title: Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain.
abstract: 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. NMR 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 N- and 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.
authors.affiliation: Centre for Systems Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Department of Medical Genetics, University of Toronto, ON, Canada M5G 1X5.
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Korzhnev
authors.forename: Derek F
authors.initials: DF
authors.lastname: Ceccarelli
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Briant
authors.forename: Arash
authors.initials: A
authors.lastname: Zarrine-Afsar
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.forename: Lewis E
authors.initials: LE
authors.lastname: Kay
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D018360:Crystallography, X-Ray; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012996:Solutions; D017186:Titrimetry; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D008024:Ligands; D012996:Solutions; D025801:Ubiquitin; C111833:MARK3 protein, human; D017346:Protein Serine-Threonine Kinases