Linear directed movement, such as seen in muscle contraction, cell motility, and intracellular trafficking, is one of the fundamental characteristics of life and is mediated primarily by the myosin, kinesin, and dynein superfamilies of motor proteins. These proteins utilize the free energy of the hydrolysis of ATP to generate molecular movement. At this time, the fundamental structural folds of myosin and kinesin are known. However, many questions remain about the molecular mechanism by which the free energy of hydrolysis of ATP is converted into directed movement. The major goal of this proposal is to understand the structural and functional role of communication pathways in myosin and the minus-end directed motor Kar3. The first objective is to construct and determine the structure of a complex of actin and myosin. A knowledge of the structure of actomyosin is needed, not only to complete the structural picture of motility, but also to understand the structure of the actin filament. The proposed studies will provide insight into the fundamental mechanism of energy transduction in myosin and establish a molecular framework for the development of new small ligands to control the interaction between actin and myosin. The second major objective is to understand the molecular mechanism for Kar3 which belongs to the Kinesin-14 class of motors. Members of this class are minus-end directed, non processive kinesins that operate with a powerstroke mechanism. Kar3 is unusual in that it functions as a heterodimer, forming a complex with either one of the non-motor proteins Vik1 or Cik1. We have recently been shown that Vik1 and Cik1 contain a globular domain that exhibits the same fold as the kinesin motor domain. This domain binds tightly to microtubules but does not hydrolyze ATP. This phenomena demands the existence of intermolecular communication between the motor and non motor domains in order to generate movement. The immediate goal is to understand the nature of the interaction between the Kar3 motor domain and the Vik1 and Cik1 motor homology domains and how this facilitates their biological function. Kar3/Vik1 and Kar3/Cik1 represent an extreme example of asymmetry between motor domains in dimeric kinesins, however, functional asymmetry between kinesin motor domains is observed in other non-processive dimeric kinesins, so that these studies will provide insight into a fundamental phenomena. These investigations will utilize a combination of X-ray crystallography, electron microscopy, kinetic analysis, and functional assays to give a comprehensive view of the structural transitions in these molecular motors.
The purpose of this proposal is to understand the structure and function of biological molecular motors with a focus on myosin and kinesin. These proteins are central components of life. Indeed, defects in these proteins are implicated in a significant number of genetic diseases including familial hypertrophic cardiomyopathy, thrombocytopenia and hearing loss and neurodegenerative diseases such as ALS. Study of these motors is expected to lead to therapeutic advances such as the identification of drugs that influence the motile cycle.
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