title: Mutational analysis reveals a single binding interface between RhoA and its effector, PRK1.
abstract: Protein kinase C-related kinases (PRKs) are serine/threonine kinases that are members of the protein kinase C superfamily and can be activated by binding to members of the Rho family of small G proteins via a Rho binding motif known as an HR1 domain. The PRKs contain three tandem HR1 domains at their N-termini. The structure of the HR1a domain from PRK1 in complex with RhoA [Maesaki, R., et al. (1999) Mol. Cell 4, 793-803] identified two potential contact interfaces between the G protein and the HR1a domain. In this work, we have used an alanine scanning mutagenesis approach to identify whether both contact sites are used when the two proteins interact in solution and also whether HR1b, the second HR1 domain from PRK1, plays a role in binding to RhoA. The mutagenesis identified just one contact site as being relevant for binding of RhoA and HR1a in solution, and the HR1b domain was found not to contribute to RhoA binding. The folded state and thermal stability of the HR1a and HR1b domains were also investigated. HR1b was found to be more thermally stable than HR1a, and it is hypothesized that the differences in the biophysical properties of these two domains govern their interaction with small G proteins.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: Catherine L
authors.initials: CL
authors.lastname: Hutchinson
authors.forename: Peter N
authors.initials: PN
authors.lastname: Lowe
authors.forename: Stephen H
authors.initials: SH
authors.lastname: McLaughlin
authors.forename: Helen R
authors.initials: HR
authors.lastname: Mott
authors.forename: Darerca
authors.initials: D
authors.lastname: Owen
mesh_terms: D000465:Algorithms; D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D020742:rhoA GTP-Binding Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012996:Solutions; C061570:protein kinase N; D011493:Protein Kinase C; D020742:rhoA GTP-Binding Protein