ILPR repeats adopt diverse G‐quadruplex conformations that determine insulin binding

Abstract

The insulin-linked polymorphic region (ILPR) is a VNTR region located upstream of the insulin (INS) gene consisting of the repeat 5'-ACAGGGGTGTGGGG (repeat a) and several less abundant sequence repeats (b-n). Here, we have investigated the structural polymorphism of G-quadruplexes formed from the most common repeat sequences (a-c) and their effect on insulin protein binding. We first established that the ILPR repeats "b" and "c" can form quadruplex structures. Insulin has previously been shown to bind a G-quadruplex formed by a dimer of the repeat "a". Our findings show that insulin binds preferentially to the repeat "a" G-quadruplex (K(d) = 0.17 + or - 0.03 microM) over G-quadruplexes formed from other ILPR repeats that were tested (K(d)s from 0.71 + or - 0.15 to 1.07 + or - 0.09 microM). Additionally, the Watson-Crick complementary relationship between the loop regions of repeat "a" (ACA and TGT) seemingly play an important role in favoring a specific G-quadruplex conformation, which based on our data is critical for insulin binding. Affinity for insulin is reduced in sequences lacking the putative WC complementarity, however upon engineered restoration of complementarity, insulin binding is recovered. A DMS footprinting assay on the repeat "a" G-quadruplex in the presence of insulin, combined with binding affinities for ILPR mutants led to identification of a loop nucleotide critical for binding. Uniquely, insulin shows clear preference for binding to the G-quadruplexes with the more antiparallel feature. Collectively, our results illustrate the specific nature of insulin binding to the ILPR G-quadruplexes and begin to provide molecular details on such interactions.