Importance of long-time simulations for rare event sampling in zinc finger proteins

Molecular dynamics (MD) simulation methods have seen significant improvement since their inception in the late 1950s. Constraints of simulation size and duration that once impeded the field have lessened with the advent of better algorithms, faster processors, and parallel computing. With newer techniques and hardware available, MD simulations of more biologically relevant timescales can now sample a broader range of conformational and dynamical changes including rare events. One concern in the literature has been under which circumstances it is sufficient to perform many shorter timescale simulations and under which circumstances fewer longer simulations are necessary. Herein, our simulations of the zinc finger NEMO (2JVX) using multiple simulations of length 15, 30, 1000, and 3000 ns are analyzed to provide clarity on this point.     

The mechanism of high affinity pentasaccharide binding to antithrombin,insights from Gaussian accelerated molecular dynamics simulations

Abstract

The activity of antithrombin (AT), a serpin protease inhibitor, is enhanced by heparin and heparin analogs against its target proteases, mainly thrombin, factors Xa and IXa. Considerable amount of information is available on the multistep mechanism of the heparin pentasaccharide binding and conformational activation. However, much of the details were inferred from ‘static’ structures obtained by X-ray diffraction. Moreover, limited information is available for the early steps of binding mechanism other than kinetic studies with various ligands. To gain insights into these processes, we performed enhanced sampling molecular dynamics (MD) simulations using the Gaussian Accelerated Molecular Dynamics (GAMD) method, applied previously in drug binding studies. We were able to observe the binding of the pentasaccharide idraparinux to a ‘non-activated’ AT conformation in two separate trajectories with low root mean square deviation (RMSD) values compared to X-ray structures of the bound state. These trajectories along with further simulations of the AT-pentasaccharide complex provided insights into the mechanisms of multiple conformational transitions, including the expulsion of the hinge region, the extension of helix D and the conformational behavior of the reactive center loop (RCL). We could also confirm the high stability of helix P in non-activated AT conformations, such states might play an important role in heparin binding. ‘Generalized correlation’ matrices revealed possible paths of allosteric signal propagation to the binding sites for the target proteases, factors Xa and IXa. Enhanced MD simulations of ligand binding to AT may assist the design of new anticoagulant drugs.

Communicated by Ramaswamy H. Sarma     

Nudged elastic band calculation of minimal energy paths for the conformational change of a GG non-canonical pair

The nudged elastic band (NEB) technique has been implemented in AMBER to calculate low-energy paths for conformational changes. A novel simulated annealing protocol that does not require an initial hypothesis for the path is used to sample low-energy paths. This was used to study the conformational change of an RNA cis Watson-Crick/Hoogsteen GG non-canonical pair, with one G syn around the glycosidic bond and the other anti. A previous solution structure, determined by NMR-constrained modeling, demonstrated that the GG pairs change from (syn)G-(anti)G to (anti)G-(syn)G in the context of duplex r(GCAGGCGUGC) on the millisecond timescale. The set of low-energy paths found by NEB show that each G flips independently around the glycosidic bond, with the anti G flipping to syn first. Guanine bases flip without opening adjacent base-pairs by protruding into the major groove, accommodated by a transient change by the ribose to C2'-exo sugar pucker. Hydrogen bonds between bases and the backbone, which lower the energetic barrier to flipping, are observed along the path. The results show the plasticity of RNA base-pairs in helices, which is important for biological processes, including mismatch repair, protein recognition, and translation. The modeling of the GG conformational change also demonstrates that NEB can be used to discover non-trivial paths for macromolecules and therefore NEB can be used as an exploratory method for predicting putative conformational change paths.     

Zinc Finger-like Motif Conserved in a Family of RNA Binding Proteins

NP220s compose a family of RNA binding proteins together with matrin 3, one of major proteins of the nuclear matrix. They have repeats of RNA recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear RNPs I/L in addition to MH1 and MH3 with unknown function. In search of additional homologous sequences, we found the reported sequence of rat matrin 3 is partially incorrect. Correction of this sequence showed that the NP220 family has a fourth homologous motif with the characteristics of a Cys₂-His₂ zinc finger-like motif. The sequence of this motif is perfectly conserved in human and mouse NP220s despite their 75% overall sequence homology.     

Molecular strategies to replace the structural metal site in the prokaryotic zinc finger domain

The specific arrangement of secondary elements in a local motif often totally relies on the formation of coordination bonds between metal ions and protein ligands. This is typified by the ~ 30 amino acid eukaryotic zinc finger motif in which a β-sheet and an α-helix are clustered around a zinc ion by various combinations of four ligands.     

The dynamic properties of the Hepatitis C Virus E2 envelope protein unraveled by molecular dynamics

Hepatitis C Virus (HCV) is one of the most persistent human viruses. Although effective therapeutic approaches have been recently discovered, their use is limited by the elevated costs. Therefore, the development of alternative/complementary strategies is an urgent need. The E2 glycoprotein, the most immunogenic HCV protein, and its variants represent natural candidates to achieve this goal. Here we report an extensive molecular dynamics (MD) analysis of the intrinsic properties of E2. Our data provide interesting clues on the global and local intrinsic dynamic features of the protein. Present MD data clearly indicate that E2 combines a flexible structure with a network of covalent bonds. Moreover, the analysis of the two most important antigenic regions of the protein provides some interesting insights into their intrinsic structural and dynamic properties. Our data indicate that a fluctuating β-hairpin represents a populated state by the region E2^412?423. Interestingly, the analysis of the epitope E2^427?446 conformation, that undergoes a remarkable rearrangement in the simulation, has significant similarities with the structure that the E2^430?442 fragment adopts in complex with a neutralizing antibody. Present data also suggest that the strict conservation of Gly436 in E2 protein of different HCV genotypes is likely dictated by structural restraints. Moreover, the analysis of the E2^412?423 flexibility provides insights into the mechanisms that some antibodies adopt to anchor Trp437 that is fully buried in E2. Finally, the present investigation suggests that MD simulations should systematically complement crystallographic studies on flexible proteins that are studied in combination with antibodies.     

Positive and negative cooperativity of modularly assembled zinc fingers

One simple and widespread method to create engineered zinc fingers targeting the desired DNA sequences is to modularly assemble multiple finger modules pre-selected to recognize each DNA triplet. However, it has become known that a sufficient DNA binding affinity is not always obtained. In order to create successful zinc finger proteins, it is important to understand the context-dependent contribution of each finger module to the DNA binding ability of the assembled zinc finger proteins. Here, we have created finger-deletion mutants of zinc finger proteins and examined the DNA bindings of these zinc fingers to clarify the contributions of each finger module. Our results indicate that not only a positive cooperativity but also a context-dependent reduction in the DNA binding activity can be induced by assembling zinc finger modules.