Zhiwei Feng

Modeling, molecular dynamics simulation, and mutation validation for structure of cannabinoid receptor 2 based on known crystal structures of GPCRs.

The cannabinoid receptor 2 (CB2) plays an important role in the immune system. Although a few of GPCRs crystallographic structures have been reported, it is still challenging to obtain functional transmembrane proteins and high resolution X-ray crystal structures, such as for the CB2 receptor. In the present work, we used 10 reported crystal structures of GPCRs which had high sequence identities with CB2 to construct homology-based comparative CB2 models. We applied these 10 models to perform a prescreen by using a training set consisting of 20 CB2 active compounds and 980 compounds randomly selected from the National Cancer Institute (NCI) database. We then utilized the known 170 cannabinoid receptor 1 (CB1) or CB2 selective compounds for further validation. Based on the docking results, we selected one CB2 model (constructed by β1AR) that was most consistent with the known experimental data, revealing that the defined binding pocket in our CB2 model was well-correlated with the training and testing data studies. Importantly, we identified a potential allosteric binding pocket adjacent to the orthosteric ligand-binding site, which is similar to the reported allosteric pocket for sodium ion Na+ in the A2AAR and the δ-opioid receptor. Our studies in correlation of our data with others suggested that sodium may reduce the binding affinities of endogenous agonists or its analogs to CB2. We performed a series of docking studies to compare the important residues in the binding pockets of CB2 with CB1, including antagonist, agonist, and our CB2 neutral compound (neutral antagonist) XIE35-1001. Then, we carried out 50 ns molecular dynamics (MD) simulations for the CB2 docked with SR144528 and CP55940, respectively. We found that the conformational changes of CB2 upon antagonist/agonist binding were congruent with recent reports of those for other GPCRs. Based on these results, we further examined one known residue, Val1133.32, and predicted two new residues, Phe183 in ECL2 and Phe2817.35, that were important for SR144528 and CP55940 binding to CB2. We then performed site-directed mutation experimental study for these residues and validated the predictions by radiometric binding affinity assay.

Mechanism of graphene oxide as an enzyme inhibitor from molecular dynamics simulations.

Graphene and its water-soluble derivative, graphene oxide (GO), have attracted huge attention because of their interesting physical and chemical properties, and they have shown wide applications in various fields including biotechnology and biomedicine. Recently, GO has been shown to be the most efficient inhibitor for α-chymotrypsin (ChT) compared with all other artificial inhibitors. However, how GO interacts with bioactive proteins and its potential in enzyme engineering have been rarely explored. In this study, we investigate the interactions between ChT and graphene/GO by using molecular dynamics (MD) simulation. We find that ChT is adsorbed onto the surface of GO or graphene during 100 ns MD simulations. The α-helix of ChT plays as an important anchor to interact with GO. The cationic and hydrophobic residues of ChT form strong interactions with GO, which leads to the deformation of the active site of ChT and the inhibition of ChT. In comparison, the active site of ChT is only slightly affected after ChT adsorbed onto the graphene surface. In addition, the secondary structure of ChT is not affected after it is adsorbed onto GO or graphene surface. Our results illustrate the mechanism of the interaction between GO/graphene and enzyme and provide guidelines for designing efficient artificial inhibitors.

Studies on the Interactions between β2 Adrenergic Receptor and Gs Protein by Molecular Dynamics Simulations

The β2 adrenergic receptor (β2AR) plays a key role in the control of smooth muscle relaxation in airways, the therapy of asthma, and a series of other basic physiological functions. Recently, the crystal structure of the β2AR-Gs protein complex was reported, which facilitates study of the activation mechanism of the β2AR and G-protein-coupled receptors (GPCRs). In this work, we perform 20 ns molecular dynamics (MD) simulations of the β2AR-Gs protein complex with its agonist in an explicit lipid and water environment to investigate the activation mechanism of β2AR. We find that during 20 ns MD simulation with a nanobody bound the interaction between the β2AR and the Gs protein is stable and the whole system is equilibrated within 6 ns. However, without a nanobody stabilizing the complex, the agonist triggers conformational changes of β2AR sequentially from the extracellular region to the intracellular region, especially the intracellular parts of TM3, TM5, TM6, and TM7, which directly interact with the Gs protein. Our results show that the β2AR-Gs protein complex makes conformational changes in the following sequence: (1) an agonist-bound part of β2AR, (2) the intracellular region of β2AR, and (3) the Gs protein.

p62/SQSTM1/Sequestosome-1 is an N-recognin of the N-end rule pathway which modulates autophagosome biogenesis

Macroautophagy mediates the selective degradation of proteins and non-proteinaceous cellular constituents. Here, we show that the N-end rule pathway modulates macroautophagy. In this mechanism, the autophagic adapter p62/SQSTM1/Sequestosome-1 is an N-recognin that binds type-1 and type-2 N-terminal degrons (N-degrons), including arginine (Nt-Arg). Both types of N-degrons bind its ZZ domain. By employing three-dimensional modeling, we developed synthetic ligands to p62 ZZ domain. The binding of Nt-Arg and synthetic ligands to ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62 interaction with LC3, leading to the delivery of p62 and its cargoes to the autophagosome. Upon binding to its ligand, p62 acts as a modulator of macroautophagy, inducing autophagosome biogenesis. Through these dual functions, cells can activate p62 and induce selective autophagy upon the accumulation of autophagic cargoes. We also propose that p62 mediates the crosstalk between the ubiquitin-proteasome system and autophagy through its binding Nt-Arg and other N-degrons.Soluble misfolded proteins that fail to be degraded by the ubiquitin proteasome system (UPS) are redirected to autophagy via specific adaptors, such as p62. Here the authors show that p62 recognises N-degrons in these proteins, acting as a N-recognin from the proteolytic N-end rule pathway, and targets these cargos to autophagosomal degradation.

Structural Insight into Tetrameric hTRPV1 from Homology Modeling, Molecular Docking, Molecular Dynamics Simulation, Virtual Screening, and Bioassay Validations

The transient receptor potential vanilloid type 1 (TRPV1) is a heat-activated cation channel protein, which contributes to inflammation, acute and persistent pain. Antagonists of human TRPV1 (hTRPV1) represent a novel therapeutic approach for the treatment of pain. Developing various antagonists of hTRPV1, however, has been hindered by the unavailability of a 3D structure of hTRPV1. Recently, the 3D structures of rat TRPV1 (rTRPV1) in the presence and absence of ligand have been reported as determined by cryo-EM. rTRPV1 shares 85.7% sequence identity with hTRPV1. In the present work, we constructed and reported the 3D homology tetramer model of hTRPV1 based on the cryo-EM structures of rTRPV1. Molecular dynamics (MD) simulations, energy minimizations, and prescreen were applied to select and validate the best model of hTRPV1. The predicted binding pocket of hTRPV1 consists of two adjacent monomers subunits, which were congruent with the experimental rTRPV1 data and the cyro-EM structures of rTRPV1. The detailed interactions between hTRPV1 and its antagonists or agonists were characterized by molecular docking, which helped us to identify the important residues. Conformational changes of hTRPV1 upon antagonist/agonist binding were also explored by MD simulation. The different movements of compounds led to the different conformational changes of monomers in hTRPV1, indicating that TRPV1 works in a concerted way, resembling some other channel proteins such as aquaporins. We observed that the selective filter was open when hTRPV1 bound with an agonist during MD simulation. For the lower gate of hTRPV1, we observed large similarities between hTRPV1 bound with antagonist and with agonist. A five-point pharmacophore model based on several antagonists was established, and the structural model was used to screen in silico for new antagonists for hTRPV1. By using the 3D TRPV1 structural model above, the pilot in silico screening has begun to yield promising hits with activity as hTRPV1 antagonists, several of which showed substantial potency.

The flexibility of P-glycoprotein for its poly-specific drug binding from molecular dynamics simulations

The multidrug efflux pump P-glycoprotein (P-gp) contributes to multidrug resistance in about half of human cancers. Recently, high resolution X-ray crystal structures of mouse P-gp (inward-facing) were reported, which significantly facilitates the understanding of the function of P-gp and the structure-based design of inhibitors for P-gp. Here we perform 20 ns molecular dynamics simulations of inward-facing P-gp with/without ligand in explicit lipid and water to investigate the flexibility of P-gp for its poly-specific drug binding. By analyzing the interactions between P-gp and QZ59-RRR or QZ59-SSS, we summarize the important residues and the flexibility of different parts of P-gp. Particularly, the flexibility of the side chains of aromatic residues (Phe and Tyr) allows them to form rotamers with different orientations in the binding pocket, which plays a critical role for the poly-specificity of the drug-binding cavity of P-gp. MD simulations reveal that trans-membrane (TM) TM12 and TM6 are flexible and contribute to the poly-specific drug binding, while TM4 and TM5 are rigid and stabilize the whole structure. We also construct outward-facing P-gp based on the MsbA structure and perform 20 ns MD simulations. The comparison between the MD results for outward-facing P-gp and those for inward-facing P-gp shows that the TM parts in outward-facing P-gp undergo significant conformational change to facilitate the export of small molecules.

The Selective Interaction between Silica Nanoparticles and Enzymes from Molecular Dynamics Simulations

Nanoscale particles have become promising materials in many fields, such as cancer therapeutics, diagnosis, imaging, drug delivery, catalysis, as well as biosensors. In order to stimulate and facilitate these applications, there is an urgent need for the understanding of the interaction mode between the nano-particles and proteins. In this study, we investigate the orientation and adsorption between several enzymes (cytochrome c, RNase A, lysozyme) and 4 nm/11 nm silica nanoparticles (SNPs) by using molecular dynamics (MD) simulation. Our results show that three enzymes are adsorbed onto the surfaces of both 4 nm and 11 nm SNPs during our MD simulations and the small SNPs induce greater structural stabilization. The active site of cytochrome c is far away from the surface of 4 nm SNPs, while it is adsorbed onto the surface of 11 nm SNPs. We also explore the influences of different groups (-OH, -COOH, -NH2 and CH3) coated onto silica nanoparticles, which show significantly different impacts. Our molecular dynamics results indicate the selective interaction between silicon nanoparticles and enzymes, which is consistent with experimental results. Our study provides useful guides for designing/modifying nanomaterials to interact with proteins for their bio-applications.

Hologram quantitative structure activity relationship, docking, and molecular dynamics studies of inhibitors for CXCR4.

CXCR4 plays a crucial role as a co‐receptor with CCR5 for HIV‐1 anchoring to mammalian cell membrane and is implicated in cancer metastasis and inflammation. In the current work, we study the relationship of structure and activity of AMD11070 derivatives and other inhibitors of CXCR4 using HQSAR, docking and molecular dynamics (MD) simulations. We obtain an HQSAR model (q2 = 0.779), and the HQSAR result illustrates that AMD11070 shows a high antiretroviral activity. As HQSAR only provides 2D information, we perform docking and MD to study the interaction of It1t, AMD3100, and AMD3465 with CXCR4. Our results illustrate that the binding are affected by two crucial residues Asp97 and Glu288. The butyl amine moiety of AMD11070 contributes to its high antiretroviral activity. Without a butyl amine moiety, (2,7a‐Dihydro‐1H‐benzoimidazol‐2‐ylmethyl)‐methyl‐(5,6,7,8‐tetrahydro‐quinolin‐8‐yl)‐amine (compound 5a) shows low antiretroviral activity. Our results provide structural details about the interactions between the inhibitors and CXCR4, which are useful for rational drug design of CXCR4.

Concerted Movement in pH-Dependent Gating of FocA from Molecular Dynamics Simulations

FocA, a member of the formate-nitrite transporter (FNT) family, transports formate and nitrite across biological membranes in cellular organisms. The export and uptake of formate in bacteria are both mediated by FocA, which undergoes a pH-dependent functional switch. Recently, the crystal structures of Escherichia coli FocA (EcFocA), Vibrio cholerae FocA (VcFocA), and Salmonella typhimurium FocA (StFocA) were reported. We performed molecular dynamics (MD) on StFocA and EcFocA with different states of His209 (protonated and unprotonated), representing different pH conditions of FocA. The N-terminal helix in each protomer of StFocA covers and blocks the formate channel. At neutral or high pH (MD simulations with unprotonated His209), the concerted movement of the N-terminal helices of pairs of protomers of StFocA opens its formate channel. At low pH (MD simulations with protonated His209), protonated His209 interacts tightly with its neighboring residue Asn262, and the channel becomes narrower, so that the formate can hardly pass through the channel. We obtained similar results for EcFocA. Our study shows that pairs of protomers of FocA move in a concerted way to achieve its pH-dependent gating function, which provides information on the dynamics of the gating mechanism of FNT proteins and aquaporins.

Computational Advances for the Development of Allosteric Modulators and Bitopic Ligands in G Protein-Coupled Receptors

Under the section BITOPIC LIGANDS, the authors have inadvertently omitted the citation of the original work and specific sentences byMohr et al. (101), who havemade significant contributions in this field. The authors regret this error. Specifically, the following sentence strings should have been placed within quotes and directly attributed to Mohr et al. (101) from which the sentences originated: “...Steinfeld et al. (94) generated the compound THR160209 by connecting an orthosteric 3-benzyhydrylpyrrolinyl building partner via a heptane chain to an allosteric 4aminobenzylpiperidine motif, and they gained a considerably higher receptor affinity than seen with individual components (pKi (total) = 9.5 vs. pKi = 5.5 for the single compounds) and a certain preference for the M2mAChR” (101). “This can be achieved if two parts bind into their corresponding binding pockets in an ideal manner without inducing an unfavorable conformational rearrangement of the receptor protein, which can be explained by the lower total entropy cost of the ligand-receptor complex. The mostly favorable spatial proximity between the orthosteric and the allosteric binding areas contributes to a decrease in entropy (96). Conformational changes in the two binding events can either be (locally) entropic or enthalpic. This can differ case by case. The general rule is that an increase in entropy is the driving force for the docking of a polar agonist and a decrease in enthalpy in the driving force for the docking of a mostly hydrophobic antagonist (97)”(101). “...several parts of the molecule—the orthossteric and the allosteric parts as well as the linker—must be optimized and linked in appropriate ways.”(101)