Crystal structural studies of destripeptide (B28-B30) insulin

Abstract

Single crystals of destripeptide (B28-B30) insulin (DTRI) in three forms were obtained by hanging-drop vapor diffusion method. Form 1 belongs to P2 1 space group with cell parameters a=4.77 nm b=6.19 nm c=6.12 nm β=110.3 o . Form 2 belongs to P4 1 22 or P4 3 22 space group with cell parameters a= 6.45 nm, c=12.07 nm. Form 3 belongs to P2 1 2 1 2 1 space group with cell parameters a=4.98 nm b=5.16 nm c=10.06 nm. The structure of form 1 crystal was determined by molecular replacement method and refined at 0.23 nm resolution. The R-factor of the final model is 18.8% with r.m.s. deviations of 0.001 5 nm and 3.3 o for the bond lengths and the bond angles, respectively. Studies on the crystal structure show that the removal of B28 Pro has brought DTRI structural changes which made it dissociate more easily than native insulin although DTRI can still form a hexamer.Insulin has been used successfully in the treatment of diabetes, but its self-association property is always a disturbing problem. Subcutaneously injected insulin exists as oligomers [1] and it takes a long time for insulin to migrate into the bloodstream, consequently hindering its effective absorption of insulin. It is important to find a fast-acting insulin that exists as monomer or dissociates to monomer rapidly in treating diabetes.The interactions between the dimerizing monomers are crucial in insulin association [2] . B28 Pro is located in the dimerization surface and it is very important for dimer formation and stabilization. Brems et al. [3] found that the association ability of mutants at B28 Pro were all significantly reduced whenever B28 Pro was substituted by acid amino acid Asp, neutral Ala or basic Lys. Their results also showed that the removal of B30 and B29 were not critical for insulin association but the additional removal of B28 Pro reduced the dimerization constant by a factor of 300. Lys B28 Pro B29 insulin (Humalog®) is a commercially available monomer insulin analogue [4] . It is obtained by simple amino acid reversion of B28 and B29. The crystal structures of B28 Pro Asp insulin [5] and Lys B28 Pro B29 insulin [6] have been solved. The results show that the hydrophobic interaction between B28 Pro and the other monomer in a dimer is the dominant reason of dimer formation and stabilization. Jrgensen et al. [7] also indicated that the monomerization of Des-[B25 Phe] insulin in solution was caused by the lack of hydrophobic interaction with the other molecule when B28 Pro moved to B27. All studies show that B28 Pro is crucial for insulin association.

Crystal structure of destripeptide (B28–B30) insulin: implications for insulin dissociation

“…The preparation and crystallization of recombinant DTRI was as described earlier [11,17]. The crystals were grown by the hanging drop vapor di¡usion method.…”