that of the CYP2E1-dodecanoic acid analog complex. Based on the binding free energy calculations and analyses of the gating residues during the unbinding process, the underlying molecular origin for the tunnel gating mechanism is considered to be associated with the intermolecular electrostatic interactions, which are generally presented as hydrogen bonds between AA and three key residues, His107, Ala108, and His109. Taken together, our work could contributes to a better understanding about the related different tunnel gating mechanism for fatty acids and small weight compounds for other CYPs and help guide future experimental and computational work on CYPs.3. Molecular origin of the experimentally observed decrease of enzymatic activity by single point mutation.CYP17A1(also known as cytochrome P450c17) is a dualfunction monooxygenase with a vital role in both adrenal and gonadal steroidogenesis in mammals. As a key enzyme in the steroidogenic pathway, the17a-hydroxylase/17,20-lyase activities of CYP17A1are required for the biosynthesis of cortisol in the adrenal zona fasciculata and the generation of androgenie steroids and estrogens in the adrenal zona reticularis and in gonads. To date, more than50CYP17A1mutations have been identified, and most of these were identified in patients with17-hydroxylase deficiencies. The biochemical effects of many clinical mutations are directly associated with the generation of steroidal hormones.On the basis of MD simulations and SMD simulations, We have investigated the atomic-level structural variations and its two mutants of experimental interests (Y201N and E305G) to understand the molecular origin for the decreased enzymatic activity. We present an "access mechanism" which encapsulates the effects of mutations on the changes in both structural flexibility and tunnel dynamics, bridging the gap between the theory and the experimentally observed results of enzymatic activity changes. The observed local F-G helices structural changes upon single point mutations are directly projected onto the opening of major pathway for ligand ingress and egress. Both mutations cause the decrease of ligand binding affinity, mainly attributed to the wider opening of their respective major tunnels and the more exposure of ligand to the aqueous solution. The underlying molecular mechanism is considered to be associated with the changes of the HC1. Moreover, the MM-GBSA calculations and the SMD simulations give a reasonable agreement with the experimental results and provide evidence for previous conjecture that conformational changes induced by mutations correlate very well with the ligand binding affinity and enzymatic activity. Our work provides detailed atomistic insights into the structural motif and molecular origin to rationalize the experimentally observed decrease of enzymatic activity by single point mutation.4. Structural features and dynamic investigations of the membrane-bound CYP17A1Eukaryotic CYPs are generally membrane-bound proteins. The heme tilt angle with respect to the membrane has been estimated experimentally to be between38°and78°for different CYPs isoforms. To date, an important caveat for the investigation of CYPs is the fact that most studies appear to provide simulations in their solution form without taking into account the interaction of the enzyme with membrane. Thus, it is necessary to provide high-resolution structural determination of membrane-associated state of CYPs.To this end, we have performed the full-atomistic MD simulations with lipid bilayer to study the binding and interaction of CYP17A1to the membrane. With multiple independent simulations, a dynamic interacting procedure of the globular domain of CYP17A1with the membrane was captured, characterized by the depths of insertion and orientations of the enzyme to the membrane surface. Membrane binding of CYP17A1significantly reshapes the protein at the membrane interface, causing conformational changes relevant to the access tunnels leading to the active site of the enzyme. The detailed hydrogen-bonding and hydrophobic interactions provide an important suggestion that the CYP17A1may interact with the membrane through specific membrane-enzyme interactions that stabilize the binding mode of the protein with the membrane, rather than simply through the adsorption to the surface. In summary, our present work provides detailed atomistic insights into the effects of the presence of membrane on the structural and dynamical properties of CYP17A1. The knowledge of membrane binding characteristics could guide future experimental and computational work on membrane-bound CYPs so that various investigations of CYPs in their native, membrane-bound cellular environment may be achieved.