Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 104, Issue 36, Pages 14336-14341Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0703012104
Keywords
catalytic domain; dynamics; par-1; relaxation; ubiquitin-associated
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The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NIMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for mono-ubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the Nand C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.
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