Loop 1 of Transducer Region in Mammalian Class I Myosin, Myo1b, Modulates Actin Affinity, ATPase Activity, and Nucleotide Access

Maqsood Ali Ansari,Michael A. Geeves,Richard Clark,Sheffali Dash,Lynne M. Coluccio

doi:10.1074/jbc.m504698200

Abstract

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.

Highlights

Myo1b is a mammalian class I myosin that is expressed in many different tissues (9 –11)
Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis [1]
We proposed that Pi release causes a rotation of the converter domain of the molecule in which the motor domain connects to the calmodulin-binding region, resulting in the power stroke, but that this is insufficient to open the nucleotide pocket and release ADP

Summary

EXPERIMENTAL PROCEDURES

Preparation of Chimeras—We prepared a series of constructs in which loop 1 of Myo1b was replaced with loops from other myosins or alanine substitutions in the original loop were made. Data Interpretation—The kinetics of the interaction of expressed wild-type Myo1b1IQ and the loop 1 mutants with nucleotide (T, ATP; D, ADP) were interpreted in terms of the model we described previously [19] (Scheme 1) In this model, we assume that actin-Myo1b exists in two conformations in the absence of nucleotide (A.M and A.MЈ, equilibrium defined by K␣ ϭ kϩ␣/kϪ␣) and in the presence of ADP (A.M.D and A.MЈ.D, equilibrium defined by K␣D ϭ kϩ␣D/kϪ␣D). Where Ampfast is the observed amplitude of the fast phase of the reaction; Amp0 ϭ amplitude at zero ADP concentration; [ADP] ϭ the concentration of ADP before mixing; KAD ϭ the dissociation equilibrium constant of ADP for actin-Myo1b; and C ϭ end point. Where amplitude, expressed as %, equals the change in fluorescence observed during the reaction (⌬F) divided by the fluorescence at the end point of the reaction (Ftρ), multiplied by 100

RESULTS

AAAAAA

DISCUSSION