Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the \"inactive\" state 1 and the \"active\" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras\' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).

Full-length Bruton\'s tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology [PHTH] domain and proline-rich regions [PRR] contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveal only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. Cryo-electron microscopy (cryoEM) data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with phosphatidylinositol (3,4,5)-trisphosphate. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

By assuming that a stepwise outward movement of the four S4 segments of the hERG potassium channel determines a concomitant progressive increase in the flow of the permeant potassium ions, the inward and outward potassium currents can be simulated by using only one or two adjustable (i.e., free) parameters. This deterministic kinetic model differs from the stochastic models of hERG available in the literature, which usually require more than 10 free parameters. The K+ outward current of hERG contributes to the repolarization of the cardiac action potential. On the other hand, the K+ inward current increases with a positive shift in the transmembrane potential, in apparent contrast to both the electric and osmotic forces, which would concur in moving K+ ions outwards. This peculiar behavior can be explained by the appreciable constriction of the central pore midway along its length, with a radius < 1 Å and hydrophobic sacks surrounding it, as reported in an open conformation of the hERG potassium channel. This narrowing raises a barrier to the outward movement of K+ ions, inducing them to move increasingly inwards under a gradually more positive transmembrane potential.

The COVID-19 pandemic has spread worldwide. However, as soon as the first vaccines-the only scientifically verified and efficient therapeutic option thus far-were released, mutations combined into variants of SARS-CoV-2 that are more transmissible and virulent emerged, raising doubts about their efficiency. This study aims to explain possible molecular mechanisms responsible for the increased transmissibility and the increased rate of hospitalizations related to the new variants. A combination of theoretical methods was employed. Constant-pH Monte Carlo simulations were carried out to quantify the stability of several spike trimeric structures at different conformational states and the free energy of interactions between the receptor-binding domain (RBD) and angiotensin-converting enzyme II (ACE2) for the most worrying variants. Electrostatic epitopes were mapped using the PROCEEDpKa method. These analyses showed that the increased virulence is more likely to be due to the improved stability to the S trimer in the opened state, in which the virus can interact with the cellular receptor, ACE2, rather than due to alterations in the complexation RBD-ACE2, since the difference observed in the free energy values was small (although more attractive in general). Conversely, the South African/Beta variant (B.1.351), compared with the SARS-CoV-2 wild type (wt), is much more stable in the opened state with one or two RBDs in the up position than in the closed state with three RBDs in the down position favoring the infection. Such results contribute to understanding the natural history of disease and indicate possible strategies for developing new therapeutic molecules and adjusting the vaccine doses for higher B-cell antibody production.

UNASSIGNED: E-selectin is a member of the selectin family of cell adhesion molecules expressed on the plasma membrane of inflamed endothelium and facilitates initial leukocyte tethering and subsequent cell rolling during the early stages of the inflammatory response via binding to glycoproteins expressing sialyl LewisX and sialyl LewisA (sLeX/A). Existing crystal structures of the extracellular lectin/EGF-like domain of E-selectin complexed with sLeX have revealed that E-selectin can exist in two conformation states, a low affinity (bent) conformation, and a high affinity (extended) conformation. The differentiating characteristic of the two conformations is the interdomain angle between the lectin and the EGF-like domain.
UNASSIGNED: Using molecular dynamics (MD) simulations we observed that in the absence of tensile force E-selectin undergoes spontaneous switching between the two conformational states at equilibrium. A single amino acid substitution at residue 2 (serine to tyrosine) on the lectin domain favors the extended conformation.
UNASSIGNED: Steered molecular dynamics (SMD) simulations of E-selectin and PSGL-1 in conjunction with experimental cell adhesion assays show a longer binding lifetime of E-selectin (S2Y) to PSGL-1 compared to wildtype protein.
UNASSIGNED: The findings in this study advance our understanding into how the structural makeup of E-selectin allosterically influences its adhesive dynamics.

We present an approach that, by integrating structural data with Direct Coupling Analysis, is able to pinpoint most of the interaction hotspots (i.e. key residues for the biological activity) across very sparse protein families in a single run. An application to the Class A G-protein coupled receptors (GPCRs), both in their active and inactive states, demonstrates the predictive power of our approach. The latter can be easily extended to any other kind of protein family, where it is expected to highlight most key sites involved in their functional activity.

During HIV-1 entry into target cells, binding of the virus to host receptors, CD4 and CCR5/CXCR4, triggers serial conformational changes in the envelope glycoprotein (Env) trimer that result in the fusion of the viral and cell membranes. Recent discoveries have refined our knowledge of Env conformational states, allowing characterization of the targets of small-molecule HIV-1 entry inhibitors and neutralizing antibodies, and identifying a novel off-pathway conformation (State 2A). Here, we provide an overview of the current understanding of these conformational states, focusing on (i) the events during HIV-1 entry; (ii) conformational preferences of HIV-1 Env ligands; (iii) evasion of the host antibody response; and (iv) potential implications for therapy and prevention of HIV-1 infection.

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that plays a key role in the regulation of the inflammatory pathway. While inhibition of TNFR1 has been the focus of many studies for the treatment of autoimmune diseases such as rheumatoid arthritis, activation of the receptor is important for the treatment of immunodeficiency diseases such as HIV and neurodegenerative diseases such as Alzheimer\'s disease where a boost in immune signaling is required. In addition, activation of other TNF receptors such as death receptor 5 or FAS receptor is important for cancer therapy. Here, we used a previously established TNFR1 fluorescence resonance energy transfer (FRET) biosensor together with a fluorescence lifetime technology as a high-throughput screening platform to identify a novel small molecule that activates TNFR1 by increasing inter-monomeric spacing in a ligand-independent manner. This shows that the conformational rearrangement of pre-ligand assembled receptor dimers can determine the activity of the receptor. By probing the interaction between the receptor and its downstream signaling molecule (TRADD) our findings support a new model of TNFR1 activation in which varying conformational states of the receptor act as a molecular switch in determining receptor function.

In this work, we report on the development of a newly synthesized photoactive reversible azobenzene derived surfactant polymer, which enables active and fast control of the merging of microdroplets in microfluidic chambers, driven by a pulsed UV laser optical stimulus and the well known cis-trans photo-isomerisation of azobenzene groups. We show for the first time that merging of microdroplets can be achieved optically based on a photo-isomerization process with a high spatio-temporal resolution. Our results show that the physical process lying behind the merging of microdroplets is not driven by a change in surface activity of the droplet stabilizing surfactant under UV illumination (as originally expected), and they suggest an original mechanism for the merging of droplets based on the well-known opto-mechanical motion of azobenzene molecules triggered by light irradiation.

Solid-state NMR (ssNMR) is an ideal tool to study structure and dynamics of membrane proteins in their native lipid environment. In principle, ssNMR has no size limitations. However, this feature is rarely exploited as large membrane proteins display severe resonance overlap. In addition, dismal yields from recombinant bacterial expression systems limit severely spectroscopic characterization of membrane proteins. For very large mammalian membrane proteins, extraction from the original organism remains the most viable approach. In this case, NMR-observable nuclei must be introduced post-translationally, but the approaches developed so far are rather scarce. Here, we detail the synthesis and engineering of a reactive 13C-ethylmethanethiosulfonate (13C-EMTS) reagent for the post-translational alkylation of cysteine sidechains of a 110kDa sarcoplasmic reticulum Ca2+-ATPase (SERCA) extracted from rabbit skeletal muscle tissue. When reconstituted into liposomes, it is possible to resolve the resonances of the engineered ethyl groups by magic-angle spinning (MAS) 2D [13C,13C]-DARR experiments. Notably, the ethyl-group modification does not perturb the function of SERCA, yielding well-resolved 13C-13C fingerprints that are used to image its structural states in the catalytic cycle and filtering out overwhelming naturally-abundant 13C nuclei signals arising from the enzyme and lipids. We anticipate that this approach will be used together with 19F NMR to monitor conformational transitions of enzymes and proteins that are difficult to express recombinantly.