Refinement of recombinant oncomodulin at 1.30 A resolution.

A refinement of the oncomodulin crystal structure at 1.30 A resolution has been carried out with X-ray data from the recombinant protein. The crystallographic R-factor values are 0.169 for 19,995 reflections in the range 6.0 to 1.30 A, which were used for the restrained least-squares refinement, and 0.176 for 20,186 observed reflections in the range 10.0 to 1.30 A. This high resolution refinement has enabled us to make more definitive statements about the molecular structure than was possible heretofore. The present model includes residues 1 to 108, the two Ca2+ of the CD and EF loops, two intermolecular Ca2+, and 103 water molecules per oncomodulin molecule. The electron density maps indicate disordered orientations for ten residues on the hydrophilic surface of the molecule. The pattern of molecular aggregation via intermolecular Ca2+, which occurs in the native rat oncomodulin structure, is also present in the recombinant oncomodulin structure. The Cys18 side-chain is not in a position that would be easily accessible for molecular dimerization via a disulphide bond. The substitution of Glu59, which is preserved in all the determined species of parvalbumin, by Asp59 in oncomodulin seems to break a stabilizing hydrogen bond in the CD loop and render the main-chain in positions 59 to 60 somewhat unstable. This instability in the CD loop, and the strong tendency of oncomodulin for molecular aggregation via intermolecular Ca2+, appear to be the two outstanding features that may account for oncomodulin's biological peculiarities.

Structure of oncomodulin refined at 1.85 A resolution. An example of extensive molecular aggregation via Ca2+.

The crystal structure of oncomodulin, a 12,000 Mr protein isolated from rat tumours, has been determined by molecular replacement using the carp parvalbumin structure as a starting model. Refinement was performed by cycles of molecular fitting and restrained least-squares, using area-detector intensity data to 1.85 A resolution. For the 5770 reflections in the range 6.0 to 1.85 A, which were used in the refinement, the crystallographic R-factor is 0.166. The refined model includes residues 2 to 108, three Ca2+ and 87 water molecules per oncomodulin molecule. The oncomodulin backbone is closely related to that of parvalbumin; however, some differences are found after a least-squares fit of the two backbones, with root-mean-square (r.m.s.) deviations of 1 to 2 A in residues 2 to 6, 59 to 61 of the CD loop, 87, 90 and 108. The overall r.m.s. deviation of the backbone residues 5 to 108 is 0.62 A. Each of the two Ca2+ atoms that are bound to the CD and EF loops is co-ordinated to seven oxygen atoms, including one water molecule. The third Ca2+ is also seven-co-ordinated, to five oxygen atoms belonging to three different oncomodulin molecules and to two water molecules which form hydrogen bonds to a fourth oncomodulin; thus, this intermolecular Ca2+ and its equivalents interlink the molecules into zigzag layers normal to the b axis with a spacing of b/2 or 32.14 A. No such extensive molecular aggregation has been reported for any of the related Ca-binding regulatory proteins of the troponin-C family studied thus far. The Ca-O distances in all three polyhedra are in the range 2.07 A to 2.64 A, indicating tightly bound Ca polyhedra.