Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs.

The first identified covalent flavoprotein, a component of mammalian succinate dehydrogenase, was reported 42 years ago. Since that time, more than 20 covalent flavoenzymes have been described, each possessing one of five modes of FAD or FMN linkage to protein. Despite the early identification of covalent flavoproteins, the mechanisms of covalent bond formation and the roles of the covalent links are only recently being appreciated. The main focus of this review is, therefore, one of mechanism and function, in addition to surveying the types of linkage observed and the methods employed for their identification. Case studies are presented for a variety of covalent flavoenzymes, from which general findings are beginning to emerge.     

Structural insights into the interactions of xpt riboswitch with novel guanine analogues: a molecular dynamics simulation study

Ligand recognition in purine riboswitches is a complex process requiring different levels of conformational changes. Recent efforts in the area of purine riboswitch research have focused on ligand analogue binding studies. In the case of the guanine xanthine phosphoribosyl transferase (xpt) riboswitch, synthetic analogues that resemble guanine have the potential to tightly bind and subsequently influence the genetic expression of xpt mRNA in prokaryotes. We have carried out 25 ns Molecular Dynamics (MD) simulation studies of the aptamer domain of the xpt G-riboswitch in four different states: guanine riboswitch in free form, riboswitch bound with its cognate ligand guanine, and with two guanine analogues SJ1 and SJ2. Our work reveals novel interactions of SJ1 and SJ2 ligands with the binding core residues of the riboswitch. The ligands proposed in this work bind to the riboswitch with greater overall stability and lower root mean square deviations and fluctuations compared to guanine ligand. Reporter gene assay data demonstrate that the ligand analogues, upon binding to the RNA, lower the genetic expression of the guanine riboswitch. Our work has important implications for future ligand design and binding studies in the exciting field of riboswitches.     

Dissociation mechanism of GDP from Cdc42 via DOCK9 revealed by molecular dynamics simulations

Cell division control protein 42 homolog (Cdc42) influences a variety of cellular responses such as cell migration and polarity. Deregulation of Cdc42 has been associated with several human diseases and developmental disorders. Over-activation of Cdc42 through guanine nucleotide exchange factor (GEF) is a critical event for Cdc42 involved cancer metastasis. Members of DOCK family of GEF are important activators of Cdc42. However, this activation mechanism is still unknown. Molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) calculations were employed to investigate the central step of the activation of Cdc42: the dissociation mechanism of GDP from Cdc42 via DOCK9. Simulation results show that Mg²⁺ ion has a remarkable influence on the conformational change of switch I of Cdc42 through residue Pro34 which functions as a “clasp” to control the flexibility of switch I. In the GDP dissociation process, the Mg²⁺ ion leave first to result in a suitable conformation of Cdc42 for following DOCK9 binding to. When DOCK9 binds to Cdc42, it changes the orientations of residues Lys16, Thr17, Cys18 and Phe28 of Cdc42 to weaken the interactions between Cdc42 and GDP to release GDP. This study first elucidates the dissociation mechanism of GDP from Cdc42 via DOCK9 and identifies the essential residues of Cdc42 in this process. These simulation results are consistent with the recent findings of biochemical and amino acid mutational studies, and the observations are beneficial to understand the activation mechanism of Cdc42 and to provide insights for designing compounds targeting on Cdc42 related cancer metastasis.     

Investigation on the binding mechanism of loratinib with the c-ros oncogene 1 (ROS1) receptor tyrosine kinase via molecular dynamics simulation and binding free energy calculations

The c-ros oncogene 1 (ROS1) has proven to be an important cancer target for the treatment of various human cancers. The anaplastic lymphoma kinase inhibitor crizotinib has been granted approval for the treatment of patients with ROS1 positive metastatic non-small-cell lung cancer by the Food and Drug Administration on 2016. However, serious resistance due to the secondary mutation of glycine 2032 to arginine (G2032R) was developed in clinical studies. Loratinib (PF-06463922), a macrocyclic analog of crizotinib, showed significantly improved inhibitory activity against wild–type (WT) ROS1 and ROS1^G2032R mutant. To provide insights into the inhibition mechanism, molecular dynamics simulations and free energy calculations were carried out for the complexes of loratinib with WT and G2032R mutated ROS1. The apo-ROS1^WT and apo-ROS1^G2032R systems showed similar RMSF distributions, while ROS1^G2032R-loratinib showed significantly higher than that of WT ROS1-loratinib, which revealed that the binding of loratinib to ROS1^G2032R significantly interfered the ?uctuation of protein. Calculations of binding free energies indicate that G2032R mutation significantly reduces the binding affinity of loratinib for ROS1, which arose mostly from the increase of conformation entropy and the decrease of solvation energy. Furthermore, detailed per-residue binding free energies highlighted the increased and decreased contributions of some residues in the G2032R mutated systems. The present study revealed the detailed inhibitory mechanism of loratinib as potent WT and G2032R mutated ROS1 inhibitor, which was expected to provide a basis for rational drug design.     

Structural features of cholesteryl ester transfer protein: A molecular dynamics simulation study

Cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl esters (CEs) from atheroprotective high‐density lipoproteins (HDLs) to atherogenic low‐density lipoproteins (LDLs) or very‐low‐density lipoproteins (VLDLs). Inhibition of CETP raises HDL cholesterol (good cholesterol) levels and reduces LDL cholesterol (bad cholesterol) levels, making it a promising drug target for the prevention and treatment of coronary heart disease. Although the crystal structure of CETP has been determined, the molecular mechanism mediating CEs transfer is still unknown, even the structural features of CETP in a physiological environment remain elusive. We performed molecular dynamics simulations to explore the structural features of CETP in an aqueous solution. Results show that the distal portion flexibility of N‐terminal β‐barrel domain is considerably greater in solution than in crystal; conversely, the flexibility of helix X is slightly less. During the simulations the distal end of C‐terminal β‐barrel domain expanded while the hydrophilic surface increasing more than the hydrophobic surface. In addition, a new surface pore was generated in this domain. This surface pore and all cavities in CETP are stable. These results suggest that the formation of a continuous tunnel within CETP by connecting cavities is permitted in solution. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Molecular dynamics simulation of interleukin-2 and its complex and determination of the binding free energy

Interleukin-2 (IL-2) protein belongs to the signal modulator cytokine's family and therefore it is prevalent for immunological responses. It has been identified as a centrally important potential drug target for the inhibition of protein-protein interactions; so as to suppress the immunological responses associated with autoimmune, inflammatory and immunological diseases, and cancer. In the present work, we have performed two independent 100?ns of molecular dynamics (MD) simulations on the apo IL-2 protein and its ligand-bound complex (with a potent inhibitor FRG), to study the effect of inhibitor binding on the dynamics and stability of the protein. The calculation of binding free energy via post-processing end state method of Molecular Mechanics Poisson Boltzmann Surface Area (MM-PBSA) and Molecular Mechanics Generalised Born Surface Area (MM-GBSA) has inferred a good correlation in accordance with the already reported experimental data, demonstrating that the free energy of binding calculated by the two methods has no significant difference. The investigation of individual components of free energy revealed that the association of IL-2 protein with FRG ligand is primarily driven by the van der Waals energy contribution that represents the non-polar/hydrophobic energy contribution as dominant in this case of ligand binding.     

Insights into the binding mode of sulphamates and sulphamides to hCA II: crystallographic studies and binding free energy calculations

Sulphamate and sulphamide derivatives have been largely investigated as carbonic anhydrase inhibitors (CAIs) by means of different experimental techniques. However, the structural determinants responsible for their different binding mode to the enzyme active site were not clearly defined so far. In this paper, we report the X-ray crystal structure of hCA II in complex with a sulphamate inhibitor incorporating a nitroimidazole moiety. The comparison with the structure of hCA II in complex with its sulphamide analogue revealed that the two inhibitors adopt a completely different binding mode within the hCA II active site. Starting from these results, we performed a theoretical study on sulphamate and sulphamide derivatives, demonstrating that electrostatic interactions with residues within the enzyme active site play a key role in determining their binding conformation. These findings open new perspectives in the design of effective CAIs using the sulphamate and sulphamide zinc binding groups as lead compounds.     

A computational analysis of pyrazole-based inhibitors binding to Hsp90 using molecular dynamics simulation and the MM-GBSA method

Owing to the key role of heat-shock protein 90 (Hsp90) in the evolution, development and disease pathogenesis of cancer, it has been an important target for anti-cancer chemotherapy over the years. A five-nanosecond molecular dynamics simulation combined with the calculation of the binding free energy was carried out to investigate the binding mechanisms of three Hsp90 inhibitors 4BH, 2E1 and 2D9 to Hsp90. The binding free energy of each complex was computed using the molecular mechanics–generalised Born surface area method. Detailed binding free energies between each inhibitor and residues of Hsp90 were calculated using a per-residue basis decomposition method. The detailed inhibitor–residue interaction provides insights into binding mechanisms and in-depth understanding of the structure–affinity relationship. This study suggests that van der Waals energy is primarily responsible for driving the binding of the inhibitors to Hsp90, and the three inhibitors bind to Hsp90 in a similar binding mode. However, a substituent in 2D9 leads to higher binding free energy than the other two inhibitors. These data may assist in designing new potent drugs to combat cancer.     

Insights into scFv:drug binding using the molecular dynamics simulation and free energy calculation

Molecular dynamics simulations and free energy calculation have been performed to study how the single-chain variable fragment (scFv) binds methamphetamine (METH) and amphetamine (AMP). The structures of the scFv:METH and the scFv:AMP complexes are analyzed by examining the time-dependence of their RMSDs, by analyzing the distance between some key atoms of the selected residues, and by comparing the averaged structures with their corresponding crystallographic structures. It is observed that binding an AMP to the scFv does not cause significant changes to the binding pocket of the scFv:ligand complex. The binding free energy of scFv:AMP without introducing an extra water into the binding pocket is much stronger than scFv:METH. This is against the first of the two scenarios postulated in the experimental work of Celikel et al. (Protein Science 18, 2336 (2009)). However, adding a water to the AMP (at the position of the methyl group of METH), the binding free energy of the scFv:AMP-H2O complex, is found to be significantly weaker than scFv:METH. This is consistent with the second of the two scenarios given by Celikel et al. Decomposition of the binding energy into ligand-residue pair interactions shows that two residues (Tyr175 and Tyr177) have nearly-zero interactions with AMP in the scFv:AMP-H2O complex, whereas their interactions with METH in the scFv:METH complex are as large as -0.8 and -0.74 kcal mol^-1. The insights gained from this study may be helpful in designing more potent antibodies in treating METH abuse.     

Interaction investigations of HipA binding to HipB dimer and HipB dimer + DNA complex: a molecular dynamics simulation study

We carried out molecular dynamics simulations and free energy calculations for a series of ternary and diplex models for the HipA protein, HipB dimer, and DNA molecule to address the mechanism of HipA sequestration and the binding order of events from apo HipB/HipA to 2HipA + HipB dimer + DNA complex. The results revealed that the combination of DNA with the HipB dimer is energetically favorable for the combination of HipB dimer with HipA protein. The binding of DNA to HipB dimer induces a long‐range allosteric communication from the HipB₂‐DNA interface to the HipA–HipB₂ interface, which involves the closeness of α1 helices of HipB dimer to HipA protein and formations of extra hydrogen bonds in the HipA–HipB₂ interface through the extension of α2/3 helices in the HipB dimer. These simulated results suggested that the DNA molecule, as a regulative media, modulates the HipB dimer conformation, consequently increasing the interactions of HipB dimer with the HipA proteins, which explains the mechanism of HipA sequestration reported by the previous experiment. Simultaneously, these simulations also explored that the thermodynamic binding order in a simulated physiological environment, that is, the HipB dimer first bind to DNA to form HipB dimer + DNA complex, then capturing strongly the HipA proteins to form a ternary complex, 2HipA + HipB dimer + DNA, for sequestrating HipA in the nucleoid. Copyright © 2013 John Wiley & Sons, Ltd.     

A molecular dynamics simulation of the flavin mononucleotide–RNA aptamer complex

We report on an unrestrained molecular dynamics simulation of the flavin mononucleotide (FMN)–RNA aptamer. The simulated average structure maintains both cross‐strand and intermolecular FMN–RNA nuclear Overhauser effects from the nmr experiments and has all qualitative features of the nmr structure including the G10–U12–A25 base triple and the A13–G24, A8–G28, and G9–G27 mismatches. However, the relative orientation of the hairpin loop to the remaining part of the molecule differs from the nmr structure. The simulation predicts that the flexible phosphoglycerol part of FMN moves toward G27 and forms hydrogen bonds. There are structurally long‐lived water molecules in the FMN binding pocket forming hydrogen bonds within FMN and between FMN and RNA. In addition, long‐lived water is found bridging primarily RNA backbone atoms. A general feature of the environment of long‐lived “structural” water is at least two and in most cases three or four potential acceptor atoms. The 2′‐OH group of RNA usually acts as an acceptor in interactions with the solvent. There are almost no intrastrand O2′H(n)⋮O4′(n + 1) hydrogen bonds within the RNA backbone. In the standard case the preferred orientation of the 2′‐OH hydrogen atoms is approximately toward O3′ of the same nucleotide. However, a relatively large number of conformations with the backbone torsional angle γ in the trans orientation is found. A survey of all experimental RNA x‐ray structures shows that this backbone conformation occurs but is less frequent than found in the simulation. Experimental nmr RNA aptamer structures have a higher fraction of this conformation as compared to the x‐ray structures. The backbone conformation of nucleotide n + 1 with the torsional angle γ in the trans orientation leads to a relatively short distance between 2′‐OH(n) and O5′(n + 1), enabling hydrogen‐bond formation. In this case the preferred orientation of the 2′‐OH hydrogen atom is approximately toward O5′(n + 1). We find two relatively short and dynamically stable types of backbone–backbone next‐neighbor contacts, namely C2′(H)(n)⋮O4′(n + 1) and C5′(H)(n + 1)⋮O2′(n). These interactions may affect both backbone rigidity and thermodynamic stability of RNA helical structures. © 1999 John Wiley & Sons, Inc. Biopoly 50: 287–302, 1999     

Insight into binding mechanisms of inhibitors MKP56, MKP73, MKP86, and MKP97 to HIV-1 protease by using molecular dynamics simulation

HIV-1 protease (PR) has been a significant target for design of potent inhibitors curing acquired immunodeficiency syndrome. Molecular dynamics simulations coupled with molecular mechanics Poisson–Boltzmann surface area method were performed to study interaction modes of four inhibitors MKP56, MKP73, MKP86, and MKP97 with PR. The results suggest that the main force controlling interactions of inhibitors with PR should be contributed by van der Waals interactions between inhibitors and PR. The cross-correlation analyses based on MD trajectories show that inhibitor binding produces significant effect on the flap dynamics of PR. Hydrogen bond analyses indicate that inhibitors can form stable hydrogen bonding interactions with the residues from the catalytic strands of PR. The contributions of separate residues to inhibitor bindings are evaluated by using residue-based free energy decomposition method and the results demonstrate that the CH–π and CH–CH interactions between the hydrophobic groups of inhibitors with residues drive the associations of inhibitors with PR. We expect that this study can provide a significant theoretical aid for design of potent inhibitors targeting PR.     

Insights into internal dynamics of 6-phosphogluconolactonase from Trypanosoma brucei studied by nuclear magnetic resonance and molecular dynamics

Nuclear magnetic resonance is used to investigate the backbone dynamics in 6-phosphogluconolactonase from Trypanosoma brucei (Tb6PGL) with (holo-) and without (apo-) 6-phosphogluconic acid as ligand. Relaxation data were analyzed using the model-free approach and reduced spectral density mapping. Comparison of predictions, based on 77 ns molecular dynamics simulations, with the observed relaxation rates gives insight into dynamical properties of the protein and their alteration on ligand binding. Data indicate dynamics changes in the vicinity of the binding site. More interesting is the presence of perturbations located in remote regions of this well-structured globular protein in which no large-amplitude motions are involved. This suggests that delocalized changes in dynamics that occur upon binding could be a general feature of protein-target interactions.     

Insight derived from molecular dynamics simulation into dynamics and molecular motions of cuticle-degrading serine protease Ver112

Cuticle-degrading serine protease Ver112, which derived from a nematophagous fungus Lecanicillium psalliotae, has been exhibited to have high cuticle-degrading and nematicidal activities. We have performed molecular dynamics (MD) simulation based on the crystal structure of Ver112 to investigate its dynamic properties and large-scale concerted motions. The results indicate that the structural core of Ver112 shows a small fluctuation amplitude, whereas the substrate binding sites, and the regions close to and opposite the substrate binding sites experience significant conformational fluctuations. The large concerted motions obtained from essential dynamics (ED) analysis of MD trajectory can lead to open or close of the substrate binding sites, which are proposed to be linked to the functional properties of Ver112, such as substrate binding, orientation, catalytic, and release. The significant motion in the loop regions that is located opposite the binding sites are considered to play an important role in modulating the dynamics of the substrate binding sites. Furthermore, the bottom of free energy landscape (FEL) of Ver112 are rugged, which is mainly caused by the fluctuations of substrate binding regions and loops located opposite the binding site. In addition, the mechanism underlying the high flexibility and catalytic activity of Ver112 was also discussed. Our simulation study complements the biochemical and structural studies, and provides insight into the dynamics-function relationship of cuticle-degrading serine protease Ver112.     

Substrate binding and catalytic mechanism in phospholipase C from Bacillus Cereus: A molecular mechanics and molecular dynamics study

For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate, phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B. cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme–substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 Å) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme–substrate–water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AM1-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AM1-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure. © 1997 John Wiley and Sons, Inc. Biopoly 42: 319–336, 1997     

Metal binding sites of human H‐chain ferritin and iron transport mechanism to the ferroxidase sites: A molecular dynamics simulation study

We study via all atom classical molecular dynamics (MD) simulation the process of uptake of ferrous ions (Fe²⁺) into the human ferritin protein and the catalytic ferroxidase sites via pores (“channels”) in the interior of the protein. We observe that the three‐fold hydrophilic channels serve as the main entrance pathway for the Fe²⁺ ions. The binding sites along the ion pathway are investigated. Two strong binding sites, at the Asp131 and Glu134 residues and two weak binding sites, at the His118 and Cys130 are observed inside the three‐fold channel. We also identify an explicit pathway for an ion exiting the channel into the central core of the protein as it moves to the ferroxidase site. The diffusion of an Fe²⁺ ion from the inner opening of the channel to a ferroxidase site located in the interior region of the protein coat is assisted by Thr135, His136 and Tyr137. The Fe²⁺ ion binds preferentially to site A of the ferroxidase site. © 2013 Wiley Periodicals, Inc.     

Membrane association and selectivity of the antimicrobial peptide NK‐2: a molecular dynamics simulation study

In an effort to better understand the initial mechanism of selectivity and membrane association of the synthetic antimicrobial peptide NK‐2, we have applied molecular dynamics simulation techniques to elucidate the interaction of the peptide with the membrane interfaces. A homogeneous dipalmitoylphosphatidylglycerol (DPPG) and a homogeneous dipalmitoylphosphatidylethanolamine (DPPE) bilayers were taken as model systems for the cytoplasmic bacterial and human erythrocyte membranes, respectively. The results of our simulations on DPPG and DPPE model membranes in the gel phase show that the binding of the peptide, which is considerably stronger for the negatively charged DPPG lipid bilayer than for the zwitterionic DPPE, is mostly governed by electrostatic interactions between negatively charged residues in the membrane and positively charged residues in the peptide. In addition, a characteristic distribution of positively charged residues along the helix facilitates a peptide orientation parallel to the membrane interface. Once the peptides reside close to the membrane surface of DPPG with the more hydrophobic side chains embedded into the membrane interface, the peptide initially disturbs the respective bilayer integrity by a decrease of the order parameter of lipid acyl chain close to the head group region, and by a slightly decrease in bilayer thickness. We found that the peptide retains a high content of helical structure on the zwitterionic membrane‐water interface, while the loss of α‐helicity is observed within a peptide adsorbed onto negatively charged lipid membranes. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Investigation of the induced-fit mechanism and catalytic activity of the human cytomegalovirus protease homodimer via molecular dynamics simulations

Human cytomegalovirus (HCMV) is a highly species-specific DNA virus infecting up to 80% of the general population. The viral genome contains the open reading frame UL80, which encodes the full-length 80 kDa HCMV serine protease and its substrate. Full-length HCMV protease is composed of an N-terminal 256-amino-acid proteolytic domain, called assemblin, a linker region, and a C-terminal structural domain, the assembly protein precursor. Biochemical studies have shown that dimerization activates assemblin because of an induced stabilization of the oxyanion hole (Arg166). Thus, we performed here molecular dynamics (MD) simulations on HCMV protease models to study the induced-fit mechanism of the enzyme upon the binding of substrates and peptidyl inhibitors, and structural and energetic factors that are responsible for the catalytic activity of the enzyme dimer. Long and stable trajectories were obtained for the models of the monomeric and dimeric states, free in solution and bound to a peptidyl-activated carbonyl inhibitor, with very good agreement between theoretical and experimental results. Our results suggest that HCMV protease is indeed a novel example of serine protease that operates by an induced-fit mechanism. Also, in agreement with mutagenesis studies, our MD simulations suggest that the dimeric form is necessary to activate the enzyme because of an induced stabilization of the oxyanion hole.