Structural basis for recruitment of RILP by small GTPase Rab7

Rab7 regulates vesicle traffic from early to late endosomes, and from late endosomes to lysosomes. The crystal structure of Rab7-GTP in complex with the Rab7 binding domain of RILP reveals that Rab7 interacts with RILP specifically via two distinct areas, with the first one involving the switch and interswitch regions and the second one consisting of RabSF1 and RabSF4. Disruption of these interactions by mutations abrogates late endosomal/lysosomal targeting of Rab7 and RILP. The Rab7 binding domain of RILP forms a coiled-coil homodimer with two symmetric surfaces to interact with two separate Rab7-GTP molecules, forming a dyad configuration of Rab7-RILP(2)-Rab7. Mutations that disrupt RILP dimerization also abolish its interactions with Rab7-GTP and late endosomal/lysosomal targeting, suggesting that the dimeric form of RILP is a functional unit. Structural comparison suggests that the combined use of RabSF1 and RabSF4 with the switch regions may be a general mode of action for most Rab proteins in regulating membrane trafficking.     

Insight into the molecular switch mechanism of human Rab5a from molecular dynamics simulations

Rab5a is currently a most interesting target because it is responsible for regulating the early endosome fusion in endocytosis and possibly the budding process. We utilized longtime-scale molecular dynamics simulations to investigate the internal motion of the wild-type Rab5a and its A30P mutant. It was observed that, after binding with GTP, the global flexibility of the two proteins is increasing, while the local flexibility in their sensitive sites (P-loop, switch I and II regions) is decreasing. Also, the mutation of Ala30 to Pro30 can cause notable flexibility variations in the sensitive sites. However, this kind of variations is dramatically reduced after binding with GTP. Such a remarkable feature is mainly caused by the water network rearrangements in the sensitive sites. These findings might be of use for revealing the profound mechanism of the displacements of Rab5a switch regions, as well as the mechanism of the GDP dissociation and GTP association.     

Structural and mechanistic insights into the oxy form of tyrosinase from molecular dynamics simulations

The first, long time scale (16-ns) ligand field molecular dynamics (LFMD) simulations of the oxy form of tyrosinase are reported. The calculations use our existing type 3 copper force field for the peroxido-bridged [Cu₂O₂]²⁺ unit which is here translated from MMFF into the AMBER format together with a new charge scheme. The protein secondary and tertiary structures are not significantly altered by removing the ‘caddie’ protein, ORF378, which must be bound to tyrosinase before crystals will grow. A comprehensive principal component analysis of the Cartesian coordinates from the final 8 ns shows that the protein backbone is relatively rigid. However, the significant butterfly fold of the [Cu₂O₂]²⁺ moiety observed in the X-ray structure, presumably due to the caddie protein tyrosine at the active site, is absent in the simulations. LFMD gives a clear and persistent distinction between equatorial and axial Cu–N distances, with the latter about 0.2 Å longer and remaining syn to each other. However, the two coordination spheres display important differences. LFMD simulations of the symmetric model complex [μ-η²:μ²-O₂{Cu(Meim)₃}₂]²⁺ (Meim is 5-methyl-1H-imidazole) provide a mechanism for syn–anti interchange of axial ligands which suggests, in combination with the old experimental X-ray data, the new LFMD simulations and traditional coordination chemistry arguments, that His₅₄ on Cu_A is ‘insipiently axial’ and that a combination of a butterfly distortion of the [Cu₂O₂]²⁺ group and a rotation of the Cu_A(His)₃ moiety converts the vacant, initially axial, binding site on Cu_A into a much more favourable equatorial site.     

Structural insights into ligand binding features of dual FABP4/5 inhibitors by molecular dynamics simulations

Abstract

The fatty acid binding protein (FABP) 4 and 5 have been considered as potential targets for the treatment of metabolic diseases. A compensatory upregulation of FABP5 due to the gene ablation of FABP4 in adipocytes indicated the importance of dual FABP4/5 inhibitors. A few compounds have been discovered as dual FABP4/5 inhibitors. However, none exhibited equivalent inhibitory activity against both FABP4 and FABP5, and almost all compounds showed weaker inhibition against FABP5. To provide a better structural understanding for the design of potent dual FABP4/5 inhibitors, molecular dynamics simulations have been performed for 100?ns to disclose the ligand binding features in FABP4 and FABP5 using Amber14, respectively. Key residues were identified by analysis of close contact, hydrogen bond occupancy, binding free energy and alanine scanning mutagenesis. In addition, induced-fit effects have been observed upon ligand binding in the process of simulations. The shifted alkyl chain of ligand in FABP4 was significantly different from that in FABP5 due to the corresponding residues (Phe58^FABP4 and Leu60^FABP5). Thus, to avoid different steric effects made by these two residues, hydrophobic groups of suitable size should be taken into account. Besides, electrostatic and steric effects with Arg107^FABP4 and Arg109^FABP5 should be paid more attention to. The results will facilitate the rational design of dual FABP4/5 inhibitors.     

Rab35/ACAP2 and Rab35/RUSC2 Complex Structures Reveal Molecular Basis for Effector Recognition by Rab35 GTPase

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Structural insights by molecular dynamics simulations into differential repair efficiency for ethano-A versus etheno-A adducts by the human alkylpurine-DNA N-glycosylase

1,N⁶-ethenoadenine adducts (εA) are formed by known environmental carcinogens and found to be removed by human alkylpurine-DNA N-glycosylase (APNG). 1,N⁶-ethanoadenine (ΕA) adducts differ from εA by change of a double bond to a single bond in the 5-member exocyclic ring and are formed by chloroethyl nitrosoureas, which are used in cancer therapy. In this work, using purified recombinant human APNG, we show that ΕA is a substrate for the enzyme. However, the excision efficiency of ΕA was 65-fold lower than that of εA. Molecular dynamics simulation produced similar structural motifs for εA and ΕA when incorporated into a DNA duplex, suggesting that there are no specific conformational features in the DNA duplex which can account for the differences in repair efficiency. However, when ΕA was modeled into the APNG active site, based on the APNG/εA-DNA crystallographic coordinates, in structures produced by 2 ns molecular dynamics simulation, we observed weakening in the stacking interaction between ΕA and aromatic side chains of the key amino acids in the active site. In contrast, the planar εA is better stacked at the enzyme active site. We propose that the observed destabilization of the ΕA adduct at the active site, such as reduced stacking interactions, could account for the biochemically observed weaker recognition of ΕA by APNG as compared to εA.     

Probing the druggability of membrane-bound Rab5 by molecular dynamics simulations

Rab5 is a small GTPase and a key regulator in early endosomal trafficking. Rab5 and its effectors are involved in a large number of infectious diseases and certain types of cancer. We performed µs atomistic molecular dynamics simulations of inactive and active full-length Rab5 anchored to a complex model bilayer with composition of the early endosome membrane. Direct interactions between the Rab5?G domain and the bilayer were observed. We found two dominant nucleotide-dependent orientations characterised by a different accessibility of the switch regions. The “buried switch” orientation was mainly associated with inactive Rab5 accompanied with a rather extended structure of the hypervariable C-terminal region. Active Rab5 preferred an orientation in which the switch regions are accessible to effector proteins. These structural differences may provide an opportunity to selectively target one Rab5 state and lead to new approaches in the development of Rab5-specific therapies.     

The RAB Family GTPase Rab1A from Plasmodium falciparum Defines a Unique Paralog Shared by Chromalveolates and Rhizaria

ABSTRACT. The RAB GTPases, which are involved in regulation of endomembrane trafficking, exhibit a complex but incompletely understood evolutionary history. We elucidated the evolution of the RAB1 subfamily ancestrally implicated in the endoplasmic reticulum-to-Golgi traffic. We found that RAB1 paralogs have been generated over the course of eukaryotic evolution, with some duplications coinciding with the advent of major eukaryotic lineages (e.g. Metazoa, haptophytes). We also identified a unique, derived RAB1 paralog, orthologous to the Plasmodium Rab1A, that occurs in stramenopiles, alveolates, and Rhizaria, represented by the chlorarachniophyte Gymnochlora stellata . This finding is consistent with the recently documented existence of a major eukaryotic clade ("SAR") comprising these three lineages. We further found a Rab1A-like protein in the cryptophyte Guillardia theta , but it exhibits unusual features among RAB proteins: absence of a C-terminal prenylation motif and an N-terminal extension with two MSP domains; and its phylogenetic relationships could not be established convincingly due to its divergent nature. Our results nevertheless point to a unique membrane trafficking pathway shared by at least some lineages of chromalveolates and Rhizaria, an insight that has implications towards interpreting the early evolution of eukaryotes and the endomembrane system.     

Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations

Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc-containing IMP-1 metallo-beta-lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal-containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc-zinc distance increased by approximately 1 A in the substrate complex compared with the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum-mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc-containing metallo-beta-lactamases as free proteins, in complex with inhibitors and in complex with substrates.     

Structural insight into antisense gapmer-RNA oligomer duplexes through molecular dynamics simulations

There is an extensive research carrying out on antisense technology and the molecules entering into clinical trials are increasing rapidly. Phosphorothioate (PS) is a chemical modification in which nonbridged oxygen is replaced with a sulfur, consequently providing resistance against nuclease activity. The 2'-4' conformationally restricted nucleoside has the structural features of both 2'-O-methoxy ethyl RNA (MOE), which shows good toxicity profile, and locked nucleic acid (LNA), which shows good binding affinity towards the target RNA. These modifications have been studied and suggested that they can be a potential therapeutic agents in antisense therapy. Mipomersen (ISIS 301012), which contains the novel nucleoside modification has been used to target to apolipoprotein (Apo B), which reduces LDL cholesterol by 6–41%. In this study, classical molecular dynamics (MD) simulations were performed on six different antisense gapmer/target-RNA oligomer duplexes (LNA-PS-LNA/RNA, RcMOE-PS-RcMOE/RNA, ScMOE-PS-ScMOE/RNA, MOE-PS-MOE/RNA, PS-DNA/RNA and DNA/RNA) to investigate the structural dynamics, stability and solvation properties. The LNA, MOE nucleotides present in respective duplexes are showing the structure of A-form and the PS-DNA nucleotides resemble the structure of B-form helix with respect to some of the helical parameters. Free energy calculations suggest that the oligomer, which contains LNA binds to the RNA strongly than other modifications as shown in experimental results. The MOE modified nucleotide, which although had a lower binding affinity but higher solvent accessible surface area (SASA) compared to the other modifications, may be influencing the toxicity and hence may be used it in Mipomersen, the second antisense molecule which is approved by FDA.

Communicated by Ramaswamy H. Sarma     

Structural insights into the anti-cancer activity of quercetin on G-tetrad,mixed G-tetrad,and G-quadruplex DNA using quantum chemical and molecular dynamics simulations

Abstract

Human telomerase referred as ‘terminal transferase’ is a nucleoprotein enzyme which inhibits the disintegration of telomere length and act as a drug target for the anticancer therapy. The tandem repeating structure of telomere sequence forms the guanine-rich quadruplex structures that stabilize stacked tetrads. In our present work, we have investigated the interaction of quercetin with DNA tetrads using DFT. Geometrical analysis revealed that the influence of quercetin drug induces the structural changes into the DNA tetrads. Among DNA tetrads, the quercetin stacked with GCGC tetrad has the highest interaction energy of ?88.08?kcal/mol. The binding mode and the structural stability are verified by the absorption spectroscopy method. The longer wavelength was found at 380?nm and it exhibits bathochromic shift. The findings help us to understand the binding nature of quercetin drug with DNA tetrads and it also inhibits the telomerase activity. Further, the quercetin drug interacted with G-quadruplex DNA by using molecular dynamics (MD) simulation studies for 100?ns simulation at different temperatures and different pH levels (T?=?298 K, 320?K and pH = 7.4, 5.4). The structural stability of the quercetin with G-quadruplex structure is confirmed by RMSD. For the acidic condition (pH = 5.4), the binding affinity is higher toward G-quadruplex DNA, this result resembles that the quercetin drug is well interacted with G-quadruplex DNA at acidic condition (pH = 7.4) than the neutral condition. The obtained results show that quercetin drug stabilizes the G-quadruplex DNA, which regulates telomerase enzyme and it potentially acts as a novel anti-cancer agent.

Communicated by Ramaswamy H. Sarma     

Structural insights by molecular dynamics simulations into specificity of the major human AP endonuclease toward the benzene-derived DNA adduct, pBQ-C

The benzetheno exocyclic adduct of the cytosine (C) base (pBQ-C) is a product of reaction between DNA and a stable metabolite of the human carcinogen benzene, p-benzoquinone (pBQ). We reported previously that the pBQ-C-containing duplex is a substrate for the human AP endonuclease (APE1), an enzyme that cleaves an apurinic/apyrimidinic (AP) site from double stranded DNA. In this work, using molecular dynamics simulation (MD), we provided a structural explanation for the recognition of the pBQ-C adduct by APE1. Molecular modeling of the DNA duplex containing pBQ-C revealed significant displacement of this adduct toward the major groove with pronounced kinking of the DNA at the lesion site, which could serve as a structural element recognized by the APE1 enzyme. Using 3 ns MD it was shown that the position of the pBQ-C adduct is stabilized by two hydrogen bonds formed between the adduct and the active site amino acids Asp 189 and Ala 175. The pBQ-C/APE1 complex, generated by MD, has a similar hydrogen bond network between target phosphodiester bond at the pBQ-C site and key amino acids at the active site, as in the crystallographically determined APE1 complexed with an AP site-containing DNA duplex. The position of the adduct at the enzyme active site, together with the hydrogen bond network, suggests a similar reaction mechanism for phosphodiester bond cleavage of oligonucleotide containing pBQ-C as reported for the AP site.     

A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p

Rab GTPases, the largest subgroup in the superfamily of Ras-like GTPases, play regulatory roles in multiple steps of intracellular vesicle trafficking. They are activated by guanine nucleotide exchange factors (GEFs), which catalyze the interconversion of the GDP-bound, or inactive, form of Rab to the GTP-bound, or active, form. Relatively little is known of the mechanisms by which GEFs activate Rabs. Here, we present the crystal structure of the GEF domain of Sec2p in complex with its Rab partner Sec4p. The Sec2p GEF domain is a 220 Angstroms long coiled coil, striking in its simplicity and in the use of the coiled-coil motif for catalysis. The structure suggests a mechanism whereby Sec2p induces extensive structural rearrangements in the Sec4p switch regions and phosphate-binding loop that are incompatible with nucleotide binding. We show that Sec2p is specific for Sec4p and that specificity determinants reside in the two switch regions of Sec4p.     

Structural insights of Staphylococcus aureus FtsZ inhibitors through molecular docking, 3D-QSAR and molecular dynamics simulations

Filamentous temperature-sensitive protein Z (FtsZ) is a protein encoded by the FtsZ gene that assembles into a Z-ring at the future site of the septum of bacterial cell division. Structurally, FtsZ is a homolog of eukaryotic tubulin but has low sequence similarity; this makes it possible to obtain FtsZ inhibitors without affecting the eukaryotic cell division. Computational studies were performed on a series of substituted 3-arylalkoxybenzamide derivatives reported as inhibitors of FtsZ activity in Staphylococcus aureus. Quantitative structure-activity relationship models (QSAR) models generated showed good statistical reliability, which is evident from r²_ncv and r²_loo values. The predictive ability of these models was determined and an acceptable predictive correlation (r²_Pred) values were obtained. Finally, we performed molecular dynamics simulations in order to examine the stability of protein-ligand interactions. This facilitated us to compare free binding energies of cocrystal ligand and newly designed molecule B1. The good concordance between the docking results and comparative molecular field analysis (CoMFA)/comparative molecular similarity indices analysis (CoMSIA) contour maps afforded obliging clues for the rational modification of molecules to design more potent FtsZ inhibitors.     

Cofactor activation and substrate binding in pyruvate decarboxylase. Insights into the reaction mechanism from molecular dynamics simulations

We have performed long-term molecular dynamics simulations of pyruvate decarboxylase from Zymomonas mobilis. Nine structures were modeled to investigate mechanistic questions related to binding of the cofactor, thiamin diphosphate (ThDP), and the substrate in the active site. The simulations reveal that the proposed three ThDP-tautomers all can bind in the active site and indicate that the equilibrium is shifted toward 4'-aminopyrimidine ThDP in the absence of substrate. 4'-Aminopyrimidinium ThDP is found to be a likely intermediate in the equilibrium. Mutations of important active site residues, Glu473Ala and Glu50Ala, were modeled to further elucidate their catalytic role. Formation of the catalytic important ylide by deprotonation of ThDP(C2) is investigated. Only the less favored tautomer, 1',4'-iminopyrimidine ThDP (imino-ThDP), could be deprotonated. The two other tautomers of ThDP could not be activated at the C2-position, thus, explaining the mechanistic importance of the less stable imino-ThDP. Finally, binding of pyruvate in the active site with the cofactor modeled as the nucleophilic ylide (ylide-ThDP) is studied. The carbonyl group of the substrate forms a hydrogen bond to Tyr290(OH). No hydrogen bond could be identified between ThDP(N4') and the substrate. The geometry of the substrate binding is well-suited for a nucleophilic attack by ylide-ThDP(C2). We propose that a proton relay from His113 via Asp27 and Tyr290 to the carbonyl oxygen atom of the substrate may be involved in the mechanism.     

Some insights into the binding mechanism of Aurora B kinase gained by molecular dynamics simulation

Aurora B kinase is essential in the process of mitosis, and its overexpression has been reported to be associated with a number of solid tumors. We therefore carried out molecular docking, molecular dynamics, and molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations on several structurally diverse inhibitors (pentacyclic, pyrimidine, quinazoline, and pyrrolopyridine derivatives) and Aurora B kinase to explore the structural and chemical features responsible for the binding recognition mechanism. Molecular simulations reveal that the binding site mainly consists of six binding regions (sites A-F). We have identified that sites B and C are required for optimum binding in Aurora B-inhibitor complexes, sites A and F are needed to improve pharmacokinetic properties, while sites D and E lead to enhanced stability. We verified that hydrogen bonding to the hinge region and hydrophobic contact with the conserved hydrophobic pocket are of critical importance in the systems studied. Specifically, the amino acids Glu171, Phe172, and Ala173 in the hinge region and Leu99, Val107, and Leu223 in the conserved hydrophobic pocket probably account for the high binding affinities of these systems, as shown by hydrogen-bonding analysis and energy decomposition analysis. Hydrophobic contact with Phe172 is also in agreement with experimental data. In addition, the MM-PBSA calculations reveal that the binding of these inhibitors to Aurora B kinase is mainly driven by van der Waals/nonpolar interactions. The findings of this study should help to elucidate the binding pattern of Aurora B inhibitors and aid in the design of novel active ligands.     

Structural reorganization of molecular sheets derived from cellulose II by molecular dynamics simulations

We investigated structural reorganization of two different kinds of molecular sheets derived from the cellulose II crystal using molecular dynamics (MD) simulations, in order to identify the initial structure of the cellulose crystal in the course of its regeneration process from solution. After a one-nanosecond simulation, the molecular sheet formed by van der Waals forces along the () crystal plane did not change its structure in an aqueous environment, while the other one formed by hydrogen bonds along the (1 1 0) crystal plane changed into a van der Waals-associated molecular sheet, such as the former. The two structures that were calculated showed substantial similarities such as the high occupancy of intramolecular hydrogen bonds between O3^H and O5 of over 0.75, few intermolecular hydrogen bonds, and the high occurrence of hydrogen bonding with water. The convergence of the two structures into one denotes that the van der Waals-associated molecular sheet can be the initial structure of the cellulose crystal formed in solution. The main chain conformations were almost the same as those in the cellulose II crystal except for a −16° shift of φ (dihedral angle of O5-C1-O1-C4) and the gauche-gauche conformation of the hydroxymethyl side group appears probably due to its hydrogen bonding with water. These results suggest that the van der Waals-associated molecular sheet becomes stable in an aqueous environment with its hydrophobic inside and hydrophilic periphery. Contrary to this, a benzene environment preferred a hydrogen-bonded molecular sheet, which is expected to be the initial structure formed in benzene.     

Structural insights into substrate binding by the molecular chaperone DnaK

How substrate affinity is modulated by nucleotide binding remains a fundamental, unanswered question in the study of 70 kDa heat shock protein (Hsp70) molecular chaperones. We find here that the Escherichia coli Hsp70, DnaK, lacking the entire alpha-helical domain, DnaK(1-507), retains the ability to support lambda phage replication in vivo and to pass information from the nucleotide binding domain to the substrate binding domain, and vice versa, in vitro. We determined the NMR solution structure of the corresponding substrate binding domain, DnaK(393-507), without substrate, and assessed the impact of substrate binding. Without bound substrate, loop L3,4 and strand beta3 are in significantly different conformations than observed in previous structures of the bound DnaK substrate binding domain, leading to occlusion of the substrate binding site. Upon substrate binding, the beta-domain shifts towards the structure seen in earlier X-ray and NMR structures. Taken together, our results suggest that conformational changes in the beta-domain itself contribute to the mechanism by which nucleotide binding modulates substrate binding affinity.