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New and improved drugs are required for the treatment and ultimate eradication of malaria. The efficacy of front-line therapies is now threatened by emerging drug resistance; thus, new tools to support the development of drugs with a lower propensity for resistance are needed. Here, we describe the development of a RESistance Mapping And Profiling (ResMAP) platform for the identification of resistance-conferring mutations in Plasmodium drug targets. Proof-of-concept studies focused on interrogating the antimalarial drug target, Plasmodium falciparum lysyl tRNA synthetase ( Pf KRS). Saturation mutagenesis was used to construct a plasmid library encoding all conceivable mutations within a 20-residue span at the base of the Pf KRS ATP-binding pocket. The superior transfection efficiency of Plasmodium knowlesi was exploited to generate a high coverage parasite library expressing Pf KRS bearing all possible amino acid changes within this region of the enzyme. The selection of the library with Pf KRS inhibitors, cladosporin and DDD01510706, successfully identified multiple resistance-conferring substitutions. Genetic validation of a subset of these mutations confirmed their direct role in resistance, with computational modeling used to dissect the structural basis of resistance. The application of ResMAP to inform the development of resistance-resilient antimalarials of the future is discussed., Importance: 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., Competing Interests: The authors declare no conflict of interest.

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., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

While treatment options for human African trypanosomiasis (HAT) have improved significantly, there is still a need for new drugs with eradication now a realistic possibility. Here, we report the development of 2,4-diaminothiazoles that demonstrate significant potency against Trypanosoma brucei , the causative agent of HAT. Using phenotypic screening to guide structure-activity relationships, potent drug-like inhibitors were developed. Proof of concept was established in an animal model of the hemolymphatic stage of HAT. To treat the meningoencephalitic stage of infection, compounds were optimized for pharmacokinetic properties, including blood-brain barrier penetration. However, in vivo efficacy was not achieved, in part due to compounds evolving from a cytocidal to a cytostatic mechanism of action. Subsequent studies identified a nonessential kinase involved in the inositol biosynthesis pathway as the molecular target of these cytostatic compounds. These studies highlight the need for cytocidal drugs for the treatment of HAT and the importance of static-cidal screening of analogues.

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., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: AvdM declares ownership of shares in BioNTech SE.

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., Competing Interests: The authors have declared that no competing interests exist.

Tetratricopeptide repeat (TPR) proteins belong to the class of α-solenoid proteins, in which repetitive units of α-helical hairpin motifs stack to form superhelical, often highly flexible structures. TPR domains occur in a wide variety of proteins, and perform key functional roles including protein folding, protein trafficking, cell cycle control and post-translational modification. Here, we look at the TPR domain of the enzyme O-linked GlcNAc-transferase (OGT), which catalyses O-GlcNAcylation of a broad range of substrate proteins. A number of single-point mutations in the TPR domain of human OGT have been associated with the disease Intellectual Disability (ID). By extended steered and equilibrium atomistic simulations, we show that the OGT-TPR domain acts as an elastic nanospring, and that each of the ID-related local mutations substantially affect the global dynamics of the TPR domain. Since the nanospring character of the OGT-TPR domain is key to its function in binding and releasing OGT substrates, these changes of its biomechanics likely lead to defective substrate interaction. We find that neutral mutations in the human population, selected by analysis of the gnomAD database, do not incur these changes. Our findings may not only help to explain the ID phenotype of the mutants, but also aid the design of TPR proteins with tailored biomechanical properties., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The Dundee Resource for Sequence Analysis and Structure Prediction (DRSASP; http://www.compbio.dundee.ac.uk/drsasp.html) is a collection of web services provided by the Barton Group at the University of Dundee. DRSASP's flagship services are the JPred4 webserver for secondary structure and solvent accessibility prediction and the JABAWS 2.2 webserver for multiple sequence alignment, disorder prediction, amino acid conservation calculations, and specificity-determining site prediction. DRSASP resources are available through conventional web interfaces and APIs but are also integrated into the Jalview sequence analysis workbench, which enables the composition of multitool interactive workflows. Other existing Barton Group tools are being brought under the banner of DRSASP, including NoD (Nucleolar localization sequence detector) and 14-3-3-Pred. New resources are being developed that enable the analysis of population genetic data in evolutionary and 3D structural contexts. Existing resources are actively developed to exploit new technologies and maintain parity with evolving web standards. DRSASP provides substantial computational resources for public use, and since 2016 DRSASP services have completed over 1.5 million jobs., (© 2019 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

The structural data for the Fenna-Matthews-Olson (FMO) protein indicate that the bacteriochlorophylls (BChls) display a significant degree of conformational heterogeneity of their peripheral substituents and the protein-induced nonplanar skeletal deformations of the tetrapyrrole macrocycle. As electronic properties of chromophores are altered by such differences, a conformational effect may influence the site-energies of specific pigments and thus play a role in mediating the excitation energy transfer dynamics, but this has not yet been established. The difficulty of assessing this question is shown to be partly the result of the inability of the sequential truncation approach usually employed to account for interactions between the conformations of the macrocycle and its substituents and an alternative approach is suggested. By assigning the BChl atoms to meaningful atom groups and performing all possible permutations of partial optimizations in a full-factorial design, where each group is either frozen in the crystal geometry or optimized in vacuo, followed by excited state calculations on each resulting structure (PM6//ZIndo/S), the specific effects of the conformations of each BChl component as well as mutual interactions between the molecular fragments on the site-energy can be delineated. This factorial relaxation procedure gives different estimates of the macrocycle conformational perturbation than the approach of sequentially truncating the BChl periphery. The results were evaluated in the context of published site-energies for the FMO pigments from three species to identify how conformational effects contribute to their distribution and instances of cross-species conservation and functional divergence of the BChl nonplanarity conformational contribution are described., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tetrapyrrole-containing proteins are one of the most fundamental classes of enzymes in nature and it remains an open question to give a chemical rationale for the multitude of biological reactions that can be catalyzed by these pigment-protein complexes. There are many fundamental processes where the same (i.e., chemically identical) porphyrin cofactor is involved in chemically quite distinct reactions. For example, heme is the active cofactor for oxygen transport and storage (hemoglobin, myoglobin) and for the incorporation of molecular oxygen in organic substrates (cytochrome P450). It is involved in the terminal oxidation (cytochrome c oxidase) and the metabolism of H2O2 (catalases and peroxidases) and catalyzes various electron transfer reactions in cytochromes. Likewise, in photosynthesis the same chlorophyll cofactor may function as a reaction center pigment (charge separation) or as an accessory pigment (exciton transfer) in light harvesting complexes (e.g., chlorophyll a). Whilst differences in the apoprotein sequences alone cannot explain the often drastic differences in physicochemical properties encountered for the same cofactor in diverse protein complexes, a critical factor for all biological functions must be the close structural interplay between bound cofactors and the respective apoprotein in addition to factors such as hydrogen bonding or electronic effects. Here, we explore how nature can use the same chemical molecule as a cofactor for chemically distinct reactions using the concept of conformational flexibility of tetrapyrroles. The multifaceted roles of tetrapyrroles are discussed in the context of the current knowledge on distorted porphyrins. Contemporary analytical methods now allow a more quantitative look at cofactors in protein complexes and the development of the field is illustrated by case studies on hemeproteins and photosynthetic complexes. Specific tetrapyrrole conformations are now used to prepare bioengineered designer proteins with specific catalytic or photochemical properties.

A facile, experimentally calibrated computational procedure is described that affords the relative ordering of heme cofactor reduction potentials with respect to intrinsic shifts brought about by apoprotein induced heme-macrocycle distortion. The method utilizes heme-Fe partial atomic charges and is useful with the computationally inexpensive B3LYP/3-21g method calculated for simplified heme models extracted from the Protein Data Bank incorporating only the effects of varying macrocycle conformations and thereby delineating their physicochemical effects. The procedure was successfully calibrated using the atomic coordinates and published midpoint potentials from the heme cofactors in wild-type and a series of heme-NO and -O(2) binding domain mutants and thus confirmed the sole conformational modulation of the redox potentials in these complexes. This technique was also applied to the reaction center tetraheme cytochrome subunit of Blastochloris viridis to build upon previous work elucidating the role that conformational control plays in photosynthetic systems, and it was found that this effect may account for up to 70% (54mv) of the observed differences in the reduction potentials of the four hemes. We validate the approach using larger basis sets up to and including the triple-ζ, doubly polarized and augmented 6-311+g** basis and discuss the specific conformational origins of the effect.

Chemically identical tetrapyrrole cofactors such as hemes and chlorophylls participate in functionally diverse biological roles. An analysis of the available protein structural data for the bacteriochlorophylls in the photosynthetic reaction center gives statistically reliable evidence of the hypothesis that the protein induced cofactor conformation is a modulator of the bio-molecular function of each reaction center. The results serve as a general model to illustrate conformational control of tetrapyrrole cofactors in other proteins., (This journal is © The Royal Society of Chemistry 2011)