3D Methodology for Interpreting Disease-Associated Genomic Variation in RAG2

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PROJECT SUMMARY The current proposal seeks to advance mechanisms of interpretation of genomic variation found in RAG2. We leverage advanced computational techniques with existing and new experimental data, to develop a novel approach for characterizing and interpreting inter-individual genetic variation, and mutations observed in patients with immunodeficiency syndromes. Our central hypothesis is that structural calculations predict functional changes for RAG2 mutations via capture of specific biochemical and molecular mechanic features. Our approach investigates RAG2 mutations in a domain-specific manner, where each Aim investigates one of the two RAG2 structured domains: 1) we interrogate the effects of β-domain mutations on the RAG heterotetramer complex across its enzymatic cycle; 2) we computationally and functionally characterize how mutations in the plant homeodomain alter stability and chromatin binding capacity. Thus, between the two domains, we will investigate alteration of the RAG enzyme and its ability to be regulated by differentially targeting to the correct places in the genome, via its histone reading function. Our Aims are independent (using different approaches for different domains), yet synergistic due to each providing new information about RAG2 mutations observed in immunodeficiency patients. Both domains will be characterized in structure-dynamics-function paradigm, to elucidate details for each mutation in high-resolution, and to identify subgroups of mutations that have similar effects on RAG function. The subgroups we anticipate identifying will serve for follow-up studies into cellular effects and how to potentially address each type of dysfunction. When completed, the proposed studies will generate new data with clear biomedical relevance for diagnosis of immunodeficiency syndromes and enabling future research in how to differently address each group of mutations that modulate specific dimensions of RAG complex function. At a higher level, our proposal addresses a broad unmet need in genomics for new computational approaches to mechanistically interpret the wide landscape of human variation. We anticipate that the approach used here, will be generalizable to other proteins for how the computational tools can be applied in a robust manner to determine the underlying protein structure-function relationship for interpreting the structural biology of genetic disease pathogenesis. The data generated in this pilot award will thereby seed future applications by the current scientific and multi-disciplinary team, to further expand our understanding of RAG genetic variation and its effects on the immune system.