Identification and characterization of an 8-kDa light chain associated with Dictyostelium discoideum MyoB, a class I myosin.
Dictyostelium discoideum MyoB is a single-headed class I myosin. Analysis of purified MyoB by SDS-PAGE indicated the presence of an approximately 9-kDa light chain. A tryptic digest of MyoB yielded a partial sequence for the light chain that exactly matched a sequence in a 73-amino acid, 8,296-Da protein (dictyBase number DDB0188713). This protein, termed MlcB, contains two EF-hand motifs and shares approximately 30% sequence identity with the N- and C-terminal lobes of calmodulin. FLAG-MlcB expressed in Dictyostelium co-immunoprecipitated with MyoB but not with the related class myosins and MyoD. Recombinant MlcB bound Ca2+ with a Kd value of 0.2 microm and underwent a Ca2+-induced change in conformation that increased alpha-helical content and surface hydrophobicity. Mutational analysis showed that the first EF-hand was responsible for Ca2+ binding. In the presence and absence of Ca2+ MlcB was a monomer in solution and bound to a MyoB IQ motif peptide with a Kd value of approximately 0.5 microm. A MyoB head-neck construct with a Ser to Glu mutation at the TEDS site bound MlcB and displayed an actin-activated Mg2+ ATPase activity that was insensitive to Ca2+. We conclude that MlcB represents a novel type of small myosin light chain that binds to IQ motifs in a manner comparable with a single lobe of a typical four-EF-hand protein.
Crawley
Scott W
SW
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
de la Roche
Marc A
MA
Lee
Sheu-Fen
SF
Li
Zhihao
Z
Chitayat
Seth
S
Smith
Steven P
SP
Côté
Graham P
GP
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004023:Dictyostelium; D020832:EF Hand Motifs; D004591:Electrophoresis, Polyacrylamide Gel; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018994:Myosin Light Chains / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D024461:Myosin Type I / Q000737:chemistry* / Q000235:genetics; D009842:Oligopeptides; D010455:Peptides; D011485:Protein Binding; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
D018994:Myosin Light Chains; D009842:Oligopeptides; D010455:Peptides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C085706:FLAG peptide; D024461:Myosin Type I; D002118:Calcium