Mutation Analysis of the Interactions Between RPGR, RBD4–CY and Kinesin–Binding Domains of RanBP2, and Their Molecular Partners

Abstract

Abstract: : Purpose: Structural determinants govern the interaction between proteins. Among other effects, natural occurring mutations often affect directly the molecular determinants underlying specific protein interactions. In addition, introduction of artificial mutations helps to define functional modules important for protein function. Genetic lesions in RPGR and its molecular partner, RPGRIP1, lead to heterogeneous and disparity of retinal phenotypes in the human and among species. Likewise, mutations in opsin exhibit allelic heterogeneity that is manifested often by the variable expressivity of the disease. We have shown also that the RBD4–CY and kinesin–binding domains (KBD) of RanBP2 interact, respectively, with the red opsin and KIF5B/KIF5C but not KIF5A. Our goal was to define the molecular determinants governing the interactions between RPGR, RBD4–CY and KBD domains of RanBP2 and their molecular partners, the molecular basis of allelic heterogeneity and the pathogenesis caused by mutations in some of the cognate genes. Methods: A systematic and targeted mutagenesis analysis in RPGR, RPGRIP1, red opsin, CY and KDB domains of RanBP2 and KIF5B/KIF5C was performed. The effect of these on the activity of the proteins was investigated. Results: A number of mutations seem to affect directly the molecular determinants governing the interaction between the proteins investigated. The N–terminal end of red opsin by itself retains binding activity towards RBD4–CY of RanBP2. The equivalent P23 of rhodopsin in red opsin and some of its flanking residues are critical for conferring substrate specificity towards RBD4–CY of RanBP2. Finally, we defined two minimal RanBP2 KBD domains in KIF5B/KIF5C and narrowed down the molecular basis of the KIF5–isotype binding specificity towards RanBP2 to a single amino acid conserved in KIF5B/KIF5C but not in KIF5A. Conclusions: We have generated a series of mutations in sets of protein partners to delineate further the molecular determinants underlying the specificity of their interactions, to develop tools to manipulate the interactions and understand the allelic heterogeneity allied to mutations in their cognate genes.

Keywords: protein structure/function • proteins encoded by disease genes • retinal degenerations: hereditary