Structure of an insulin dimer in an orthorhombic crystal: the structure analysis of a human insulin mutant (B9 Ser -> Glu)

The structure of human insulin mutant. B9 (Ser-->Glu) was determined by an X-ray crystallographic method at 2.5 Angstrom resolution with an R factor o f 0.165 under non-crystallographic restraints. The crystals were grown at l ow pH (<3.8) and belong to the orthorhombic P2(1)2(1)2(1) space group with unit-cell dimensions a = 44.54, b = 46.40, c = 51.85 Angstrom and one dimer per asymmetric unit without further aggregation. The structure in this cry stal form can be regarded as a model for a discrete insulin dimer and displ ays the following features compared with the structure of 2Zn insulin. (i) The overall dimer is expanded and more symmetric. The two A chains are abou t 2 Angstrom more distant from each other, while the two B chains are about 0.8 Angstrom further apart. Both monomers are more similar to molecule 1 t han molecule 2 of the 2Zn insulin dimer. (ii) The dimer structure is stabil ized by protonation and neutralization of the carboxyl groups at lower pH a nd, in addition, by formation of a hydrogen-bond network among the side cha ins of residues GluB9, HisB13 and HisB10 on the dimer-forming surface of bo th monomers, resulting from a structural rearrangement. (iii) The B-chain a mino-terminal segment is in an open state (O state), i.e. a state different from the well known R and T states found in the insulin hexamer. In the O state, the B-chain N-terminal segment is in an extended conformation and is detached from the rest of the molecule. This conformational state has also been observed in the monomeric crystal structure of despentapeptide (B26-B 30) and desheptapeptide (B24-B30) insulin, as well as in the solution struc ture of an engineered insulin monomer. It suggests that the O state may be the characteristic conformation of insulin in lower aggregation forms and m ay be relevant to the formation of insulin fibrils. In addition, based on t he crystallization process, the smallest possible building blocks of insuli n crystal are also discussed.