Deep mutational scanning of hepatitis B virus reveals a mechanism for cis-preferential reverse transcription.

Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.

The SpoVA membrane complex is required for dipicolinic acid import during sporulation and export during germination.

In response to starvation, endospore-forming bacteria differentiate into stress-resistant spores that can remain dormant for years yet rapidly germinate and resume growth in response to nutrients. The small molecule dipicolinic acid (DPA) plays a central role in both the stress resistance of the dormant spore and its exit from dormancy during germination. The spoVA locus is required for DPA import during sporulation and has been implicated in its export during germination, but the molecular bases are unclear. Here, we define the minimal set of proteins encoded in the Bacillus subtilis spoVA operon required for DPA import and demonstrate that these proteins form a membrane complex. Structural modeling of these components combined with mutagenesis and in vivo analysis reveal that the C and Eb subunits form a membrane channel, while the D subunit functions as a cytoplasmic plug. We show that point mutations that impair the interactions between D and the C-Eb membrane complex reduce the efficiency of DPA import during sporulation and reciprocally accelerate DPA release during germination. Our data support a model in which DPA transport into spores involves cycles of unplugging and then replugging the C-Eb membrane channel, while nutrient detection during germination triggers DPA release by unplugging it.

The relaxin receptor RXFP1 signals through a mechanism of autoinhibition.

The relaxin family peptide receptor 1 (RXFP1) is the receptor for relaxin-2, an important regulator of reproductive and cardiovascular physiology. RXFP1 is a multi-domain G protein-coupled receptor (GPCR) with an ectodomain consisting of a low-density lipoprotein receptor class A (LDLa) module and leucine-rich repeats. The mechanism of RXFP1 signal transduction is clearly distinct from that of other GPCRs, but remains very poorly understood. In the present study, we determine the cryo-electron microscopy structure of active-state human RXFP1, bound to a single-chain version of the endogenous agonist relaxin-2 and the heterotrimeric Gs protein. Evolutionary coupling analysis and structure-guided functional experiments reveal that RXFP1 signals through a mechanism of autoinhibition. Our results explain how an unusual GPCR family functions, providing a path to rational drug development targeting the relaxin receptors.

Structure and mutagenic analysis of the lipid II flippase MurJ from Escherichia coli.

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.

Protein structure prediction assisted with sparse NMR data in CASP13.

CASP13 has investigated the impact of sparse NMR data on the accuracy of protein structure prediction. NOESY and 15 N-1 H residual dipolar coupling data, typical of that obtained for 15 N,13 C-enriched, perdeuterated proteins up to about 40 kDa, were simulated for 11 CASP13 targets ranging in size from 80 to 326 residues. For several targets, two prediction groups generated models that are more accurate than those produced using baseline methods. Real NMR data collected for a de novo designed protein were also provided to predictors, including one data set in which only backbone resonance assignments were available. Some NMR-assisted prediction groups also did very well with these data. CASP13 also assessed whether incorporation of sparse NMR data improves the accuracy of protein structure prediction relative to nonassisted regular methods. In most cases, incorporation of sparse, noisy NMR data results in models with higher accuracy. The best NMR-assisted models were also compared with the best regular predictions of any CASP13 group for the same target. For six of 13 targets, the most accurate model provided by any NMR-assisted prediction group was more accurate than the most accurate model provided by any regular prediction group; however, for the remaining seven targets, one or more regular prediction method provided a more accurate model than even the best NMR-assisted model. These results suggest a novel approach for protein structure determination, in which advanced prediction methods are first used to generate structural models, and sparse NMR data is then used to validate and/or refine these models.

A Hybrid Approach for Protein Structure Determination Combining Sparse NMR with Evolutionary Coupling Sequence Data.

While 3D structure determination of small (<15 kDa) proteins by solution NMR is largely automated and routine, structural analysis of larger proteins is more challenging. An emerging hybrid strategy for modeling protein structures combines sparse NMR data that can be obtained for larger proteins with sequence co-variation data, called evolutionary couplings (ECs), obtained from multiple sequence alignments of protein families. This hybrid "EC-NMR" method can be used to accurately model larger (15-60 kDa) proteins, and more rapidly determine structures of smaller (5-15 kDa) proteins using only backbone NMR data. The resulting structures have accuracies relative to reference structures comparable to those obtained with full backbone and sidechain NMR resonance assignments. The requirement that evolutionary couplings (ECs) are consistent with NMR data recorded on a specific member of a protein family, under specific conditions, potentially also allows identification of ECs that reflect alternative allosteric or excited states of the protein structure.

Bacterial spore germination receptors are nutrient-gated ion channels.

Bacterial spores resist antibiotics and sterilization and can remain metabolically inactive for decades, but they can rapidly germinate and resume growth in response to nutrients. Broadly conserved receptors embedded in the spore membrane detect nutrients, but how spores transduce these signals remains unclear. Here, we found that these receptors form oligomeric membrane channels. Mutations predicted to widen the channel initiated germination in the absence of nutrients, whereas those that narrow it prevented ion release and germination in response to nutrients. Expressing receptors with widened channels during vegetative growth caused loss of membrane potential and cell death, whereas the addition of germinants to cells expressing wild-type receptors triggered membrane depolarization. Therefore, germinant receptors act as nutrient-gated ion channels such that ion release initiates exit from dormancy.

Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices.

The transmembrane (TM) channel-like 1 (TMC1) and TMC2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of 12 mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining TM4 and TM6 helices modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

Natural and Designed Proteins Inspired by Extremotolerant Organisms Can Form Condensates and Attenuate Apoptosis in Human Cells.

Many organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically induced apoptosis. We show that several proteins from tardigrades, nematodes, and the Chinese giant salamander are apoptosis-protective. Notably, we identify a region of the human ApoE protein with similarity to extremotolerance-associated proteins that also protects against apoptosis. This region mirrors the phase separation behavior seen with such proteins, like the tardigrade protein CAHS2. Moreover, we identify a synthetic protein, DHR81, that shares this combination of elevated phase separation propensity and apoptosis protection. Finally, we demonstrate that driving protective proteins into the condensate state increases apoptosis protection, and highlights the ability of DHR81 condensates to sequester caspase-7. Taken together, this work draws a link between extremotolerance-associated proteins, condensate formation, and designing human cellular protection.

Large-scale discovery of protein interactions at residue resolution using co-evolution calculated from genomic sequences.

Increasing numbers of protein interactions have been identified in high-throughput experiments, but only a small proportion have solved structures. Recently, sequence coevolution-based approaches have led to a breakthrough in predicting monomer protein structures and protein interaction interfaces. Here, we address the challenges of large-scale interaction prediction at residue resolution with a fast alignment concatenation method and a probabilistic score for the interaction of residues. Importantly, this method (EVcomplex2) is able to assess the likelihood of a protein interaction, as we show here applied to large-scale experimental datasets where the pairwise interactions are unknown. We predict 504 interactions de novo in the E. coli membrane proteome, including 243 that are newly discovered. While EVcomplex2 does not require available structures, coevolving residue pairs can be used to produce structural models of protein interactions, as done here for membrane complexes including the Flagellar Hook-Filament Junction and the Tol/Pal complex.