MDFF_NM: Improved Molecular Dynamics Flexible Fitting into Cryo-EM Density Maps with a Multireplica Normal Mode-Based Search.

Molecular Dynamics Flexible Fitting (MDFF) is a widely used tool to refine high-resolution structures into cryo-EM density maps. Despite many successful applications, MDFF is still limited by its high computational cost, overfitting, accuracy, and performance issues due to entrapment within wrong local minima. Modern ensemble-based MDFF tools have generated promising results in the past decade. In line with these studies, we present MDFF_NM, a stochastic hybrid flexible fitting algorithm combining Normal Mode Analysis (NMA) and simulation-based flexible fitting. Initial tests reveal that, besides accelerating the fitting process, MDFF_NM increases the diversity of fitting routes leading to the target, uncovering ensembles of conformations in closer agreement with experimental data. The potential integration of MDFF_NM with other existing methods and integrative modeling pipelines is also discussed.

Assessment of Efficacy and Safety of Zagotenemab: Results From PERISCOPE-ALZ, a Phase 2 Study in Early Symptomatic Alzheimer Disease.

BACKGROUND AND OBJECTIVES: Zagotenemab (LY3303560), a monoclonal antibody that preferentially targets misfolded, extracellular, aggregated tau, was assessed in the PERISCOPE-ALZ phase 2 study to determine its ability to slow cognitive and functional decline relative to placebo in early symptomatic Alzheimer disease (AD). METHODS: Participants were enrolled across 56 sites in North America and Japan. Key eligibility criteria included age of 60-85 years, Mini-Mental State Examination score of 20-28, and intermediate levels of brain tau on PET imaging. In this double-blind study, participants were equally randomized to 1,400 mg or 5,600 mg of zagotenemab, or placebo (IV infusion every 4 weeks for 100 weeks). The primary outcome was change on the Integrated AD Rating Scale (iADRS) assessed by a Bayesian Disease Progression model. Secondary measures include mixed model repeated measures analysis of additional cognitive and functional endpoints as well as biomarkers of AD pathology. RESULTS: A total of 360 participants (mean age = 75.4 years; female = 52.8%) were randomized, and 218 completed the treatment period. Demographics and baseline characteristics were reasonably balanced among arms. The mean disease progression ratio (proportional decline in the treated vs placebo group) with 95% credible intervals for the iADRS was 1.10 (0.959-1.265) for the zagotenemab low-dose group and 1.05 (0.907-1.209) for the high-dose, where a ratio less than 1 favors the treatment group. Secondary clinical endpoint measures failed to show a drug-placebo difference in favor of zagotenemab. No treatment effect was demonstrated by flortaucipir PET, volumetric MRI, or neurofilament light chain (NfL) analyses. A dose-related increase in plasma phosphorylated tau181 and total tau was demonstrated. Zagotenemab treatment groups reported a higher incidence of adverse events (AEs) (85.1%) compared with the placebo group (74.6%). This difference was not attributable to any specific AE or category of AEs. DISCUSSION: In participants with early symptomatic AD, zagotenemab failed to achieve significant slowing of clinical disease progression compared with placebo. Imaging biomarker and plasma NfL findings did not show evidence of pharmacodynamic activity or disease modification. TRIAL REGISTRATION INFORMATION: ClinicalTrials.gov: NCT03518073. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with early symptomatic AD, zagotenemab does not slow clinical disease progression.

Safety, Tolerability, and Pharmacokinetics of Zagotenemab in Participants with Symptomatic Alzheimer's Disease: A Phase I Clinical Trial.

Background: Zagotenemab (LY3303560), a monoclonal antibody, preferentially binds to extracellular, misfolded, aggregated tau that has been implicated in Alzheimer's disease (AD). Objective: The goal of this study was to assess the safety and pharmacokinetics of multiple doses of zagotenemab in participants with AD. Methods: This was a Phase Ib, multi-site, participant- and investigator-blind, placebo-controlled, parallel-group study in participants with mild cognitive impairment due to AD or mild to moderate AD. After screening, participants were randomized to zagotenemab 70 mg, 210 mg, or placebo every 4 weeks for up to 49 weeks and were followed up for 16 weeks. Results: A total of 13 males and 9 females, aged 59 to 84 years, were dosed. No deaths occurred during this study. A total of 4 serious adverse events occurred in 2 participants who then discontinued the study. The most commonly reported (3 or more participants) treatment-emergent adverse events were sinus bradycardia, headache, fall, and bronchitis. The pharmacokinetics profile showed generally linear exposures across the dose range studied with a clearance of ~8 mL/h. The half-life of zagotenemab in serum was ~20 days. A dose-dependent increase in plasma tau was observed. No other significant pharmacodynamic differences were observed due to low dose levels and limited treatment duration. Conclusions: No dose-limiting adverse events were observed with zagotenemab treatment. Pharmacokinetics of zagotenemab were typical for a monoclonal antibody. Meaningful pharmacodynamic differences were not observed.Clinicaltrials.gov: NCT03019536.

Small GTPase Ran: Depicting the nucleotide-specific conformational landscape of the functionally important C-terminus.

The small GTPase Ran is the main regulator of the nucleo-cytoplasmic import and export through the nuclear pore complex. It functions as a molecular switch cycling between the GDP-bound inactive and GTP-bound active state. It consists of a globular (G) domain and a C-terminal region, which is bound to the G-domain in the inactive, GDP-bound states. Crystal structures of the GTP-bound active form complexed with Ran binding proteins (RanBP) show that the C-terminus undergoes a large conformational change, embracing Ran binding domains (RanBD). Whereas in the crystal structures of macromolecular complexes not containing RanBDs the structure of the C-terminal segment remains unresolved, indicating its large conformational flexibility. This movement could not have been followed either by experimental or simulation methods. Here, starting from the crystal structure of Ran in both GDP- and GTP-bound forms we show how rigid the C-terminal region in the inactive structure is during molecular dynamics (MD) simulations. Furthermore, we show how MD simulations of the active form are incapable of mapping the open conformations of the C-terminus. By using the MDeNM (Molecular Dynamics with excited Normal Modes) method, we were able to widely map the conformational surface of the C-terminus of Ran in the active GTP-bound form, which allows us to envisage how it can embrace RanBDs.

MDexciteR: Enhanced Sampling Molecular Dynamics by Excited Normal Modes or Principal Components Obtained from Experiments.

Molecular dynamics with excited normal modes (MDeNM) is an enhanced sampling method for exploring conformational changes in proteins with minimal biases. The excitation corresponds to injecting kinetic energy along normal modes describing intrinsic collective motions. Herein, we developed a new automated open-source implementation, MDexciteR (https://github.com/mcosta27/MDexciteR), enabling the integration of MDeNM with two commonly used simulation programs with GPU support. Second, we generalized the method to include the excitation of principal components calculated from experimental ensembles. Finally, we evaluated whether the use of coarse-grained normal modes calculated with elastic network representations preserved the performance and accuracy of the method. The advantages and limitations of these new approaches are discussed based on results obtained for three different protein test cases: two globular and a protein/membrane system.

Machine learning and structure-based modeling for the prediction of UDP-glucuronosyltransferase inhibition.

UDP-glucuronosyltransferases (UGTs) are responsible for 35% of the phase II drug metabolism. In this study, we focused on UGT1A1, which is a key UGT isoform. Strong inhibition of UGT1A1 may trigger adverse drug/herb-drug interactions, or result in disorders of endobiotic metabolism. Most of the current machine learning methods predicting the inhibition of drug metabolizing enzymes neglect protein structure and dynamics, both being essential for the recognition of various substrates and inhibitors. We performed molecular dynamics simulations on a homology model of the human UGT1A1 structure containing both the cofactor- (UDP-glucuronic acid) and substrate-binding domains to explore UGT conformational changes. Then, we created models for the prediction of UGT1A1 inhibitors by integrating information on UGT1A1 structure and dynamics, interactions with diverse ligands, and machine learning. These models can be helpful for further prediction of drug-drug interactions of drug candidates and safety treatments.

Cryo-electron microscopy unveils unique structural features of the human Kir2.1 channel.

We present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen's syndrome. The structural analysis (cryo-electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP2-binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP2. Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel.

Sampling of Protein Conformational Space Using Hybrid Simulations: A Critical Assessment of Recent Methods.

Recent years have seen several hybrid simulation methods for exploring the conformational space of proteins and their complexes or assemblies. These methods often combine fast analytical approaches with computationally expensive full atomic molecular dynamics (MD) simulations with the goal of rapidly sampling large and cooperative conformational changes at full atomic resolution. We present here a systematic comparison of the utility and limits of four such hybrid methods that have been introduced in recent years: MD with excited normal modes (MDeNM), collective modes-driven MD (CoMD), and elastic network model (ENM)-based generation, clustering, and relaxation of conformations (ClustENM) as well as its updated version integrated with MD simulations (ClustENMD). We analyzed the predicted conformational spaces using each of these four hybrid methods, applied to four well-studied proteins, triosephosphate isomerase (TIM), 3-phosphoglycerate kinase (PGK), HIV-1 protease (PR) and HIV-1 reverse transcriptase (RT), which provide extensive ensembles of experimental structures for benchmarking and comparing the methods. We show that a rigorous multi-faceted comparison and multiple metrics are necessary to properly assess the differences between conformational ensembles and provide an optimal protocol for achieving good agreement with experimental data. While all four hybrid methods perform well in general, being especially useful as computationally efficient methods that retain atomic resolution, the systematic analysis of the same systems by these four hybrid methods highlights the strengths and limitations of the methods and provides guidance for parameters and protocols to be adopted in future studies.

Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor.

There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Unexpected Gating Behaviour of an Engineered Potassium Channel Kir.

In this study, we investigated the dynamics and functional characteristics of the KirBac3.1 S129R, a mutated bacterial potassium channel for which the inner pore-lining helix (TM2) was engineered so that the bundle crossing is trapped in an open conformation. The structure of this channel has been previously determined at high atomic resolution. We explored the dynamical characteristics of this open state channel using an in silico method MDeNM that combines molecular dynamics simulations and normal modes. We captured the global and local motions at the mutation level and compared these data with HDX-MS experiments. MDeNM provided also an estimation of the probability of the different opening states that are in agreement with our electrophysiological experiments. In the S129R mutant, the Arg129 mutation releases the two constriction points in the channel that existed in the wild type but interestingly creates another restriction point.

Insights into the substrate binding mechanism of SULT1A1 through molecular dynamics with excited normal modes simulations.

Sulfotransferases (SULTs) are phase II drug-metabolizing enzymes catalyzing the sulfoconjugation from the co-factor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to a substrate. It has been previously suggested that a considerable shift of SULT structure caused by PAPS binding could control the capability of SULT to bind large substrates. We employed molecular dynamics (MD) simulations and the recently developed approach of MD with excited normal modes (MDeNM) to elucidate molecular mechanisms guiding the recognition of diverse substrates and inhibitors by SULT1A1. MDeNM allowed exploring an extended conformational space of PAPS-bound SULT1A1, which has not been achieved up to now by using classical MD. The generated ensembles combined with docking of 132 SULT1A1 ligands shed new light on substrate and inhibitor binding mechanisms. Unexpectedly, our simulations and analyses on binding of the substrates estradiol and fulvestrant demonstrated that large conformational changes of the PAPS-bound SULT1A1 could occur independently of the co-factor movements that could be sufficient to accommodate large substrates as fulvestrant. Such structural displacements detected by the MDeNM simulations in the presence of the co-factor suggest that a wider range of drugs could be recognized by PAPS-bound SULT1A1 and highlight the utility of including MDeNM in protein-ligand interactions studies where major rearrangements are expected.

Multimolecular complexes of the phosphodiester anion with Zn(II) or Mg(II) and water molecules-Preliminary validations of a polarizable potential by ab initio quantum chemistry.

Dimethyl phosphate (DMP- ) is a model for the phosphodiester backbone of DNA, RNA, and phospholipids. It is central for the binding of divalent cations and water along the backbone of nucleic acids. Significant polarization and charge-transfer contributions and nonadditivity come into play in the multimolecular complexes organized around phosphate. Prior to large-scale molecular dynamics (MD) with advanced polarizable potentials, it is essential to evaluate how well the values and trends of intermolecular interaction energies (ΔE) from ab initio quantum chemistry (QC) and their individual contributions are reproduced in a diversity of such complexes. These differ by the starting binding modes of a divalent cation, Zn(II), namely direct, bi- or mono-dentate to anionic and/or ester oxygens, versus through-water binding. We present first the results from automated refinements of the individual contributions of the SIBFA potential with respect to their QC counterparts using a Zn(II) or a water probe. This is followed by validations on eight relaxed multimolecular complexes of DMP- with Zn(II) or Mg(II) and seven waters, then on sixteen complexes of DMP- with Zn(II) and eight waters in arrangements extracted from MD or energy-minimization on a droplet of sixty-four waters. This monitors the compared evolutions of SIBFA and QC ΔE and their individual contributions in the competing arrangements. Some waters, bridging Zn(II) and DMP- , were found to have exceptionally large dipole moments, of up to 3.8 Debye. The perspectives of extension to a flexible phosphodiester backbone are discussed in the context of the SIBFA potential for DNA and RNA.

Revealing the activation mechanism of autoinhibited RalF by integrated simulation and experimental approaches.

RalF is an Arf GEF from Legionella pneumophilia, the bacterium that causes severe pneumonia. In its crystal structure, RalF is in the autoinhibited form. A large-scale domain motion is expected to lift the autoinhibition, the mechanism of which is still unknown. Since RalF is activated in the presence of the membrane, its active structure and the structure of the RalF-Arf1 complex could not have been determined experimentally. On the simulation side, it has been proven that classical Molecular Dynamics (MD) alone is not efficient enough to map motions of such amplitude and determine the active conformation of RalF. In this article, using Molecular Dynamics with excited Normal Modes (MDeNM) combined with previous experimental findings we were able to determine the active RalF structure and the structure of the RalF-Arf1 complex in the presence of the membrane, bridging the gap between experiments and simulation.

Integrative Study of the Structural and Dynamical Properties of a KirBac3.1 Mutant: Functional Implication of a Highly Conserved Tryptophan in the Transmembrane Domain.

ATP-sensitive potassium (K-ATP) channels are ubiquitously expressed on the plasma membrane of cells in several organs, including the heart, pancreas, and brain, and they govern a wide range of physiological processes. In pancreatic ß-cells, K-ATP channels composed of Kir6.2 and SUR1 play a key role in coupling blood glucose and insulin secretion. A tryptophan residue located at the cytosolic end of the transmembrane helix is highly conserved in eukaryote and prokaryote Kir channels. Any mutation on this amino acid causes a gain of function and neonatal diabetes mellitus. In this study, we have investigated the effect of mutation on this highly conserved residue on a KirBac channel (prokaryotic homolog of mammalian Kir6.2). We provide the crystal structure of the mutant KirBac3.1 W46R (equivalent to W68R in Kir6.2) and its conformational flexibility properties using HDX-MS. In addition, the detailed dynamical view of the mutant during the gating was investigated using the in silico method. Finally, functional assays have been performed. A comparison of important structural determinants for the gating mechanism between the wild type KirBac and the mutant W46R suggests interesting structural and dynamical clues and a mechanism of action of the mutation that leads to the gain of function.

The allosteric activation mechanism of a phospholipase A2-like toxin from Bothrops jararacussu venom: a dynamic description.

The activation process of phospholipase A2-like (PLA2-like) toxins is a key step in their molecular mechanism, which involves oligomeric changes leading to the exposure of specific sites. Few studies have focused on the characterization of allosteric activators and the features that distinguish them from inhibitors. Herein, a comprehensive study with the BthTX-I toxin from Bothrops jararacussu venom bound or unbound to α-tocopherol (αT) was carried out. The oligomerization state of BthTX-I bound or unbound to αT in solution was studied and indicated that the toxin is predominantly monomeric but tends to oligomerize when complexed with αT. In silico molecular simulations showed the toxin presents higher conformational changes in the absence of αT, which suggests that it is important to stabilize the structure of the toxin. The transition between the two states (active/inactive) was also studied, showing that only the unbound BthTX-I system could migrate to the inactive state. In contrast, the presence of αT induces the toxin to leave the inactive state, guiding it towards the active state, with more regions exposed to the solvent, particularly its active site. Finally, the structural determinants necessary for a molecule to be an inhibitor or activator were analyzed in light of the obtained results.

Nucleotide-Specific Autoinhibition of Full-Length K-Ras4B Identified by Extensive Conformational Sampling.

K-Ras is one of the most frequently mutated oncogenes in human tumor cells. It consists of a well-conserved globular catalytic domain and a flexible tail-like hypervariable region (HVR) at its C-terminal end. It plays a key role in signaling networks in proliferation, differentiation, and survival, undergoing a conformational switch between the active and inactive states. It is regulated through the GDP-GTP cycle of the inactive GDP-bound and active GTP-bound states. Here, without imposing any prior constraints, we mapped the interaction pattern between the catalytic domain and the HVR using Molecular Dynamics with excited Normal Modes (MDeNM) starting from an initially extended HVR conformation for both states. Our sampling captured similar interaction patterns in both GDP- and GTP-bound states with shifted populations depending on the bound nucleotide. In the GDP-bound state, the conformations where the HVR interacts with the effector lobe are more populated than in the GTP-bound state, forming a buried thus autoinhibited catalytic site; in the GTP-bound state conformations where the HVR interacts with the allosteric lobe are more populated, overlapping the α3/α4 dimerization interface. The interaction of the GTP with Switch I and Switch II is stronger than that of the GDP in line with a decrease in the fluctuation upon GTP binding.

Towards gaining sight of multiscale events: utilizing network models and normal modes in hybrid methods.

With the explosion of normal mode analyses (NMAs) based on elastic network models (ENMs) in the last decade, and the proven precision of MD simulations for visualizing interactions at atomic scale, many hybrid methods have been proposed in recent years. These aim at exploiting the best of both worlds: the atomic precision of MD that often fall short of exploring time and length scales of biological interest, and the capability of ENM-NMA to predict the cooperative and often functional rearrangements of large structures and assemblies, albeit at low resolution. We present an overview of recent progress in the field with examples of successful applications highlighting the utility of such hybrid methods and pointing to emerging future directions guided by advances in experimental characterization of biomolecular systems structure and dynamics.

Unveiling functional motions based on point mutations in biased signaling systems: A normal mode study on nerve growth factor bound to TrkA.

Many receptors elicit signal transduction by activating multiple intracellular pathways. This transduction can be triggered by a non-specific ligand, which simultaneously activates all the signaling pathways of the receptors. However, the binding of one biased ligand preferentially trigger one pathway over another, in a process called biased signaling. The identification the functional motions related to each of these distinct pathways has a direct impact on the development of new effective and specific drugs. We show here how to detect specific functional motions by considering the case of the NGF/TrkA-Ig2 complex. NGF-mediated TrkA receptor activation is dependent on specific structural motions that trigger the neuronal growth, development, and survival of neurons in nervous system. The R221W mutation in the ngf gene impairs nociceptive signaling. We discuss how the large-scale structural effects of this mutation lead to the suppression of collective motions necessary to induce TrkA activation of nociceptive signaling. Our results suggest that subtle changes in the NGF interaction network due to the point mutation are sufficient to inhibit the motions of TrkA receptors putatively linked to nociception. The methodological approach presented in this article, based jointly on the normal mode analysis and the experimentally observed functional alterations due to point mutations provides an essential tool to reveal the structural changes and motions linked to the disease, which in turn could be necessary for a drug design study.

New Structural insights into Kir channel gating from molecular simulations, HDX-MS and functional studies.

Inward rectifier potassium (Kir) channels play diverse and important roles in shaping action potentials in biological membranes. An increasing number of diseases are now known to be directly associated with abnormal Kir function. However, the gating of Kir still remains unknown. To increase our understanding of its gating mechanism, a dynamical view of the entire channel is essential. Here the gating activation was studied using a recent developped in silico method, MDeNM, which combines normal mode analysis and molecular dynamics simulations that showed for the very first time the importance of interrelated collective and localized conformational movements. In particular, we highlighted the role played by concerted movements of the different regions throughout the entire protein, such as the cytoplasmic and transmembrane domains and the slide helices. In addition, the HDX-MS analysis achieved in these studies provided a comprehensive and detailed view of the dynamics associated with open/closed transition of the Kir channel in coherence with the theoretical results. MDeNM gives access to the probability of the different opening states that are in agreement with our electrophysiological experiments. The investigations presented in this article are important to remedy dysfunctional channels and are of interest for designing new pharmacological compounds.