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

Area of Science:

• Genetics and Molecular Biology
• Biochemistry and Biophysics

Background:

• The insulin-linked polymorphic region (ILPR) contains variable number tandem repeats (VNTRs) upstream of the insulin (INS) gene.
• Specific repeat sequences within the ILPR, particularly repeat 'a', are known to form G-quadruplex structures.

Purpose of the Study:

• To investigate the structural polymorphism of G-quadruplexes formed by common ILPR repeat sequences (a-c).
• To determine the effect of these G-quadruplex structures on insulin protein binding affinity and specificity.

Main Methods:

• G-quadruplex formation analysis for ILPR repeat sequences 'a', 'b', and 'c'.
• Insulin binding assays using surface plasmon resonance to determine binding affinities (K(d)).
• Site-directed mutagenesis and DMS footprinting to identify critical nucleotide residues and structural features for binding.

Main Results:

• ILPR repeats 'b' and 'c' form quadruplex structures, but repeat 'a' forms a higher affinity G-quadruplex for insulin (K(d) = 0.17 µM).
• Watson-Crick complementarity in the loop regions of repeat 'a' G-quadruplex is crucial for high-affinity insulin binding.
• Insulin exhibits a preference for antiparallel G-quadruplex conformations.

Conclusions:

• Insulin binding to ILPR G-quadruplexes is specific, with repeat 'a' being the preferred binding site.
• The structural conformation, including loop complementarity and antiparallel orientation, dictates insulin binding affinity.
• These findings provide molecular insights into the specific interactions between insulin and ILPR G-quadruplexes.