Unraveling the symmetry ambiguity in a hexamer: Calculation of the R-6 human insulin structure

Crystallographic and NMR studies of insulin have revealed a highly flexible molecule with a range of different aggregation and structural states; the importance of these states for the function of the hormone is still unclear . To address this question, we have studied the solution structure of the i nsulin R-6 symmetric hexamer using NMR spectroscopy. Structure determinatio n of symmetric oligomers by NMR is complicated due to `symmetry ambiguity' between intra- and intermonomer NOEs, and between different classes of inte rmonomer NOEs. Hence, to date, only two symmetric tetramers and one symmetr ic pentamer (VTB, B subunit of verotoxin) have been solved by NMR; there ha s been no other symmetric hexamer or higher-order oligomer. Recently, we re ported a solution structure for R-6 insulin hexamer. However, in that study , a crystal structure was used as a reference to resolve ambiguities caused by the threefold symmetry; the same method was used in solving VTB. Here, we have successfully recalculated R-6 insulin using the symmetry-ADR method , a computational strategy in which ambiguities are resolved using the NMR data alone. Thus the obtained structure is a refinement of the previous R-6 solution structure. Correlated motions in the final structural ensemble we re analysed using a recently developed principal component method; this sug gests the presence of two major conformational substates. The study demonst rates that the solution structure of higher-order symmetric oligomers can b e determined unambiguously from NMR data alone, using the symmetry-ADR meth od. This success bodes well for future NMR studies of higher-order symmetri c oligomers. The correlated motions observed in the structural ensemble sug gest a new insight into the mechanism of phenol exchange and the T-6 <----> R-6 transition of insulin in solution.