Abstract
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.
Highlights
(MyoK) that has no tail but that contains a TH2 domain-like insert in the motor domain
The cellular functions of the Dictyostelium myosin I isozymes have the subject of extensive investigation, only the 16-kDa light chain associated with MyoD, termed MlcD, has been identified [14]
SDS-PAGE Analysis of MyoB—MyoB and MyoD were purified from Dictyostelium using established methods and analyzed by SDS-PAGE [15]
Summary
(MyoK) that has no tail but that contains a TH2 domain-like insert in the motor domain. ITC was used to monitor the binding of MlcB and MlcD to a 24-residue peptide (Ac-KDFDCTAKIQKAFRNWKAKKHSLE-NH2) corresponding to the IQ motif sequence in the MyoB neck.
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