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The interaction between the SARS-CoV-2 virus Spike protein receptor binding domain (RBD) and the ACE2 cell surface protein is required for viral infection of cells. Mutations in the RBD are present in SARS-CoV-2 variants of concern that have emerged independently worldwide. For example, the B.1.1.7 lineage has a mutation (N501Y) in its Spike RBD that enhances binding to ACE2. There are also ACE2 alleles in humans with mutations in the RBD binding site. Here we perform a detailed affinity and kinetics analysis of the effect of five common RBD mutations (K417N, K417T, N501Y, E484K, and S477N) and two common ACE2 mutations (S19P and K26R) on the RBD/ACE2 interaction. We analysed the effects of individual RBD mutations and combinations found in new SARS-CoV-2 Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P1) variants. Most of these mutations increased the affinity of the RBD/ACE2 interaction. The exceptions were mutations K417N/T, which decreased the affinity. Taken together with other studies, our results suggest that the N501Y and S477N mutations enhance transmission primarily by enhancing binding, the K417N/T mutations facilitate immune escape, and the E484K mutation enhances binding and immune escape. As the COVID-19 pandemic has progressed, new variants of the virus SARS-CoV-2 have emerged that are more infectious than the original form. The variants known as Alpha, Beta and Gamma have mutations in a protein on the virus’s surface that is vital for attaching to cells and infecting them. This protein, called Spike, carries out its role by binding to ACE2, a protein on the surface of human cells. Mutations on Spike are found on the region where it binds to ACE2. The interaction between these two proteins appears to be important to the behaviour of SARS-CoV-2, but the impact of individual mutations in Spike is unknown. In addition, some people have different variants of ACE2 with mutations in the region that interacts with Spike, but it is not known whether this affects these people’s risk of contracting COVID-19. To answer these questions, Barton et al. measured the precise effect of mutations in Spike and ACE2 on the strength of the interaction between the two proteins. The experiments showed that three of the five common Spike mutations in the Alpha, Beta and Gamma SARS-CoV-2 variants strengthened binding to ACE2. The two mutations that weakened binding were only found together with other mutations that strengthened binding. This meant that the Spike proteins in all three of these SARS-CoV-2 variants bind to ACE2 more strongly than the original form. The experiments also showed that two common variants of ACE2 also increased the strength of binding to Spike. Interestingly, one of these ACE2 variants reversed the effect of a specific SARS-CoV-2 mutation, suggesting that carriers would be resistant to SARS-CoV-2 variants with this mutation. Identifying the precise effects of Spike mutations on ACE2 binding helps understand why new variants of SARS-CoV-2 spread more rapidly. This could help to identify concerning new variants before they spread widely and inform the response by health authorities. The finding that two common ACE2 variants bind more strongly to Spike suggests that people with these mutations could be more susceptible to SARS-CoV-2.

SARS-CoV-2 Spike (Spike) binds to human angiotensin-converting enzyme 2 (ACE2) and the strength of this interaction could influence parameters relating to virulence. To explore whether population variants in ACE2 influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities and kinetics for Spike receptor binding domain through surface plasmon resonance (SPR) at 37°C. We discovered variants that reduce and enhance binding, including three ACE2 variants that strongly inhibited (p.Glu37Lys, ΔΔG = –1.33 ± 0.15 kcal mol -1 and p.Gly352Val, predicted ΔΔG = –1.17 kcal mol -1 ) or abolished (p.Asp355Asn) binding. We also identified two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (ΔΔG = 0.59 ± 0.08 kcal mol -1 ) is predominant in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg (ΔΔG = 0.26 ± 0.09 kcal mol -1 ) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. We compared ACE2 variant affinities to published SARS-CoV-2 pseudotype infectivity data and confirmed that ACE2 variants with reduced affinity for Spike can protect cells from infection. The effect of variants with enhanced Spike affinity remains unclear, but we propose a mechanism whereby these alleles could cause greater viral spreading across tissues and cell types, as is consistent with emerging understanding regarding the interplay between receptor affinity and cell-surface abundance. Finally, we compared mCSM-PPI2 ΔΔG predictions against our SPR data to assess the utility of predictions in this system. We found that predictions of decreased binding were well-correlated with experiment and could be improved by calibration, but disappointingly, predictions of highly enhanced binding were unreliable. Recalibrated predictions for all possible ACE2 missense variants at the Spike interface were calculated and used to estimate the overall burden of ACE2 variants on Covid-19.

Ankyrin protein repeats bind to a wide range of substrates and are one of the most common protein motifs in nature. Here, we collate a high-quality alignment of 7,407 ankyrin repeats and examine for the first time, the distribution of human population variants from large-scale sequencing of healthy individuals across this family. Population variants are not randomly distributed across the genome but are constrained by gene essentiality and function. Accordingly, we interpret the population variants in context with evolutionary constraint and structural features including secondary structure, accessibility and protein-protein interactions across 383 three-dimensional structures of ankyrin repeats. We find five positions that are highly conserved across homologues and also depleted in missense variants within the human population. These positions are significantly enriched in intra-domain contacts and so likely to be key for repeat packing. In contrast, a group of evolutionarily divergent positions are found to be depleted in missense variants in human and significantly enriched in protein-protein interactions. Our analysis also suggests the domain has three, not two surfaces, each with different patterns of enrichment in protein-substrate interactions and missense variants. Our findings will be of interest to those studying or engineering ankyrin-repeat containing proteins as well as those interpreting the significance of disease variants.

An increase in treatment failures for malaria highlights an urgent need for new tools to understand and minimize the spread of drug resistance. We describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium spp, the causative agent of malaria. Saturation mutagenesis was used to generate a mutation library containing all conceivable mutations for a region of the antimalarial-binding site of a promising drug target, Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS). Screening of this high-coverage library with characterized Pf KRS inhibitors revealed multiple resistance-conferring substitutions including several clinically relevant mutations. Genetic validation of these mutations confirmed resistance of up to 100-fold and computational modeling dissected their role in drug resistance. We discuss potential applications of this data including the potential to design compounds that can bypass the most serious resistance mutations and future resistance surveillance.

Fragment screening is used to identify binding sites and leads in drug discovery, but it is often unclear which binding sites are functionally important. Here, data from 37 experiments, and 1309 protein structures binding to 1601 ligands were analysed. A method to group ligands by binding sites is introduced and sites clustered according to profiles of relative solvent accessibility. This identified 293 unique ligand binding sites, grouped into four clusters (C1-4). C1 includes larger, buried, conserved, and population missense-depleted sites, enriched in known functional sites. C4 comprises smaller, accessible, divergent, missense-enriched sites, depleted in functional sites. A site in C1 is 28 times more likely to be functional than one in C4. Seventeen sites, which to the best of our knowledge are novel, in 13 proteins are identified as likely to be functionally important with examples from human tenascin and 5-aminolevulinate synthase highlighted. A multi-layer perceptron, and K -nearest neighbours model are presented to predict cluster labels for ligand binding sites with an accuracy of 96% and 100%, respectively, so allowing functional classification of sites for proteins not in this set. Our findings will be of interest to those studying protein-ligand interactions and developing new drugs or function modulators. The authors classify 293 ligand binding sites from 37 proteins into four groups by their solvent accessibility profile. The groups differ in evolutionary conservation and functional enrichment, and can be predicted by a machine learning model.

Protein evolution is constrained by structure and function, creating patterns in residue conservation that are routinely exploited to predict structure and other features. Similar constraints should affect variation across individuals, but it is only with the growth of human population sequencing that this has been tested at scale. Now, human population constraint has established applications in pathogenicity prediction, but it has not yet been explored for structural inference. Here, we map 2.4 million population variants to 5885 protein families and quantify residue-level constraint with a new Missense Enrichment Score (MES). Analysis of 61,214 structures from the PDB spanning 3661 families shows that missense depleted sites are enriched in buried residues or those involved in small-molecule or protein binding. MES is complementary to evolutionary conservation and a combined analysis allows a new classification of residues according to a conservation plane. This approach finds functional residues that are evolutionarily diverse, which can be related to specificity, as well as family-wide conserved sites that are critical for folding or function. We also find a possible contrast between lethal and non-lethal pathogenic sites, and a surprising clinical variant hot spot at a subset of missense enriched positions. A proteome-scale analysis of human coding variants reveals constrained sites in thousands of domains. This highlights key structural or functional residues, including pathogenic sites, especially when integrated with evolutionary conservation.

SARS-CoV-2 invades host cells via an endocytic pathway that begins with the interaction of the SARS-CoV-2 Spike glycoprotein (S-protein) and human Angiotensin-converting enzyme 2 (ACE2). Genetic variability in ACE2 may be one factor that mediates the broad-spectrum severity of SARS-CoV-2 infection and COVID-19 outcomes. We investigated the capacity of ACE2 variation to influence SARS-CoV-2 infection with a focus on predicting the effect of missense variants on the ACE2 SARS-CoV-2 S-protein interaction. We validated the mCSM-PPI2 variant effect prediction algorithm with 26 published ACE2 mutant SARS-CoV S-protein binding assays and found it performed well in this closely related system (True Positive Rate = 0.7, True Negative Rate = 1). Application of mCSM-PPI2 to ACE2 missense variants from the Genome Aggregation Consortium Database (gnomAD) identified three that are predicted to strongly inhibit or abolish the S-protein ACE2 interaction altogether (p.Glu37Lys, p.Gly352Val and p.Asp355Asn) and one that is predicted to promote the interaction (p.Gly326Glu). The S-protein ACE2 inhibitory variants are expected to confer a high degree of resistance to SARS-CoV-2 infection whilst the S-protein ACE2 affinity enhancing variant may lead to additional susceptibility and severity. We also performed in silico saturation mutagenesis of the S-protein ACE2 interface and identified a further 38 potential missense mutations that could strongly inhibit binding and one more that is likely to enhance binding (Thr27Arg). A conservative estimate places the prevalence of the strongly protective variants between 12-70 per 100,000 population but there is the possibility of higher prevalence in local populations or those underrepresented in gnomAD. The probable interplay between these ACE2 affinity variants and ACE2 expression polymorphisms is highlighted as well as gender differences in penetrance arising from ACE2's situation on the X-chromosome. It is also described how our data can help power future genetic association studies of COVID-19 phenotypes and how the saturation mutant predictions can help design a mutant ACE2 with tailored S-protein affinity, which may be an improvement over a current recombinant ACE2 that is undergoing clinical trial. Competing Interest Statement The authors have declared no competing interest.

Abstract Ankyrin protein repeats bind to a wide range of substrates and are one of the most common protein motifs in nature. Here, we collate a high-quality alignment of 7,407 ankyrin repeats and examine for the first time, the distribution of human population variants from large-scale sequencing of healthy individuals across this family. Population variants are not randomly distributed across the genome but are constrained by gene essentiality and function. Accordingly, we interpret the population variants in context with evolutionary constraint and structural features including secondary structure, accessibility and protein-protein interactions across 383 three-dimensional structures of ankyrin repeats. We find five positions that are highly conserved across homologs and also depleted in missense variants within the human population. These positions are significantly enriched in intra-domain contacts and so likely to be key for repeat packing. In contrast, a group of evolutionarily divergent positions are found to be depleted in missense variants in human but significantly enriched in protein-protein interactions. Our analysis also suggests the domain has three, not two surfaces, each with different patterns of enrichment in protein-substrate interactions and missense variants. Our findings will be of interest to those studying or engineering ankyrin-repeat containing proteins as well as those interpreting the significance of disease variants. Author Summary Comparison of variation at each position of the amino acid sequence for a protein across different species is a powerful way to identify parts of the protein that are important for its structure and function. Large-scale DNA sequencing of healthy people has recently made it possible to study normal genetic variation within just one species. Our work combines information on genetic differences between over 100,000 people with in-depth analysis of all available three-dimensional structures for Ankyrin repeats which are a widespread family of binding proteins formed by units with similar amino acid sequence that are found in tandem. Our combined analysis identifies sites critical for ankyrin stability as well as the positions most important for substrate interactions and hence function. Although focused only on the Ankyrins, the principles developed in our work are general and can be applied to any protein family. Competing Interest Statement The authors have declared no competing interest.

SARS-CoV-2 infection begins with the interaction of the SARS-CoV-2 Spike (Spike) and human angiotensin-converting enzyme 2 (ACE2). To explore whether population variants in ACE2 might influence Spike binding and hence infection, we selected 10 ACE2 variants based on affinity predictions and prevalence in gnomAD and measured their affinities for Spike receptor binding domain through surface plasmon resonance (SPR). We discovered variants that enhance and reduce binding, including two variants with distinct population distributions that enhanced affinity for Spike. ACE2 p.Ser19Pro (ΔΔG = 0.59 ± 0.08 kcal mol-1) is often seen in the gnomAD African cohort (AF = 0.003) whilst p.Lys26Arg (ΔΔG = 0.26 ± 0.09 kcal mol-1) is predominant in the Ashkenazi Jewish (AF = 0.01) and European non-Finnish (AF = 0.006) cohorts. Carriers of these alleles may be more susceptible to infection or severe disease and these variants may influence the global epidemiology of Covid-19. We also identified three rare ACE2 variants that strongly inhibited (p.Glu37Lys, ΔΔG = -1.33 ± 0.15 kcal mol-1 and p.Gly352Val, predicted ΔΔG = -1.17 kcal mol-1) or abolished (p.Asp355Asn) Spike binding. These variants may confer resistance to infection. Finally, we calibrated the mCSM-PPI2 ΔΔG prediction algorithm against our SPR data, give new predictions for all possible ACE2 missense variants at the Spike interface and estimate the overall burden of ACE2 variants on Covid-19 phenotypes. Competing Interest Statement AvdW declares ownership of shares in BioNTech SE.

Human genome sequencing has generated population variant datasets containing millions of variants from hundreds of thousands of individuals. The datasets show the genomic distribution of genetic variation to be influenced on genic and sub-genic scales by gene essentiality, protein domain architecture and the presence of genomic features such as splice donor/acceptor sites. However, the variant data are still too sparse to provide a comparative picture of genetic variation between individual protein residues in the proteome. Here, we overcome this sparsity for ~25,000 human protein domains in 1,291 domain families by aggregating variants over equivalent positions (columns) in multiple sequence alignments of sequence-similar (paralagous) domains. We then compare the resulting variation profiles from the human population to residue conservation across all species and find that the same tertiary structural and functional pressures that affect amino acid conservation during domain evolution constrain missense variant distributions. Thus, depletion of missense variants at a position implies that it is structurally or functionally important. We find such positions are enriched in known disease-associated variants (OR = 2.83, p 0) while positions that are both missense depleted and evolutionary conserved are further enriched in disease-associated variants (OR = 1.85, p = 3.3x10-17) compared to those that are only evolutionary conserved (OR = 1.29, p = 4.5x10-19). Unexpectedly, a subset of evolutionary Unconserved positions are Missense Depleted in human (UMD positions) and these are also enriched in pathogenic variants (OR = 1.74, p = 0.02). UMD positions are further differentiated from other unconserved residues in that they are enriched in ligand, DNA and protein binding interactions (OR = 1.59, p = 0.003), which suggests this stratification can identify functionally important positions. A different class of positions that are Conserved and Missense Enriched (CME) show an enrichment of ClinVar risk factor variants (OR = 2.27, p = 0.004). We illustrate these principles with the G-Protein Coupled Receptor (GPCR) family, Nuclear Receptor Ligand Binding Domain family and In Between Ring-Finger (IBR) domains and list a total of 343 UMD positions in 211 domain families. This study will have broad applications to: (a) providing focus for functional studies of specific proteins by mutagenesis; (b) refining pathogenicity prediction models; (c) highlighting which residue interactions to target when refining the specificity of small-molecule drugs.