Structural and Evolutionary Divergence of the RAG1/1L N-Terminal Zinc-Finger Domain

Abstract

RAG1/2 catalyzes V(D)J recombination to assemble antigen receptor genes, yet the zinc-finger element at the RAG1 N terminus has remained structurally and functionally uncharacterized. Using NMR spectroscopy, we determined the structure of the RAG1 N-terminal zinc-finger domain (NZD; residues 89-223). NZD adopts a compact, zinc-dependent fold comprising four -helices organized into two interdigitated zinc-finger modules, ZFa and ZFb. Structural similarity searches across protein-structure databases revealed no solved homologs, establishing NZD as a previously undescribed zinc-finger fold. Comparative analyses show that NZD is broadly conserved across RAG1 and RAG1-like (RAG1L) proteins but has undergone lineage-specific remodeling, including the jawed vertebrate-specific acquisition of an additional -helix (H2). Guided by structure prediction, we identified two NZD-like types in Chapaev transposases; one is highly similar to the RAG1L NZD, supporting an evolutionary link between RAG1/RAG1L and Chapaev NZDs. Similarity between ZFa and the single-zinc ZAD further suggests that NZD originated from an ancestral single-zinc fold. Together, these findings provide a structural framework for mechanistic and evolutionary analyses of RAG1.