Dictyostelium discoideum express seven single-headed myosin-I isozymes (MyoA-MyoE and MyoK) that drive motile processes at the cell membrane. The light chains for MyoA and MyoE were identified by expressing Flag-tagged constructs consisting of the motor domain and the two IQ motifs in the neck region in Dictyostelium. The MyoA and MyoE constructs both copurified with calmodulin. Isothermal titration calorimetry (ITC) showed that apo-calmodulin bound to peptides corresponding to the MyoA and MyoE IQ motifs with micromolar affinity. In the presence of calcium, calmodulin cross-linked two IQ motif peptides, with one domain binding with nanomolar affinity and the other with micromolar affinity. The IQ motifs were required for the actin-activated MgATPase activity of MyoA but not MyoE; however, neither myosin exhibited calcium-dependent activity. A Flag-tagged construct consisting of the MyoC motor domain and the three IQ motifs in the adjacent neck region bound a novel 8.6 kDa two EF-hand protein named MlcC, for myosin light chain for MyoC. MlcC is most similar to the C-terminal domain of calmodulin but does not bind calcium. ITC studies showed that MlcC binds IQ1 and IQ2 but not IQ3 of MyoC. IQ3 contains a proline residue that may render it nonfunctional. Each long-tailed Dictyostelium myosin-I has now been shown to have a unique light chain (MyoB-MlcB, MyoC-MlcC, and MyoD-MlcD), whereas the short-tailed myosins-I, MyoA and MyoE, have the multifunctional calmodulin as a light chain. The diversity in light chain composition is likely to contribute to the distinct cellular functions of each myosin-I isozyme.

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Myosin light chains are key regulators of class 1 myosins and typically comprise two domains, with calmodulin being the archetypal example. They bind IQ motifs within the myosin neck region and amplify conformational changes in the motor domain. A single lobe light chain, myosin light chain C (MlcC), was recently identified and shown to specifically bind to two sequentially divergent IQ motifs of the Dictyostelium myosin-1C. To provide a molecular basis of this interaction, the structures of apo-MlcC and a 2:1 MlcC⅐myosin-1C neck complex were determined. The two non-functional EF-hand motifs of MlcC pack together to form a globular four-helix bundle that opens up to expose a central hydrophobic groove, which interacts with the N-terminal portion of the divergent IQ1 and IQ2 motifs. The N-and C-terminal regions of MlcC make critical contacts that contribute to its specific interactions with the myosin-1C divergent IQ motifs, which are contacts that deviate from the traditional mode of calmodulin-IQ recognition.The class 1 myosins (myosin-1) are a widely expressed family of single-headed, non-filament-forming, membrane-binding myosins (1-3). The myosin-1 heavy chain consists of a highly conserved N-terminal motor domain with sites for binding actin and for ATP hydrolysis, a light chain-binding domain (LCBD) 5 that acts as the motor's lever arm, and a C-terminal tail. The myosin-1 tail contains a tail homology 1 (TH1) domain that interacts with acidic phospholipids and, in some cases, also contains a glycine-and proline-rich TH2 domain that binds filamentous actin and an Src homology 3 domain that forms complexes with proteins that stimulate actin filament assembly (4 -10). Myosin-1 is able to cross-link the plasma membrane to the underlying actin cytoskeleton and plays a direct role in the control of cortical and membrane tension (11,12). These motor proteins can also drive vesicle trafficking, pseudopod retraction, and the uptake of particles and fluids by phagocytosis and micropinocytosis (13-16).The myosin-1 LCBD includes one or more IQ motifs, which are ␣-helical sequences between 18 and 25 residues in length that terminate with a loosely conserved IQXXXRGXXXR consensus sequence (17, 18). IQ motifs bind calmodulin (CaM) or CaM-related light chains (LCs) in the absence of Ca 2ϩ . The LCs stabilize the ␣-helical structure of the LCBD, allowing it to function as a rigid swinging lever arm to amplify ATP-dependent conformational changes that originate within the motor domain (19). The LCs are also important regulatory sites that interact with the motor domain to influence mechanochemical properties such as step size, motility rate, and force sensing (1,20,21).The social amoeba Dictyostelium discoideum has been used extensively to study myosin-1 structure, function, and regulation. The seven Dictyostelium myosin-1 isozymes include three with short tails that contain only a TH1 domain (myosin-1A, -1E, and -1F), three with long tails that have TH1, TH2, and Src homology 3 domains (myosin-1B, -1C, and -1D), and one ...

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Background: MlcB is a MyoB-specific light chain. Results: MlcB adopts a unique fold among EF-hand calcium-binding proteins and binds the MyoB IQ motif via a hydrophobic surface that is maintained in the holo state. Conclusion: The MlcB structure and mode of IQ recognition is unique among myosin light chains. Significance: ApoMlcB structure provides a basis for MyoB IQ-motif recognition.