The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins.

A protein sequence encodes its energy landscape-all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs-questioning the differences between them and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the consensus protein derived from our full Ribonuclease H sequence alignment is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted set of sequences is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order sequence correlations using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

Stepwise activation of a metabotropic glutamate receptor.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

Domain coupling in activation of a family C GPCR.

The G protein-coupled metabotropic glutamate receptors form homodimers and heterodimers with highly diverse responses to glutamate and varying physiological function. The molecular basis for this diversity remains poorly delineated. We employ molecular dynamics, single-molecule spectroscopy, and hydrogen-deuterium exchange to dissect the pathway of activation triggered by glutamate. We find that activation entails multiple loosely coupled steps and identify a novel pre-active intermediate whose transition to the active state forms dimer interactions that set signaling efficacy. Such subunit interactions generate functional diversity that differs across homodimers and heterodimers. The agonist-bound receptor is remarkably dynamic, with low occupancy of G protein-coupling conformations, providing considerable headroom for modulation of the landscape by allosteric ligands. Sites of sequence diversity within the dimerization interface and diverse coupling between activation rearrangements may contribute to precise decoding of glutamate signals and transients over broad spatial and temporal scales.

Selection pressures on evolution of ribonuclease H explored with rigorous free-energy-based design.

Understanding natural protein evolution and designing novel proteins are motivating interest in development of high-throughput methods to explore large sequence spaces. In this work, we demonstrate the application of multisite λ dynamics (MSλD), a rigorous free energy simulation method, and chemical denaturation experiments to quantify evolutionary selection pressure from sequence-stability relationships and to address questions of design. This study examines a mesophilic phylogenetic clade of ribonuclease H (RNase H), furthering its extensive characterization in earlier studies, focusing on E. coli RNase H (ecRNH) and a more stable consensus sequence (AncCcons) differing at 15 positions. The stabilities of 32,768 chimeras between these two sequences were computed using the MSλD framework. The most stable and least stable chimeras were predicted and tested along with several other sequences, revealing a designed chimera with approximately the same stability increase as AncCcons, but requiring only half the mutations. Comparing the computed stabilities with experiment for 12 sequences reveals a Pearson correlation of 0.86 and root mean squared error of 1.18 kcal/mol, an unprecedented level of accuracy well beyond less rigorous computational design methods. We then quantified selection pressure using a simple evolutionary model in which sequences are selected according to the Boltzmann factor of their stability. Selection temperatures from 110 to 168 K are estimated in three ways by comparing experimental and computational results to evolutionary models. These estimates indicate selection pressure is high, which has implications for evolutionary dynamics and for the accuracy required for design, and suggests accurate high-throughput computational methods like MSλD may enable more effective protein design.

A helical fulcrum in eIF2B coordinates allosteric regulation of stress signaling.

The integrated stress response (ISR) enables cells to survive a variety of acute stresses, but chronic activation of the ISR underlies age-related diseases. ISR signaling downregulates translation and activates expression of stress-responsive factors that promote return to homeostasis and is initiated by inhibition of the decameric guanine nucleotide exchange factor eIF2B. Conformational and assembly transitions regulate eIF2B activity, but the allosteric mechanisms controlling these dynamic transitions and mediating the therapeutic effects of the small-molecule ISR inhibitor ISRIB are unknown. Using hydrogen-deuterium exchange-mass spectrometry and cryo-electron microscopy, we identified a central α-helix whose orientation allosterically coordinates eIF2B conformation and assembly. Biochemical and cellular signaling assays show that this 'switch-helix' controls eIF2B activity and signaling. In sum, the switch-helix acts as a fulcrum of eIF2B conformational regulation and is a highly conserved actuator of ISR signal transduction. This work uncovers a conserved allosteric mechanism and unlocks new therapeutic possibilities for ISR-linked diseases.

A Novel Platform for Evaluating Dose Rate Effects on Oxidative Damage to Peptides: Toward a High-Throughput Method to Characterize the Mechanisms Underlying the FLASH Effect.

High dose rate radiation has gained considerable interest recently as a possible avenue for increasing the therapeutic window in cancer radiation treatment. The sparing of healthy tissue at high dose rates relative to conventional dose rates, while maintaining tumor control, has been termed the FLASH effect. Although the effect has been validated in animal models using multiple radiation sources, it is not yet well understood. Here, we demonstrate a new experimental platform for quantifying oxidative damage to protein sidechains in solution as a function of radiation dose rate and oxygen availability using liquid chromatography mass spectrometry. Using this reductionist approach, we show that for both X-ray and electron sources, isolated peptides in solution are oxidatively modified to different extents as a function of both dose rate and oxygen availability. Our method provides an experimental platform for exploring the parameter space of the dose rate effect on oxidative changes to proteins in solution.

Step-wise activation of a Family C GPCR.

Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain (ECD) that is linked via a cysteine-rich domain (CRDs) to their 7-transmembrane (TM) domain. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the ECD to the G protein-coupling TM. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging we reveal distinct receptor conformations upon allosteric modulator and G protein binding.

The importance of input sequence set to consensus-derived proteins and their relationship to reconstructed ancestral proteins.

A protein sequence encodes its energy landscape - all the accessible conformations, energetics, and dynamics. The evolutionary relationship between sequence and landscape can be probed phylogenetically by compiling a multiple sequence alignment of homologous sequences and generating common ancestors via Ancestral Sequence Reconstruction or a consensus protein containing the most common amino acid at each position. Both ancestral and consensus proteins are often more stable than their extant homologs - questioning the differences and suggesting that both approaches serve as general methods to engineer thermostability. We used the Ribonuclease H family to compare these approaches and evaluate how the evolutionary relationship of the input sequences affects the properties of the resulting consensus protein. While the overall consensus protein is structured and active, it neither shows properties of a well-folded protein nor has enhanced stability. In contrast, the consensus protein derived from a phylogenetically-restricted region is significantly more stable and cooperatively folded, suggesting that cooperativity may be encoded by different mechanisms in separate clades and lost when too many diverse clades are combined to generate a consensus protein. To explore this, we compared pairwise covariance scores using a Potts formalism as well as higher-order couplings using singular value decomposition (SVD). We find the SVD coordinates of a stable consensus sequence are close to coordinates of the analogous ancestor sequence and its descendants, whereas the unstable consensus sequences are outliers in SVD space.

Nanopore molecular trajectories of a eukaryotic reverse transcriptase reveal a long-range RNA structure sensing mechanism.

Eukaryotic reverse transcriptases (RTs) can have essential or deleterious roles in normal human physiology and disease. Compared to well-studied helicases, it remains unclear how RTs overcome the ubiquitous RNA structural barriers during reverse transcription. Herein, we describe the development of a Mycobacterium smegmatis porin A (MspA) nanopore technique to sequence RNA to quantify the single-molecule kinetics of an RT from Bombyx mori with single-nucleotide resolution. By establishing a quadromer map that correlates RNA sequence and MspA ion current, we were able to quantify the RT's dwell time at every single nucleotide step along its RNA template. By challenging the enzyme with various RNA structures, we found that during cDNA synthesis the RT can sense and actively destabilize RNA structures 11-12 nt downstream of its front boundary. The ability to sequence single molecules of RNA with nanopores paves the way to investigate the single-nucleotide activity of other processive RNA translocases.

Ligand-specific changes in conformational flexibility mediate long-range allostery in the lac repressor.

Biological regulation ubiquitously depends on protein allostery, but the regulatory mechanisms are incompletely understood, especially in proteins that undergo ligand-induced allostery with few structural changes. Here we used hydrogen-deuterium exchange with mass spectrometry (HDX/MS) to map allosteric effects in a paradigm ligand-responsive transcription factor, the lac repressor (LacI), in different functional states (apo, or bound to inducer, anti-inducer, and/or DNA). Although X-ray crystal structures of the LacI core domain in these states are nearly indistinguishable, HDX/MS experiments reveal widespread differences in flexibility. We integrate these results with modeling of protein-ligand-solvent interactions to propose a revised model for allostery in LacI, where ligand binding allosterically shifts the conformational ensemble as a result of distinct changes in the rigidity of secondary structures in the different states. Our model provides a mechanistic basis for the altered function of distal mutations. More generally, our approach provides a platform for characterizing and engineering protein allostery.

Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B GPCRs. We show that RAMP2 directly interacts with the glucagon receptor (GCGR), a Family B GPCR responsible for blood sugar homeostasis, and broadly inhibits receptor-induced downstream signaling. HDX-MS experiments demonstrate that RAMP2 enhances local flexibility in select locations in and near the receptor extracellular domain (ECD) and in the 6th transmembrane helix, whereas smFRET experiments show that this ECD disorder results in the inhibition of active and intermediate states of the intracellular surface. We determined the cryo-EM structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner; however, the receptor ECD is indeed largely disordered along with rearrangements of several intracellular hallmarks of activation. Our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses.

To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of ß-coronaviruses: SARS-CoV-2 S2 apex residues 980-1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.

Evolution avoids a pathological stabilizing interaction in the immune protein S100A9.

Stability constrains evolution. While much is known about constraints on destabilizing mutations, less is known about the constraints on stabilizing mutations. We recently identified a mutation in the innate immune protein S100A9 that provides insight into such constraints. When introduced into human S100A9, M63F simultaneously increases the stability of the protein and disrupts its natural ability to activate Toll-like receptor 4. Using chemical denaturation, we found that M63F stabilizes a calcium-bound conformation of hS100A9. We then used NMR to solve the structure of the mutant protein, revealing that the mutation distorts the hydrophobic binding surface of hS100A9, explaining its deleterious effect on function. Hydrogen-deuterium exchange (HDX) experiments revealed stabilization of the region around M63F in the structure, notably Phe37. In the structure of the M63F mutant, the Phe37 and Phe63 sidechains are in contact, plausibly forming an edge-face π-stack. Mutating Phe37 to Leu abolished the stabilizing effect of M63F as probed by both chemical denaturation and HDX. It also restored the biological activity of S100A9 disrupted by M63F. These findings reveal that Phe63 creates a molecular staple with Phe37 that stabilizes a nonfunctional conformation of the protein, thus disrupting function. Using a bioinformatic analysis, we found that S100A9 proteins from different organisms rarely have Phe at both positions 37 and 63, suggesting that avoiding a pathological stabilizing interaction indeed constrains S100A9 evolution. This work highlights an important evolutionary constraint on stabilizing mutations, namely, that they must avoid inappropriately stabilizing nonfunctional protein conformations.

Saturation mutagenesis of a predicted ancestral Syk-family kinase.

Many tyrosine kinases cannot be expressed readily in Escherichia coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and zeta-chain-associated protein kinase of 70 kDa [ZAP-70]). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Syk-family kinase in bacteria, we developed a two-hybrid assay that couples the growth of E. coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.

An automated liquid jet for fluorescence dosimetry and microsecond radiolytic labeling of proteins.

X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method's overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.

Site-specific ubiquitination: Deconstructing the degradation tag.

Ubiquitin is a small eukaryotic protein so named for its cellular abundance and originally recognized for its role as the posttranslational modification (PTM) "tag" condemning substrates to degradation by the 26S proteasome. Since its discovery in the 1970s, protein ubiquitination has also been identified as a key regulatory feature in dozens of non-degradative cellular processes. This myriad of roles illustrates the versatility of ubiquitin as a PTM; however, understanding the cellular and molecular factors that enable discrimination between degradative versus non-degradative ubiquitination events has been a persistent challenge. Here, we discuss recent advances in uncovering how site-specificity - the exact residue that gets modified - modulates distinct protein fates and cellular outcomes with an emphasis on how ubiquitination site specificity regulates proteasomal degradation. We explore recent advances in structural biology, biophysics, and cell biology that have enabled a broader understanding of the role of ubiquitination in altering the dynamics of the target protein, including implications for the design of targeted protein degradation therapeutics.

A saturation-mutagenesis analysis of the interplay between stability and activation in Ras.

Cancer mutations in Ras occur predominantly at three hotspots: Gly 12, Gly 13, and Gln 61. Previously, we reported that deep mutagenesis of H-Ras using a bacterial assay identified many other activating mutations (Bandaru et al., 2017). We now show that the results of saturation mutagenesis of H-Ras in mammalian Ba/F3 cells correlate well with the results of bacterial experiments in which H-Ras or K-Ras are co-expressed with a GTPase-activating protein (GAP). The prominent cancer hotspots are not dominant in the Ba/F3 data. We used the bacterial system to mutagenize Ras constructs of different stabilities and discovered a feature that distinguishes the cancer hotspots. While mutations at the cancer hotspots activate Ras regardless of construct stability, mutations at lower-frequency sites (e.g. at Val 14 or Asp 119) can be activating or deleterious, depending on the stability of the Ras construct. We characterized the dynamics of three non-hotspot activating Ras mutants by using NMR to monitor hydrogen-deuterium exchange (HDX). These mutations result in global increases in HDX rates, consistent with destabilization of Ras. An explanation for these observations is that mutations that destabilize Ras increase nucleotide dissociation rates, enabling activation by spontaneous nucleotide exchange. A further stability decrease can lead to insufficient levels of folded Ras - and subsequent loss of function. In contrast, the cancer hotspot mutations are mechanism-based activators of Ras that interfere directly with the action of GAPs. Our results demonstrate the importance of GAP surveillance and protein stability in determining the sensitivity of Ras to mutational activation.

The SARS-CoV-2 spike reversibly samples an open-trimer conformation exposing novel epitopes.

Current COVID-19 vaccines and many clinical diagnostics are based on the structure and function of the SARS-CoV-2 spike ectodomain. Using hydrogen-deuterium exchange monitored by mass spectrometry, we have uncovered that, in addition to the prefusion structure determined by cryo-electron microscopy, this protein adopts an alternative conformation that interconverts slowly with the canonical prefusion structure. This new conformation-an open trimer-contains easily accessible receptor-binding domains. It exposes the conserved trimer interface buried in the prefusion conformation, thus exposing potential epitopes for pan-coronavirus antibody and ligand recognition. The population of this state and kinetics of interconversion are modulated by temperature, receptor binding, antibody binding, and sequence variants observed in the natural population. Knowledge of the structure and populations of this conformation will help improve existing diagnostics, therapeutics, and vaccines.