This proposal is to characterize the solution structure, dynamics, and interactions of polyubiquitin (polyUb) chains, a universal proteolytic signal in eukaryotes. The ubiquitin-proteasome proteolytic pathway is the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how Lys48-linked polyUb chains are recognized by the 26S proteasome and other downstream effector molecules is central to our understanding of the mechanisms of regulation. Remarkably, polyUb chains linked via different lysines also act as cellular signals, although regulatory rather than proteolytic. Although significant evidence suggests that the specific recognition of different polyUb chains is based on conformational determinants, as yet there is no clear view of the conformational properties of these molecules in solution. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. We will use NMR approaches to determine the three-dimensional conformation of Lys48-linked tetra-Ub chain, free in solution, and of di- and tetra-Ub bound to molecular mimics of the proteasomal receptors, hHR23A and S5a. This will be performed using a combination of long-range, orientational constraints derived from spin-relaxation and residual dipolar coupling measurements, complemented with distance information from paramagnetic relaxation enhancements and pseudocontact shifts introduced by paramagnetic labels. These novel NMR approaches will be used in combination with the conventional methods based on NOEs and mapping of the interdomain or intermolecular interface based on chemical shift perturbations and solvent accessibility measurements. We will use a representative set of UBA domains in order to identify structural determinants of the linkage specificity of various Ub-chain receptors. Domain motions play essential role in polyUb chain's ability to interact with various receptors. We will introduce mutations in the residues at the Ub-Ub interface in order to identify determining factors contributing to the interface stability and dynamics. We will measure protein dynamics in di-Ub and tetra-Ub under various pH and temperature conditions in order to determine the contribution from interdomain motions and to characterize the conformational flexibility of polyUb chains.
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