W0303

Atomic Resolution Crystallography of Insulin Allosterism. Robert H. Blessing,^a,b Benoit Guillot,^c Claude Lecomte,^c and G. David Smith.^d,b, ^aHauptman-Woodward Medical Research Institute, Inc., 73 High St., Buffalo, NY 14203, USA, ^bDept. of Structural Biology, State Univ. of New York at Buffalo, blessing@hwi.buffalo.edu, ^cLaboratoire de Cristallographie et Modélisation des Matériaux Minéraux et Biologiques, Univ. Henri Poincaré, Nancy 1, Faculté des Sciences, BP 239, 54506 Vanœuvre-lès-Nancy, France, bguillot@lcm3b.uhp-nancy.fr and lecomte@lcm3b.uhp-nancy.fr, ^dStructural Biology and Biochemistry, Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada, gdsmith@sauron.psf.sickkids.on.ca.

Insulin (Ins), the hormone that regulates glucose metabolism, is composed of two peptide chains, an A-chain of 21 amino acid residues and a B-chain of 30 residues. The two chains are cross-linked by two disulfide bonds, and the A-chain contains a third, intra-chain disulfide bond. Insulin is synthesized in the pancreas and stored there as hexameric complexes with zinc ions. The complexes, which can be formulated Zn₂Ins₆, (ZnIns₃)₂, or Zn[(Ins₂)₃]Zn, readily crystallize, and pharmaceutical preparations of insulin for diabetes therapy are physiologically compatible aqueous suspensions of microcrystalline hexameric zinc insulin. Depending on concentrations of salt and/or of phenol or a phenolic additive, zinc insulin hexamers undergo reversible, allosteric transformations between T and R conformational states of Ins monomers in three, interconvertible, stable hexamers, viz., Zn₂(T-Ins)₆← → Zn₂(T-Ins•R-Ins)₃ ← → Zn₂(R-Ins₂)₆. Since it is the Ins monomer that is hormonally active, the pharmacokinetics of insulin therapy depend critically on the rates of microcrystal dissolution and hexamer dissociation, and these, in turn, depend on the allosteric conformational states T₆ ← → T₃R₃ ← → R₆.

High-resolution structures have been determined for each of the three conformational states in a series of hexamer crystals: T₆ and T₃R₃ crystals to atomic resolutions of 1.0 and 1.2 Å, respectively, and R₆ crystals to 1.7 and 1.8 Å resolution. Our studies now focus on mapping and analyzing the water structure and charge density and electrostatic potential distributions in the central cores of the hexamers, where rearrangements of ionization/protonation, hydrogen bonding, and hydration structures within [(Glu^B13)₂(H₂O)_2x]₃ clusters around the hexamer centers appear to be implicated in the trigger mechanism of T → R transformation.