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Prediction of Function Determining and Buried Residues Through Analysis of Saturation Mutagenesis Datasets.

ABSTRACT: Mutational scanning can be used to probe effects of large numbers of point mutations on protein function. Positions affected by mutation are primarily at either buried or at exposed residues directly involved in function, hereafter designated as active-site residues. In the absence of prior structural information, it has not been easy to distinguish between these two categories of residues. We curated and analyzed a set of twelve published deep mutational scanning datasets. The analysis revealed differential patterns of mutational sensitivity and substitution preferences at buried and exposed positions. Prediction of buried-sites solely from the mutational sensitivity data was facilitated by incorporating predicted sequence-based accessibility values. For active-site residues we observed mean sensitivity, specificity and accuracy of 61, 90 and 88% respectively. For buried residues the corresponding figures were 59, 90 and 84% while for exposed non active-site residues these were 98, 44 and 82% respectively. We also identified positions which did not follow these general trends and might require further experimental re-validation. This analysis highlights the ability of deep mutational scans to provide important structural and functional insights, even in the absence of three-dimensional structures determined using conventional structure determination techniques, and also discuss some limitations of the methodology.

SUBMITTER: Bhasin M

PROVIDER: S-EPMC7991590 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Semaphorin 4A (Sema4A) exerts a stabilizing effect on human Treg cells in PBMC and CD4+ T cell cultures by engaging Plexin B1. Sema4A deficient mice display enhanced allergic airway inflammation accompanied by fewer Treg cells, while Sema4D deficient mice displayed reduced inflammation and increased Treg cell numbers even though both Sema4 subfamily members engage Plexin B1. The main objectives of this study were: 1. To compare the in vitro effects of Sema4A and Sema4D proteins on human Treg cells; and 2. To identify function-determining residues in Sema4A critical for binding to Plexin B1 based on Sema4D homology modeling. We report here that Sema4A and Sema4D display opposite effects on human Treg cells in in vitro PBMC cultures; Sema4D inhibited the CD4+CD25+Foxp3+ cell numbers and CD25/Foxp3 expression. Sema4A and Sema4D competitively bind to Plexin B1 in vitro and hence may be doing so in vivo as well. Bayesian Partitioning with Pattern Selection (BPPS) partitioned 4505 Sema domains from diverse organisms into subgroups based on distinguishing sequence patterns that are likely responsible for functional differences. BPPS groups Sema3 and Sema4 into one family and further separates Sema4A and Sema4D into distinct subfamilies. Residues distinctive of the Sema3,4 family and of Sema4A (and by homology of Sema4D) tend to cluster around the Plexin B1 binding site. This suggests that the residues both common to and distinctive of Sema4A and Sema4D may mediate binding to Plexin B1, with subfamily residues mediating functional specificity. We mutated the Sema4A-specific residues M198 and F223 to alanine; notably, F223 in Sema4A corresponds to alanine in Sema4D. Mutant proteins were assayed for Plexin B1-binding and Treg stimulation activities. The F223A mutant was unable to stimulate Treg stability in in vitro PBMC cultures despite binding Plexin B1 with an affinity similar to the WT protein. This research is a first step in generating potent mutant Sema4A molecules with stimulatory function for Treg cells with a view to designing immunotherapeutics for asthma.

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Prediction of Function Determining and Buried Residues Through Analysis of Saturation Mutagenesis Datasets.

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