Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

A Model for Allosteric Communication in Drug Transport by the AcrAB-TolC Tripartite Efflux Pump

RND family efflux pumps are complex macromolecular machines involved in multidrug resistance by extruding antibiotics from the cell. While structural studies and molecular dynamics simulations have provided insights into the architecture and conformational states of the pumps, the path followed by conformational changes from the inner membrane protein (IMP) to the periplasmic membrane fusion protein (MFP) and to the outer membrane protein (OMP) in tripartite efflux assemblies is not fully understood. Here, we investigated AcrAB-TolC efflux pump’s allostery by comparing resting and transport states using difference distance matrices supplemented with evolutionary couplings data and buried surface area measurements. Our analysis indicated that substrate binding by the IMP triggers quaternary level conformational changes in the MFP, which induce OMP to switch from the closed state to the open state, accompanied by a considerable increase in the interface area between the MFP subunits and between the OMPs and MFPs. This suggests that the pump’s transport-ready state is at a more favourable energy level than the resting state, but raises the puzzle of how the pump does not become stably trapped in a transport-intermediate state. We propose a model for pump allostery that includes a downhill energetic transition process from a proposed ‘activated’ transport state back to the resting pump.

Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9

CRISPR-Cas9 is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system's function.

Allosteric mechanism of signal transduction in the two-component system histidine kinase PhoQ

Transmembrane signaling proteins couple extracytosolic sensors to cytosolic effectors. Here, we examine how binding of Mg2+ to the sensor domain of an E. coli two component histidine kinase (HK), PhoQ, modulates its cytoplasmic kinase domain. We use cysteine-crosslinking and reporter-gene assays to simultaneously and independently probe the signaling state of PhoQ's sensor and autokinase domains in a set of over 30 mutants. Strikingly, conservative single-site mutations distant from the sensor or catalytic site strongly influence PhoQ's ligand-sensitivity as well as the magnitude and direction of the signal. Data from 35 mutants are explained by a semi-empirical three-domain model in which the sensor, intervening HAMP, and catalytic domains can adopt kinase-promoting or inhibiting conformations that are in allosteric communication. The catalytic and sensor domains intrinsically favor a constitutively 'kinase-on' conformation, while the HAMP domain favors the 'off' state; when coupled, they create a bistable system responsive to physiological concentrations of Mg2+. Mutations alter signaling by locally modulating domain intrinsic equilibrium constants and interdomain couplings. Our model suggests signals transmit via interdomain allostery rather than propagation of a single concerted conformational change, explaining the diversity of signaling structural transitions observed in individual HK domains.

Probing allosteric interactions in homo-oligomeric molecular machines using solution NMR spectroscopy

Developments in solution NMR spectroscopy have significantly impacted the biological questions that can now be addressed by this methodology. By means of illustration, we present here a perspective focusing on studies of a number of molecular machines that are critical for cellular homeostasis. The role of NMR in elucidating the structural dynamics of these important molecules is emphasized, focusing specifically on intersubunit allosteric communication in homo-oligomers. In many biophysical studies of oligomers, allostery is inferred by showing that models specifically including intersubunit communication best fit the data of interest. Ideally, however, experimental studies focusing on one subunit of a multisubunit system would be performed as an important complement to the more traditional bulk measurements in which signals from all components are measured simultaneously. Using an approach whereby asymmetric molecules are prepared in concert with NMR experiments focusing on the structural dynamics of individual protomers, we present examples of how intersubunit allostery can be directly observed in high-molecular-weight protein systems. These examples highlight some of the unique roles of solution NMR spectroscopy in studies of complex biomolecules and emphasize the important synergy between NMR and other atomic resolution biophysical methods.

Allosteric Regulation of Human Plastins

Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis, and other cellular processes by organizing actin assemblies of strikingly different geometries as in aligned bundles and branched networks. We propose that this unique ability stems from an allosteric communication between the two actin-binding domains (ABD1/2) engaged in a tight spatial association. We found that although ABD1 binds actin first, ABD2 can bind to actin three orders of magnitude stronger if not inhibited by an equally strong allosteric engagement with ABD1. Binding of ABD1 to actin lessened the inhibition, enabling weak bundling within aligned bundles. A mutation mimicking physiologically relevant phosphorylation at the ABD1-ABD2 interface strongly reduced their association, dramatically potentiating actin cross-linking. Cryo-EM reconstruction revealed the ABD1-actin interface and enabled modeling of the plastin bridge to confirm domain separation in parallel bundles. The characteristic domain organization with a strong allosteric inhibition imposed by ABD1 on ABD2 allows plastins to tune cross-linking, contributing to the assembly and remodeling of actin assemblies with different morphological and functional properties defining the unique place of plastins in actin organization.

Probing the Structural Dynamics of the Activation Gate of KcsA Using Homo-FRET Measurements

The allosteric coupling between activation and inactivation processes is a common feature observed in K+ channels. Particularly, in the prokaryotic KcsA channel the K+ conduction process is controlled by the inner gate, which is activated by acidic pH, and by the selectivity filter (SF) or outer gate, which can adopt non-conductive or conductive states. In a previous study, a single tryptophan mutant channel (W67 KcsA) enabled us to investigate the SF dynamics using time-resolved homo-Förster Resonance Energy Transfer (homo-FRET) measurements. Here, the conformational changes of both gates were simultaneously monitored after labelling the G116C position with tetramethylrhodamine (TMR) within a W67 KcsA background. At a high degree of protein labeling, fluorescence anisotropy measurements showed that the pH-induced KcsA gating elicited a variation in the homo-FRET efficiency among the conjugated TMR dyes (TMR homo-FRET), while the conformation of the SF was simultaneously tracked (W67 homo-FRET). The dependence of the activation pKa of the inner gate with the ion occupancy of the SF unequivocally confirmed the allosteric communication between the two gates of KcsA. This simple TMR homo-FRET based ratiometric assay can be easily extended to study the conformational dynamics associated with the gating of other ion channels and their modulation.

GPCR activation mechanisms across classes and macro/microscales

Two-thirds of human hormones and one-third of clinical drugs activate ~350 G-protein-coupled receptors (GPCR) belonging to four classes: A, B1, C and F. Whereas a model of activation has been described for class A, very little is known about the activation of the other classes, which differ by being activated by endogenous ligands bound mainly or entirely extracellularly. Here we show that, although they use the same structural scaffold and share several ‘helix macroswitches’, the GPCR classes differ in their ‘residue microswitch’ positions and contacts. We present molecular mechanistic maps of activation for each GPCR class and methods for contact analysis applicable for any functional determinants. This provides a superfamily residue-level rationale for conformational selection and allosteric communication by ligands and G proteins, laying the foundation for receptor-function studies and drugs with the desired modality.