OneOPES, a Combined Enhanced Sampling Method to Rule Them All

Enhanced sampling techniques have revolutionized molecular dynamics (MD) simulations, enabling the study of rare events and the calculation of free energy differences in complex systems. One of the main families of enhanced sampling techniques uses physical degrees of freedom called collective variables (CVs) to accelerate a system’s dynamics and recover the original system’s statistics. However, encoding all the relevant degrees of freedom in a limited number of CVs is challenging, particularly in large biophysical systems. Another category of techniques, such as parallel tempering, simulates multiple replicas of the system in parallel, without requiring CVs. However, these methods may explore less relevant high-energy portions of the phase space and become computationally expensive for large systems. To overcome the limitations of both approaches, we propose a replica exchange method called OneOPES that combines the power of multireplica simulations and CV-based enhanced sampling. This method efficiently accelerates the phase space sampling without the need for ideal CVs, extensive parameters fine tuning nor the use of a large number of replicas, as demonstrated by its successful applications to protein–ligand binding and protein folding benchmark systems. Our approach shows promise as a new direction in the development of enhanced sampling techniques for molecular dynamics simulations, providing an efficient and robust framework for the study of complex and unexplored problems

Subcellular location defines GPCR signal transduction

Intracellular G protein-coupled receptors (GPCRs) can be activated by permeant ligands, which contributes to agonist selectivity. Opioid receptors (ORs) provide a notable example, where opioid drugs rapidly activate ORs in the Golgi apparatus. Our knowledge on intracellular GPCR function remains incomplete, and it is unknown whether OR signaling in plasma membrane (PM) and Golgi apparatus differs. Here, we assess the recruitment of signal transducers to mu- and delta-ORs in both compartments. We find that Golgi ORs couple to Gαi/o probes and are phosphorylated but, unlike PM receptors, do not recruit β-arrestin or a specific Gα probe. Molecular dynamics simulations with OR–transducer complexes in bilayers mimicking PM or Golgi composition reveal that the lipid environment promotes the location-selective coupling. We then show that delta-ORs in PM and Golgi have distinct effects on transcription and protein phosphorylation. The study reveals that the subcellular location defines the signaling effects of opioid drugs

Polymersomes confined self-assembly: micellisation in 2D

Biological systems exploit self-assembly to create complex structures whose arrangements are finely controlled from molecular to mesoscopic level. Herein we report an example of using fully synthetic systems that mimic two levels of self-assembly. We show the formation of vesicles using amphiphilic copolymers whose chemical nature is chosen to control both membrane formation and membrane-confined interactions. We report polymersomes with patterns that emerge by engineering interfacial tension within the polymersome surface. This allows the formation of domains whose topology is tailored by the chemical synthesis paving the avenue to complex supramolecular designs functionally similar to those found in viruses and trafficking vesicles.Comment: 11 pages, 4 Figures plus supporting informatio

Revealing druggable cryptic pockets in the Nsp1 of SARS-CoV-2 and other β-coronaviruses by simulations and crystallography

Non-structural protein 1 (Nsp1) is a main pathogenicity factor of α- and β-coronaviruses. Nsp1 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suppresses the host gene expression by sterically blocking 40S host ribosomal subunits and promoting host mRNA degradation. This mechanism leads to the downregulation of the translation-mediated innate immune response in host cells, ultimately mediating the observed immune evasion capabilities of SARS-CoV-2. Here, by combining extensive molecular dynamics simulations, fragment screening and crystallography, we reveal druggable pockets in Nsp1. Structural and computational solvent mapping analyses indicate the partial crypticity of these newly discovered and druggable binding sites. The results of fragment-based screening via X-ray crystallography confirm the druggability of the major pocket of Nsp1. Finally, we show how the targeting of this pocket could disrupt the Nsp1-mRNA complex and open a novel avenue to design new inhibitors for other Nsp1s present in homologous β-coronaviruses

DNA-binding protects p53 from interactions with cofactors involved in transcription-independent functions

Binding-induced conformational changes of a protein at regions distant from the binding site may play crucial roles in protein function and regulation. The p53 tumour suppressor is an example of such an allosterically regulated protein. Little is known, however, about how DNA binding can affect distal sites for transcription factors. Furthermore, the molecular details of how a local perturbation is transmitted through a protein structure are generally elusive and occur on timescales hard to explore by simulations. Thus, we employed state-of-the-art enhanced sampling atomistic simulations to unveil DNA-induced effects on p53 structure and dynamics that modulate the recruitment of cofactors and the impact of phosphorylation at Ser215. We show that DNA interaction promotes a conformational change in a region 3 nm away from the DNA binding site. Specifically, binding to DNA increases the population of an occluded minor state at this distal site by more than 4-fold, whereas phosphorylation traps the protein in its major state. In the minor conformation, the interface of p53 that binds biological partners related to p53 transcription-independent functions is not accessible. Significantly, our study reveals a mechanism of DNA-mediated protection of p53 from interactions with partners involved in the p53 transcription-independent signalling. This also suggests that conformational dynamics is tightly related to p53 signalling

An allosteric cross-talk between the activation loop and the ATP binding site regulates the activation of Src kinase

Phosphorylation of the activation loop is a fundamental step in the activation of most protein kinases. In the case of the Src tyrosine kinase, a prototypical kinase due to its role in cancer and its historic importance, phosphorylation of tyrosine 416 in the activation loop is known to rigidify the structure and contribute to the switch from the inactive to a fully active form. However, whether or not phosphorylation is able per-se to induce a fully active conformation, that efficiently binds ATP and phosphorylates the substrate, is less clear. Here we employ a combination of solution NMR and enhanced-sampling molecular dynamics simulations to fully map the effects of phosphorylation and ATP/ADP cofactor loading on the conformational landscape of Src tyrosine kinase. We find that both phosphorylation and cofactor binding are needed to induce a fully active conformation. What is more, we find a complex interplay between the A-loop and the hinge motion where the phosphorylation of the activation-loop has a significant allosteric effect on the dynamics of the C-lobe

Epidemiologically most successful SARS-CoV-2 variant: concurrent mutations in RNA-dependent RNA polymerase and spike protein

The D614G mutation of the Spike protein is thought to be relevant for SARS-CoV-2 infection. Here we report biological and epidemiological aspects of this mutation. Using pseudotyped lentivectors, we were able to confirm that the G614 variant of the Spike protein is markedly more infectious than the ancestral D614 variant. We demonstrate by molecular modelling that the replacement of aspartate by glycine in position 614 facilitates the transition towards an open state of the Spike protein. To understand whether the increased infectivity of the D614 variant explains its epidemiological success, we analysed the evolution of 27,086 high-quality SARS-CoV-2 genome sequences from GISAID. We observed striking coevolution of D614G with the P323L mutation in the viral polymerase. Importantly, exclusive presence of G614 or L323 did not become epidemiologically relevant. In contrast, the combination of the two mutations gave rise to a viral G/L variant that has all but replaced the initial D/P variant. There was no significant correlation between reported COVID mortality in different countries and the prevalence of the Wuhan versus G/L variant. However, when comparing the speed of emergence and the ultimate predominance in individual countries, the G/L variant displays major epidemiological supremacy. Our results suggest that the P323L mutation, located in the interface domain of the RNA-dependent RNA polymerase (RdRp), is a necessary alteration that led to the epidemiological success of the present variant of SARS-CoV-2

Architecture of the MKK6-p38α complex defines the basis of MAPK specificity and activation

The mitogen-activated protein kinase (MAPK) p38α is a central component of signaling in inflammation and the immune response and is, therefore, an important drug target. Little is known about the molecular mechanism of its activation by double phosphorylation from MAPK kinases (MAP2Ks), because of the challenge of trapping a transient and dynamic heterokinase complex. We applied a multidisciplinary approach to generate a structural model of p38α in complex with its MAP2K, MKK6, and to understand the activation mechanism. Integrating cryo-electron microscopy with molecular dynamics simulations, hydrogen-deuterium exchange mass spectrometry, and experiments in cells, we demonstrate a dynamic, multistep phosphorylation mechanism, identify catalytically relevant interactions, and show that MAP2K-disordered amino termini determine pathway specificity. Our work captures a fundamental step of cell signaling: a kinase phosphorylating its downstream target kinase