Modification of loop 1 affects the nucleotide binding properties of Myo1c, the adaptation motor in the inner ear.
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 nucleotide- and 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, Myo1c(1IQ), as well as chimeras of Myo1c(1IQ) 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, or Ca(2+) sensitivity of Myo1c(1IQ). Substitution of loop 1 with that of the corresponding region from tonic smooth muscle myosin II (Myo1c(1IQ)-tonic) or replacement with a single glycine (Myo1c(1IQ)-G) accelerated the release of ADP from A.M 2-3-fold in Ca(2+), whereas substitution with loop 1 from phasic muscle myosin II (Myo1c(1IQ)-phasic) accelerated the release of ADP 35-fold. Motility assays with chimeras containing a single alpha-helix, or SAH, domain showed that Myo1c(SAH)-tonic translocated actin in vitro twice as fast as Myo1c(SAH)-WT and 3-fold faster than Myo1c(SAH)-G. The studies show that changes induced in Myo1c via modification of loop 1 showed no resemblance to the behavior of the loop donor myosins or to the changes previously observed with similar Myo1b chimeras.
Adamek
Nancy
N
University of Kent, Canterbury, Kent CT2 7NJ, UK.
Lieto-Trivedi
Alena
A
Geeves
Michael A
MA
Coluccio
Lynne M
LM
D000199:Actins / Q000378:metabolism; D000222:Adaptation, Physiological / Q000235:genetics / Q000502:physiology*; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000502:physiology; D007758:Ear, Inner / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000235:genetics; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024461:Myosin Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D011237:Predictive Value of Tests; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D051381:Rats; D018411:Spodoptera / Q000235:genetics
D000199:Actins; D020409:Molecular Motor Proteins; C088340:Myo1c protein, rat; D009711:Nucleotides; D017301:Ca(2+) Mg(2+)-ATPase; D024461:Myosin Type I; D000409:Alanine; D002118:Calcium; D005998:Glycine