Structure of a recombinant calmodulin from Drosophila melanogaster refined at 2.2-A resolution.

Abstract

The crystal structure of calmodulin (Mr 16,700,148 residues) from Drosophila melanogaster as expressed in a bac$erial system has been determined and refined at 2.2-A resolution.Starting with the structure of mammalian calmodulin, we produced an extensively refitted and refined model with a conventional crystallographic R value of 0.197 for t4e 5,239 reflections (F 2 2a(F)) within the 10.0-2.2-Aresolution range.The model includes 1,164 protein atoms, 4 calcium ions, and 78 water molecules and has root mFan square deviations from stapdard values of 0.018 A for bond lengths and 0.043 A for angle distances.The overall structure is similar to mammalian calmodulin, with a seven-turn central helix connecting the two calciumbinding domains.The "dumb-bell" shaped molecule contains seven a-helices and four "EF hand" calciumbinding sites.Although the amino acid sequences of mammalian and Drosophila calmodulins differ by only three conservative amino acid changes, the refined model reveals a number of significant differences between the two structures.Superimposition of the stpctures yields a root mean square deviation of 1.22 A for the 1,120 equivalent atoms.The calcium-binding domains have a root mean square deviation of 0.85 A for the 353 equivalent atoms.There are also differences in the amino terminus, the bend of the central a-helix, and the orientations of some of the side chains.Calmodulin is the principal calcium-dependent regulator of a variety of intracellular processes (1-3).The small, highly acidic protein (148 residues, M, of 16,700) has four Ca2+ sites, is ubiquitous in eukaryotes, and is h;:hly conserved throughout the animal kingdom.The crystal structure of Ca2'-satu: rated mammalian calmodulin has been determined to 2.2-A resolution with an R value of 0.175 (4, 5).In this form the protein consists of two globular calcium-binding domains connected by a long, 28-residue central helix.The Ca2+binding sites of calmodulin have the "EF hand" configuration first identified in parvalbumin (6) and subsequently observed in the crystal structures of intestinal calcium-binding protein (7) and troponin C (8,9).We are using Drosophila melanogaster calmodulin, expressed from bacteria (lo), in site-directed mutagenesis studand Welch Foundation grants (to F. A. Q. and K. B.).