Structural Basis for the Interaction of the Myosin Light Chain Mlc1p with the Myosin V Myo2p IQ Motifs

Abstract

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.Myosin V are processive motors that transport a variety of intracellular cargo in a manner that is timely, selectively, and reversibly regulated by its interaction with different proteins via their cargo bound domain (1, 2). Myosin V display a dimeric heavy chain containing several light chain binding sites called IQ motifs. The so-called IQ motif (ϳ23-25 residues), with a consensus sequence of the form IQXXXRGXXXR, is repeated tandemly (2-6 times) in the heavy chain of many myosins. These motifs are among the best known Ca 2ϩ -independent calmodulin (CaM) 3 and myosin light chain targets. Single or multiple repeats IQ motif-like sequences have been identified in myosin, kinases, and IQGAP-like and Ras-GEF signaling proteins in Alzheimer disease proteins (3-5). Tissue myosin V copurify with at least two light chains in addition to calmodulin, but the identity and binding sites of these light chains are unknown (6). It has been shown that either an essential light chain or CaM can occupy the first IQ motif of myosin V (7, 8). How such differential interaction is achieved is unknown. Nor is it clear if and when essential light chain and CaM compete for the same site (4, 9 -11). Recent structural and genetic studies point to a previously uncovered role for calmodulin/myosin light chain interaction with the class V myosin IQ motifs in regulating the cargo binding abiliti...

Solution Structures of Ca2+-CIB1 and Mg2+-CIB1 and Their Interactions with the Platelet Integrin αIIb Cytoplasmic Domain

“…Moreover, 1XO5 is a monomeric protein, and our previous NMR data are consistent with the protein being in a monomeric form in solution (12,42). The same protocol has been successfully employed in the structure determination of several other proteins (33,41,43,44). The 13 C-and 15 Nedited NOESY provided distance restraints for our structure calculation in addition to the dihedral angle, hydrogen bonds, and the RDC restraints for the protein backbone.…”

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase

“…On the basis of recent structural studies of Cdc4p and Mlc1p (the budding yeast ELC), Cam2p might be capable of simultaneously associating with Pik1p and Myo1p in a ternary complex. X-ray crystallography and NMR-based analysis of Cdc4p and Mlc1p have shown that these proteins are monomeric dumbbell-shaped molecules made up of two structurally distinct domains connected by a flexible linker (Amata et al, 2008;Escobar-Cabrera et al, 2005;Pennestri et al, 2007;Slupsky et al, 2001;Terrak et al, 2003). Mlc1p can adopt different conformations depending on the IQ motif it binds to (Terrak et al, 2003).…”

“…Mlc1p can adopt different conformations depending on the IQ motif it binds to (Terrak et al, 2003). In a 'compact' conformation both domains of Mlc1p interact with the IQ motif, whereas in an 'extended' conformation only one domain binds the IQ motif (Pennestri et al, 2007;Terrak et al, 2005;Terrak et al, 2003) leaving the other domain free to potentially bind another IQ motif on a different protein (Terrak et al, 2003). In the future it will be worth testing whether such a mechanism allows Cam2p to function with both Myo1p and Pik1p at the cortex to directly coordinate actomyosin forces with phospholipid modification at sites of endocytosis.…”

Myosin V from head to tail

“…A second conclusion of the crystallographic studies was that the N-lobe of Mlc1p does not interact with IQ motifs 4 and 6, leaving it free to potentially interact with another binding partner. NMR studies further showed that MLC1p also binds to the first IQ motif of Myo2p only via its C-lobe, leaving the N-lobe free to bind another target [21]. In contrast, CaM binds to IQ1 with both lobes based on fluorescence resonance It includes the last three strands of the seven-stranded b sheet (magenta and yellow) and other regions that accommodate the distortion that occurs between the rigor and post-rigor states, including loop 1 (missing residues that connect the green ends), and the bulge between b strands six and seven.…”

“…65,2008 Review Article energy transfer studies [18]. The different behavior of CaM and Mlc1p with respect to the first IQ motif of Myo2p appears to be related both to unique structural features of the N-lobe of Mlc1p, particularly the fifth D helix that stabilizes a closed lobe structure, and to the exact sequence of the IQ motif [21]. These different modes of binding may explain why Mlc1p has an essential function for vesicle targeting that is not shared by CaM.…”