Molecular Dynamics Simulations of the AAA Protein P97

Pre-B-Cell Leukemia Transcription Factor 1 Regulates Expression of Valosin-Containing Protein, a Gene Involved in Cancer Growth

Structural insight into mutations at 155 position of valosin containing protein (VCP) linked to inclusion body myopathy with Paget disease of bone and frontotemporal Dementia.

"Everything that living things do can be understood in terms of jigglings and wigglings of atoms." Richard Feynman's remarks in the early 1960's summarize what is today widely accepted, namely, that biological processes can be described by the dynamics of biomolecules. Molecular dynamics (MD) simulation, in this regard, is the main methodology employed in structural biology to explore the dynamical behavior of macromolecules at a microscopic level. Aided by MD, researchers have been able, for instance, to resolve atomic structures of multi-protein complexes from cryo-EM densities, thus unveiling the atomistic details of enzymatic mechanisms and characterize the binding of small molecules to proteins. To achieve all this, the capabilities of MD packages are constantly evolving, providing a multitude of complex simulation and analysis techniques, e.g., enhanced sampling and free energy calculations. Although applicable to a wide variety of research problems, a broader usage of MD is hindered by a steep initial learning curve imposed by nearly every MD software. To reduce this initial barrier and make the methodology more accessible to the general community of biomolecular researchers, we developed an intuitive tool named QwikMD (1), which assists the users in the preparation, execution, and analysis of biomolecular MD simulations. Among many other features, QwikMD automatically checks the initial structure for structural inconsistencies, facilitates structure manipulations such as point mutations and partial deletions, simplifies the protein insertion in lipid membranes and enables the visualization and analysis of MD simulations on the fly. The user-friendly graphical interface of QwiKMD allows the preparation of MD simulations in a point-and-click fashion, offering the user multiple MD protocols, such as unbiased MD simulations, Steered MD, MD Flexible Fitting (MDFF), and, most recently, hybrid QM/MM simulations. The latter exploits the recently developed VMD and NAMD interface to common quantum mechanics software packages. QwikMD facilitates performing MD simulations for nearly any user, novice or expert. While assisting the user, QwikMD ensures reproducibility of the results by recording all parameters and steps into two log files, one in a script-like format and another in a "methods section" format. QwikMD also serves as a learning tool, providing the theoretical background of the different MD protocols and options in many "info buttons". J. V Ribeiro et al., QwikMD — Integrative Molecular Dynamics Toolkit for Novices and Experts. Sci. Rep. 6, 26536 (2016).

Scorpion venom is a rich source of toxins which have great potential to develop new therapeutic agents. Scorpion chloride channel toxins (ClTxs), such as Chlorotoxin selectively inhibit human Matrix Methaloproteinase-2 (hMMP-2). The inhibitors of hMMP-2 have potential use in cancer therapy. Three new ClTxs, meuCl14, meuCl15 and meuCl16, derived from the venom transcriptome of Iranian scorpion, M. eupeus (Buthidea family), show high sequence identity (71.4%) with Chlorotoxin. Here, 3-D homology model of new ClTxs were constructed. The models were optimized by Molecular Dynamics simulation based on MDFF (molecular dynamics flexible fitting) method. New ClTxs indicate the presence of CSαβ folding of other scorpion toxins. A docking followed by steered molecular dynamics (SMD) simulations to investigate the interactions of meuCl14, meuCl15, and meuCl16 with hMMP-2 was applied. The current study creates a correlation between the unbinding force and the inhibition activities of meuCl14, meuCl15 and meuCl16 to shed some insights as to which toxin may be used as a drug deliverer. To this aim, SMD simulations using Constant Force Pulling method were carried out. The SMD provided useful details related to the changes of electrostatic, van de Waals (vdW), and hydrogen-bonding (H-bonding) interactions between ligands and receptor during the pathway of unbinding. According to SMD results, the interaction of hMMP-2 with meuCl14 is more stable. In addition, Arginine residue was found to contribute significantly in interaction of ClTxs with hMMP-2. All in all, the present study is a dynamical approach whose results are capable of being implemented in structure-based drug design.

Integrated computational characterization of valosin-containing protein double-psi β-barrel domain: Insights into structural stability, binding mechanisms, and evolutionary significance.

Two structure determination methods, based on the molecular dynamics flexible fitting (MDFF) paradigm, are presented that resolve sub-5 Å cryo-electron microscopy (EM) maps with either single structures or ensembles of such structures. The methods, denoted cascade MDFF and resolution exchange MDFF, sequentially re-refine a search model against a series of maps of progressively higher resolutions, which ends with the original experimental resolution. Application of sequential re-refinement enables MDFF to achieve a radius of convergence of ~25 Å demonstrated with the accurate modeling of β-galactosidase and TRPV1 proteins at 3.2 Å and 3.4 Å resolution, respectively. The MDFF refinements uniquely offer map-model validation and B-factor determination criteria based on the inherent dynamics of the macromolecules studied, captured by means of local root mean square fluctuations. The MDFF tools described are available to researchers through an easy-to-use and cost-effective cloud computing resource on Amazon Web Services.DOI: http://dx.doi.org/10.7554/eLife.16105.001

Striated muscle contraction is regulated in a calcium-dependent manner through dynamic motions of the tropomyosin/troponin polymer, a multicomponent complex wrapped around actin-containing thin filaments. Tropomyosin/troponin sterically blocks myosin-binding at low-calcium concentrations but moves to expose myosin-binding sites at high-calcium concentrations leading to force development. Understanding the key intermolecular interactions that define these dynamic motions will promote our understanding of mutation-induced contractile dysfunction that eventually leads to hypertrophic cardiomyopathy, dilated cardiomyopathy, and skeletal myopathies. Advancements in cryoelectron microscopy (cryoEM) have resulted in a partial elucidation of structures of the thin filament, revealing many atomic-level interactions between the component proteins and critical calcium-dependent conformational alterations. However, building models at the resolutions achieved can be challenging since landmarks in the maps are often missing or ambiguous. Therefore, current computational analyses including de novo structure prediction, protein-protein docking, molecular dynamics flexible fitting, and molecular dynamics simulations are needed to ensure good quality models. We review here our efforts to model the troponin T domain spanning the head-to-tail overlap domain of tropomyosin, improving previous models. Next, we refined the published cryoEM modeled structures, which had mistakenly compressed alpha helices, with a model that has expected helical parameters while matching densities in the cryoEM volume. Lastly, we used this model to reinterpret the interactions between tropomyosin and troponin I showing key features that hold the tropomyosin cable in its low-calcium, sterically blocking position. These revised thin filament models show improved intermolecular interactions in the key low- and high-calcium regulatory states, providing novel insights into function.

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage phi KZ has been determined to 2.5-A resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. phi KZ gp144 is a 260-residue alpha-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The fold of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G D-Ala-D-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu 115 in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the phi KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine)(4) has been determined to 2.6-A resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.

Calcium-dependent lectin I from Pseudomonas aeruginosa (PA-IL) binds specifically to oligosaccharides presenting an alpha-galactose residue at their nonreducing end, such as the disaccharides alphaGal1-2betaGalOMe, alphaGal1-3betaGalOMe, and alphaGal1-4betaGalOMe. This provides a unique model for studying the effect of the glycosidic linkage of the ligands on structure and thermodynamics of the complexes by means of experimental and theoretical tools. The structural features of PA-IL in complex with the three disaccharides were established by docking and molecular dynamics simulations and compared with those observed in available crystal structures, including PA-IL.alphaGal1-2betaGalOMe complex, which was solved at 2.4 A resolution and reported herein. The role of a structural bridge water molecule in the binding site of PA-IL was also elucidated through molecular dynamics simulations and free energy calculations. This water molecule establishes three very stable hydrogen bonds with O6 of nonreducing galactose, oxygen from Pro-51 main chain, and nitrogen from Gln-53 main chain of the lectin binding site. Binding free energies for PA-IL in complex with the three disaccharides were investigated, and the results were compared with the experimental data determined by titration microcalorimetry. When the bridge water molecule was included in the free energy calculations, the simulations predicted the correct binding affinity trends with the 1-2-linked disaccharide presenting three times stronger affinity ligand than the other two. These results highlight the role of the water molecule in the binding site of PA-IL and indicate that it should be taken into account when designing glycoderivatives active against P. aeruginosa adhesion.

Advances in cryoelectron microscopy (cryo-EM) have revolutionized the structural investigation of large macromolecular assemblies. In this review, we first provide a broad overview of modeling methods used for flexible fitting of molecular models into cryo-EM density maps. We give special attention to approaches rooted in molecular simulations-atomistic molecular dynamics and Monte Carlo. Concise descriptions of the methods are given along with discussion of their advantages, limitations, and most popular alternatives. We also describe recent extensions of the widely used molecular dynamics flexible fitting (MDFF) method and discuss how different model-building techniques could be incorporated into new hybrid modeling schemes and simulation workflows. Finally, we provide two illustrative examples of model-building and refinement strategies employing MDFF, cascade MDFF, and RosettaCM. These examples come from recent cryo-EM studies that elucidated transcription preinitiation complexes and shed light on the functional roles of these assemblies in gene expression and gene regulation.

Advances in the molecular dynamics flexible fitting method for cryo-EM modeling

<h3>Abstract</h3> RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The...

Cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids, including cholesteryl esters (CEs) and triglycerides (TGs), between HDL, LDL and VLDL. Lipoprotein particles contain a neutral lipid core composed of CE and TG surrounded by a surface monolayer of phospholipids (PL), free cholesterol (FC), and apolipoproteins, most notably, apo B-100 in LDL and VLDL and apo A-I in HDL. An elevated level of LDL-cholesterol (LDL-C) and/or a low level of HDL-cholesterol (HDL-C) in human plasma are major risk factors for cardiovascular disease (CVD). Since increased CETP can reduce HDL-C concentration and CETP deficiency is associated with elevated HDL-C levels, CETP inhibitors, including torcetrapib, anacetrapib and dalcetrapib have been investigated in clinical trials for treating CVD. Despite the intense clinical interest in CETP inhibition, little is known concerning the molecular mechanisms of CETP-mediated lipid transfer among lipoproteins, or even how CETP interacts with lipoproteins. CETP is a hydrophobic glycoprotein of 476 amino acids (∼53 kDa, before posttranslational modification). Its crystal structure reveals a banana-shaped molecule with N- and C-terminal β-barrel domains, a central β-sheet, and a ∼60 Å-long hydrophobic central cavity. Three CETP neutral lipid transfer hypotheses were proposed more than two decades ago: 1) a shuttle mechanism that involves CETP collecting CEs from one lipoprotein and delivering them through the aqueous phase to another lipoprotein; 2) a tunnel mechanism in which CETP bridges two lipoproteins forming a ternary complex, with lipids flowing from the donor to acceptor lipoprotein through the CETP molecule; and 3) a modified tunnel mechanism implicating a CETP dimer. One difficulty in investigating CETP mechanisms using structural methods is that interaction with CETP can alter the size, shape, and composition of lipoproteins, especially HDL. We validated an optimized negative-staining electron microscopy (NS-EM) protocol in which flash-fixation of lipoprotein particles preserves a near native-state conformation for direct visualization of individual molecular or macromolecular particles. We applied this protocol to study the mechanisms by which CETP interacts with spherical HDL, LDL and VLDL. Three-dimensional (3D) reconstructions of CETP, free and HDL-bound, were obtained by single-particle techniques. In addition, we used inhibitory CETP antibodies to identify the regions of CETP that interact with HDL and LDL. Finally molecular dynamics (MD) simulation was used to assess the molecular mobility of CETP and predict the likely conformational changes that are associated with lipid transfer. We discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into HDL and its C-terminal domain interacting with LDL or VLDL. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.

Voltage gated potassium ion (Kv) channels regulate action potentials of the nervous system by responding to changes in transmembrane voltage, enabling K+ transport across the membrane to restore cells to their resting potential. Comprised of four identical subunits, Kv channels contain four voltage sensing domains arranged on the periphery of a central pore domain. Each voltage sensor is comprised of four transmembrane helices, numbered S1 through S4. The S4 helix, containing four to six highly-conserved, positively-charged arginine or lysine residues, is responsible for voltage sensitivity in Kv channels. The pore domain consists of two transmembrane helices, S5 and S6. The S5 helix constitutes the periphery of the pore domain and is believed to be relatively immobile. The S6 helices, lining the interior of the channel, gate the protein and regulate K+ permeation. Because each subunit of Kv channels contains six transmembrane helices, they are often referred to as 6TM Kv channels. The depolarization of an action potential is initiated as sodium ions enter the cell. At the cellular resting potential of -70 mV, potassium ion channels are closed, and the S4 helix is in its “down” state. As the electrochemical gradient changes, the S4 helices of Kv channels begin to reorient within the membrane. At the peak of the action potential (roughly +20 mV), the S4 helices exist in their “up” state. This conformational transition of the S4 helix is coupled to the pore domain via the S4-S5 linker, a short, amphipathic helix along the intracellular membrane-water interface. By bridging the C-terminus of the voltage sensor to the N-terminus of the pore domain, the S4-S5 linker couples the voltage sensitivity of the voltage sensor to K+ conduction in the pore domain. Because they begin opening at voltages less than 0 mV, all crystal structures of Kv channels contain an open pore domain. With no structure in the closed conformation, the mechanism of gating in Kv channels remains unclear. Nevertheless, significant biophysical studies have revealed insights into both the closed conformation and the gating transition itself. In this dissertation, I will explore questions relevant to the gating mechanism in voltage gated potassium ion channels through fully-atomistic molecular dynamics (MD) simulations. First, in Chapter 2, I will address the potential role of the 310 helical conformation found in the C-terminal end of S4 in the crystal structures of Kv channels. Spanning eight or more residues, these 310 helices are both uncharacteristically long and conserved in K+ channel crystal structures. By simulating the Kv1.2/2.1 chimera channel’s voltage sensor embedded in a lipid bilayer, I find that an alpha to 310 helical interconversion of the S4 helix reproduces many experimental measurements of the open and closed states of Kv channels. In Chapter 3, I perform molecular dynamics simulations of the entire Kv1.2/2.1 chimera channel. First, I examine the impact of an alpha to 310 helical interconversion of the S4 helix on the pore domain of the channel. Though the results are consistent with the results in Chapter 2 (and the corresponding experimental measurements), I find that this secondary structural modification is insufficient to influence the pore domain of the channel on the timescale of my simulations. In the second half of Chapter 3, I use molecular dynamics simulations to generate a closed state model of the Kv1.2/2.1 chimera from luminescence resonance energy transfer (LRET) measurements of the closed conformation of KvAP. The resulting structure is indeed closed, and also recapitulates a number of experimentally determined measurements of the closed channel. In Chapter 4, I focus on the pore domain. First, using targeted molecular dynamics simulations, I generate a transition between a closed model of the KvAP linker and pore domain to the open conformation. Then, using an umbrella sampling method, I quantify the energetics of the gating transition in KvAP and assess the physiological implications. In agreement with experimental studies of Kv channel energetics, I find that the open pore is roughly 2.7 kcal/mol lower in free energy than the closed conformation. The targeted molecular dynamics and umbrella sampling simulations reveal additional insights into the gating mechanism of KvAP. Lastly, in Chapter 5, I use MD simulations to gain insights into the binding mechanism of VSTx1, a Kv channel inhibitor. By using the experimentally determined neutron scattering density profile of the VSTx1 toxin bound to a lipid bilayer as a restraint for molecular dynamics simulations, I recreate the experimental scattering density profile, and also offer insight into the binding of VSTx1 to a lipid membrane.

Cryo-electron microscopy (EM) and X-ray studies proposed different mechanisms for annexin-induced membrane aggregation. In this work, molecular dynamics (MD) simulation technique was utilized to gain an insight into the calcium-induced conformational changes on annexin I and their implication in membrane aggregation mechanism. MD simulations were performed on the Ca2+ -free annexin I with the N-terminal domain buried inside the core (System 1), the Ca2+ -bound annexin I without N-terminal domain (System 2) and the Ca2+ -bound annexin I with the N-terminal domain exposed (System 3). Our results indicated that calcium binding increases the flexibility of annexin I core domain residues including the calcium coordinating residues. As a result, annexin I was activated to interact with the negatively charged membrane. The exposed N-terminal domain was very flexible and gradually lost the secondary structure during MD simulation, suggesting that the N-terminal may adopt a favorable conformation to bind a second membrane and also explaining the failure of attempts to crystallize the full-length annexin I in the presence of calcium ions. The measured dimensions of the averaged simulation structure of the Ca2+ -bound annexin I with the N-terminal exposed (System 3) support the proposed membrane aggregation mechanism based on X-ray studies.