Modification of Loop 1 Affects the Nucleotide - Binding Properties of Myo 1 c ,

Myo1c is one of eight members of the mammalian myosin I family of actin-associated molecular motors. In stereocilia of the hair cells in the inner ear, Myo1c presumably serves as the adaptation motor, which regulates the opening and closing of transduction channels. Although there is conservation of sequence and structure among all myosins in the N-terminal motor domain, which contains the nucleotideand actin-binding sites, some differences include the length and composition of surface loops, including loop 1, which lies near the nucleotide-binding domain. To investigate the role of loop 1, we expressed in insect cells mutants of a truncated form of Myo1c, Myo1c1IQ, as well as chimeras of Myo1c1IQ with the analogous loop from other myosins. We found that replacement of the charged residues in loop 1 with alanines or the whole loop with a series of alanines did not alter the ATPase activity, transient kinetics properties and Ca2+-sensitivity of Myo1c1IQ. Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c1IQtonic) or replacement with a single glycine (Myo1c1IQ-G) accelerated ADP release from A.M 2-3fold in Ca2+, whereas substitution with loop 1 from phasic muscle myosin II (Myo1c1IQ-phasic) accelerated ADP release 35-fold. Motility assays with chimeras containing a single g-helix, or SAH, domain showed that Myo1cSAH-tonic translocated actin in vitro twice as fast as Myo1cSAH-WT and 3-fold faster than Myo1cSAH-G. The studies show that changes induced in Myo1c by modifying loop 1 showed no resemblance to the behaviour of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras. Myosins are a large family of molecular motors that have been subdivided into more than 30 subgroups (1,2). Different family members are involved in a wide range of motor activities in eukaryotic cells such as muscle contraction, cell division, pseudopod extension and vesicle transport (3,4). Class I myosins are a diverse group of monomeric myosins implicated in several actin-mediated processes including organization and maintenance of tension of the cytoskeleton as well as signal transduction (5-7). The mammalian class I myosin, Myo1c, consists of a heavy chain containing an N-terminal motor domain, a neck or lever arm stabilised by 3 calmodulin molecules and a C-terminal tail region implicated in membrane binding (8-10). Myo1c mediates the cycling of GLUT4 transporters in adipocytes by promoting the †This work was supported by NIH Grant R01 DC08793 to L. M. C. and Wellcome Trust Grant 070021 to M. A. G. *To whom correspondence should be addressed: Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472. Telephone: (617) 658-7784. Fax. (617) 972-1761. [email protected]. NIH Public Access Author Manuscript Biochemistry. Author manuscript; available in PMC 2011 February 9. Published in final edited form as: Biochemistry. 2010 February 9; 49(5): 958–971. doi:10.1021/bi901803j. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript fusion of GLUT4-containing vesicles with the cell membrane (11-13). In the specialized hair cells of the inner ear, Myo1c is believed to be the adaptation motor, which regulates the tension on the tip links that connect neighbouring stereocilia thereby controlling the opening and closing of transduction channels (5). We recently defined the biochemical kinetics of the ATP-driven interaction of Myo1c with actin and showed that it has an unusual calcium dependence (14). Calcium binding to the calmodulin closest to the motor domain has little effect on the ATPase or motor activity, but alters specific steps of the ATPase cycle. ATP hydrolysis was inhibited 7-fold by calcium while ADP release from acto-Myo1c was accelerated by 10-fold. These two changes together would reduce the lifetime of the actin-attached states and increase the lifetime of the actin-detached state without altering the overall cycle time. In combination these properties appear to be ideal to modulate the activity of Myo1c in response to a calcium transient of the sort expected to occur in the inner ear. The observation that certain events in the ATPase cycle are regulated oppositely in calcium is curious and in particular the Ca2+-regulation of the ATP hydrolysis step has not been reported previously for any myosin. This raises the question of whether the ATP hydrolysis step is controlled by calcium binding to the calmodulin light chain. In fact, the regulation may occur via the myosin conformational change that precedes the ATP cleavage step and is thought to position the catalytic residues to allow ATP splitting. This conformational change, known as the recovery stroke, involves the movement of switch II and the accompanying converterdomain movement to reprime the lever arm/light-chain domain (15,16). Thus, the recovery stroke results in a repositioning of the light chains and therefore could be influenced by a calcium-induced change in the calmodulin/light chain conformation. Other events regulated by calcium are ATP binding and ADP release. ADP release in smooth and scallop muscle is altered via phosphorylation or calcium binding to light chains (17,18). The exact mechanism by which the conformation of the light chains is communicated to the nucleotide-binding pocket for these dimeric myosins is not defined, but may involve some form of interaction between the two motor domains facilitated by the conformation of the neck domains (19-21). Although we previously reported a modest regulation of nucleotide release in calcium for the related myosin I, Myo1b, the behaviour of Myo1c is more dramatic (22). The communication pathway of this novel regulation via the light chains for monomeric myosins is unknown. The structure of the myosin motor domain is highly conserved across the broad myosin family; however, there are several surface loops that are less well conserved. These loops have been proposed to tune the activity of myosins to their specific cellular roles (23). One such loop is loop 1 near the entrance to the nucleotide-binding pocket. Loop 1 joins two helices, one connected to switch 1 and the other to the P-loop. These two elements form a major part of the binding site for the gamma phosphate in ATP. Loop 1 is therefore in a position to influence access to the nucleotide pocket and the interaction with the gamma Pi of ATP. Previous studies of natural variations of loop 1 in vertebrate smooth muscle myosin II (24-26) have shown that alternately expressed loops can alter the release of ADP from the nucleotide pocket. Furthermore, studies on scallop muscle myosin II (27) have established that alternate splicing of loop 1 alters the affinity of acto-myosin for ADP thereby permitting the cell to produce myosins with differing ATPase and motility properties. However, studies of engineered variations in the size and composition of loop 1 in either a smooth muscle myosin II or a Dictyostelium myosin II (28) have so far failed to resolve which features of loop 1 are responsible for this modulation of myosin activity. Adamek et al. Page 2 Biochemistry. Author manuscript; available in PMC 2011 February 9. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript Goodson and colleagues showed that the sequences of loop 1 are well conserved when myosins are grouped according to their kinetic activity and proposed that loop 1 modulates the kinetic characteristics that distinguish one myosin isoform from another (29). It has also been suggested that the flexibility of loop 1 or its length determines activity (26) and that longer loops interact with other parts of the myosin molecule including the light chains in some conformations thereby affecting regulation (30). In a previous study (22), we explored the role of loop 1 on Myo1b by replacing its charged residues with alanine, the whole loop with alanine residues or replacing the loop with either a single glycine, or loop 1 from either phasic or tonic smooth muscle myosin II, a strategy similar to that used by others to explore the effects of loop 1 on smooth muscle myosin II (24,26,27). We found that loop 1 had major effects on the coupling of actin and nucleotide-binding events in Myo1b and that it is likely to modulate the load dependence of Myo1b. The role of loop 1 can therefore have broad implications for modulating myosin motor activity; however, the same mutations in different myosin backgrounds can have quite different effects (22,31). To explore the role of loop 1 in Myo1c we investigated how loop 1 modulates the motor activity of Myo1c, specifically its effects on nucleotide binding and release in the presence and absence of calcium. We found that all the results obtained with the Myo1c chimeras stand in marked contrast to those previously obtained with similar constructs using Myo1b. Experimental Procedures Preparation of Constructs and Expression in Insect Cells Using cDNA encoding the entire open reading frame of Myo1c (previously known as myr 2; the kind gift of Dr. Martin Bähler; Institut für Allgemeine Zoologie und Genetik, WestfälischeWilhelms Universität, Münster, Germany), we prepared a series of constructs in which loop 1 of Myo1c1IQ, a truncated form of rat Myo1c representing the motor domains and first IQ domain (amino acids 1-725) (32), was replaced with loops 1 from other myosins; or in which alanine substitutions were made in the endogenous loop 1. To define the beginning and end of loop 1 in Myo1c, secondary structure assignments were made by the program DSSP (Definition of Secondary Structure of Protein; http://swift.cmbi.ru.nl/gv/dssp/descrip.html). All mutants except Myo1c1IQ-phasic were created using polymerase chain reaction (PCR). Overlapping sets of primers having the desired mutations and restriction sites and incorporating a FLAG tag at the C-terminus were used in the first set of PCR reactions. The two resulting fragments were then subjected to another round of PCR with the outside primers to generate the in-frame fusion proteins. After treatment with the appropriate restriction enzymes, the purified PCR products were ligated into the pFastBacDUAL transfer vector (Gibco BRL, Gaithersburg, MD) downstream of the polyhedrin promoter; the p10 promoter cloning site contained the gene coding for calmodulin (32). The plasmids were transformed into DH5g cells and selected by antibiotic resistance. Colonies were grown and the isolated DNA was tested for the presence of inserts by restriction analysis. Plasmids containing Myo1c inserts were sequenced with internal and vector-specific oligonucleotides using automated sequencing. Myo1c1IQ-phasic was made using the Transformer Site-Directed Mutagenesis Kit (Clontech, Mountain View, CA 94043). Briefly, a mutagenic primer and a selection primer were used to generate a mixture of mutated and unmutated plasmids. The mixture was then subjected to a selective restriction digestion, which selectively digested the unmutated plasmid. The mutated plasmid was then transformed as above into DH5g followed by selection according to antibiotic resistance. Plasmids were isolated from selected colonies and those containing inserts were subjected to automatic sequencing. In all cases, the recombinant donor plasmid was transformed into DH10Bac E. coli cells (Invitrogen, Carlsbad, Ca 92008) for transposition into bacmid. Recombinant bacmid DNA Adamek et al. Page 3 Biochemistry. Author manuscript; available in PMC 2011 February 9. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript was isolated by potassium acetate precipitation as described by the Bac-to-Bac Baculovirus Expression Systems Instruction Manual supplied by Invitrogen. Virus was produced by transfecting the recombinant bacmid DNA into Spondoptera frugiperda 9 (Sf9) insect cells with Cellfectin reagent (Invitrogen) followed by 3 days of growth. Subsequently, amplified virus was used to infect Sf9 cells in suspension. Infection was allowed to proceed for 4 days after which time cells were harvested by centrifugation. Cell pellets were either used immediately for protein isolation or frozen in liquid N2 and stored at -80°C for future use. Protein purification To isolate protein, the insect cell pellets were homogenized in 10 mM Tris, pH 7.5, 0.2 M NaCl, 4 mM MgCl2, and 2 mM ATP in the presence of protease inhibitors and then centrifuged at 183,000 × g for 50 min. The supernatant was applied to an anti-FLAG column, and after washing the expressed proteins were eluted with a step gradient of FLAG peptide. Fractions containing protein were identified by SDS-polyacrylamide gel electrophoresis, pooled and dialyzed against 10 mM Tris, 50 mM KCL and 1 mM DTT. Proteins were either used immediately or stored at -80°C for future use. Preparation of Constructs with a SAH Domain Using the clone for rat myosin X (kindly provided by Drs. Erich Boger and Thomas Friedman, NIDCD, NIH) we prepared by PCR a fusion construct (Myo1cSAH-WT) in which the SAH domain of myosin X (amino acids 805-843) followed by a myc and FLAG tag were added to wild-type Myo1c at residue 762 based on sequence analogy between Myo1c and myosin X as determined by Clustal X. This results in a LCBD consisting of two complete IQ motifs and most of the third IQ motif followed by the SAH domain. The SAH constructs express in insect cells at levels equivalent to the 1IQ forms. Similar constructs for the loop 1 mutants, tonic, phasic and G (Myo1cSAH-tonic, Myo1cSAH-phasic and Myo1cSAH-G, respectively) were also prepared and expressed in insect cells and purified by affinity purification with anti-FLAG.