The Structure of 2Zn Pig Insulin Crystals at 1.5 <latex>$\overset{\circ}{A}$</latex> Resolution

Edward N. Baker, Thomas L. Blundell, John F. Cutfield, Susan M. Cutfield, Eleanor J. Dodson, Guy G. Dodson, Dorothy M. Crowfoot Hodgkin, Roderick E. Hubbard, Neil W. Isaacs, Colin D. Reynolds, Kiwako Sakabe, Norioshi Sakabe, Numminate M. Vijayan

Abstract

The paper describes the arrangement of the atoms within rhombohedral crystals of 2Zn pig insulin as seen in electron density maps calculated from X-ray data extending to 1.5 A (1 A = 10<latex>$^{-10}$</latex> m = 10<latex>$^{-1}$</latex> nm) at room temperature and refined to R = 0.153. The unit cell contains 2 zinc ions, 6 insulin molecules and about 3 x 283 water molecules. The atoms in the protein molecules appear well defined, 7 of the 102 side chains in the asymmetric unit have been assigned alternative disordered positions. The electron density over the water molecules has been interpreted in terms of 349 sites, 217 weighted 1.0, 126 weighted 0.5, 5 at 0.33 and 1 at 0.25 giving ca. 282 molecules. The positions and contacts of all the residues belonging to the two A and B chains of the asymmetric unit are shown first and then details of their arrangement in the two insulin molecules, 1 and 2, which are different. The formation from these molecules of a compact dimer and the further aggregation of three dimers to form a hexamer around two zinc ions, follows. It appears that in the packing of the hexamers in the crystal there are conflicting influences; too-close contacts between histidine B5 residues in neighbouring hexamers are probably responsible for movements of atoms at the beginning of the A chain of one of the two molecules of the dimer that initiate movements in other parts, particularly near the end of the B chain. At every stage of the building of the protein structure, residues to chains of definite conformation, molecules, dimers, hexamers and crystals, we can trace the effect of the packing of like groups to like, aliphatic groups together, aromatic groups together, hydrogenbonded structures, positive and negative ions. Between the protein molecules, the water is distributed in cavities and channels that are continuous throughout the crystals. More than half the water molecules appear directly hydrogen bonded to protein atoms. These are generally in contact with other water molecules in chains and rings of increasing disorder, corresponding with their movement through the crystals. Within the established crystal structure we survey next the distribution of hydrogen bonds within the protein molecules and between water and protein and water and water; all but eight of the active atoms in the protein form at least one hydrogen bond. We follow with a discussion of the effect of different contacts on the observed thermal parameters and the possibility of correlating these with movements of the monomer, dimer or hexamer as a whole. The correlation seems best for molecule 1 in the dimer. Finally we examine the relation of the crystal structure as a whole to the biological activity of insulin. The large size of the insulin receptor makes it likely that when it combines to form the receptor complex, it makes a large number of contacts with the surface of the insulin molecule. Some of these points of contact, such as, for example, B24 and B25 phenylalanine, are suggested by the changes in biological activity observed when these residues are modified. The conformational changes in the insulin chains produced by crystal packing can be seen as a model for possible changes induced by insulin contacts with the receptor that eventually we may hope to discover if the insulin-receptor complex is crystallized.

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