Mechanochemistry of ATP binding cassettes and the F1 model for mechanical ATPases at large.

摘要

This thesis explores the mechanochemical properties and biological contexts of the ubiquitous ATP binding cassette (ABC) transporter superfamily of ATPases. Members of this family fall under the broader classification of proteins that are structurally related to the F1 component of ATP synthase. Chapter I reviews the thermodynamic and structural properties of these F1 related proteins. F1 ATP synthase is a well studied mechanoenzyme that provides a clear archetype for how the free energy changes associated with the binding and hydrolysis of ATP are coupled to protein conformational changes and the performance of mechanical work. The mechanochemical features of F 1 are clearly seen in other structurally related and unrelated proteins. Analyses of these features provides a simple, yet compelling model for ATP mechanoenzymes at large. Chapter I also elucidates the key structural and functional differences between mechanical ATPases and G-proteins and sets the stage for the interpretation of ABC mechanochemistry through the model developed from F1.;Members of the ABC transporter superfamily are ubiquitous ATPases most often associated with transmembrane transport processes. Prior to the work described in this thesis, a coherent mechanism for ABC protein function did not exist that was consistent with available biochemical and structural data. At the beginning of my career in graduate school, working models for ABC transporters were all derived from data that showed that cooperative interaction of two ABC domains was necessary for transport. Circa 2000, a great deal of conflicting structural data led to wildly varied mechanisms for the cooperative interactions seen in ABC domains. My efforts as a graduate student were centered around determining the structural basis for ABC cooperativity and creating a comprehensive and biochemically correct model for ABC mechanochemistry. Using hydrolysis deficient ABC domains, I was able to obtain the first physiological dimer structure of a true ABC transporter and put forward a mechanism for ABC function that is consistent with the biochemical properties of ABC domains. The details of this structure and the mechanistic model for ABC's to which it led are found in Chapter II.;Prior to obtaining this ABC transporter structure, I had been working to obtain the structures of several ABC proteins that contain two ABC domains in a single polypeptide. My original intent in crystallizing these proteins was to observe the interactions of these proteins' tandem ABC domains in hopes that they would reflect the physiologically relevant interactions that provided the basis for ABC cooperativity. Closer examination revealed that these proteins represented an entirely distinct class of ABC proteins that occur in all organisms with no obvious connection to transmembrane transport. The exact functions of the majority of these proteins was, and still is, unknown. Chapter III represents my efforts at developing a complete structure-function picture for these proteins as had been achieved for transport related ABC proteins. Results to date suggest that the majority of these proteins serve as regulators of protein synthesis involved in responses to environmental stress. Structural data reveal that these regulators contain a novel domain ABC architecture that is likely to undergo a large clamping motion driven by coordinated ATP binding at four ABC domains. The effort to establish the connection between this unique mechanochemical feature of these proteins and their apparent role in translation is ongoing, and proposals for future experimentation are found in Chapter IV.