title: Control of protein signaling using a computationally designed GTPase/GEF orthogonal pair.
abstract: Signaling pathways depend on regulatory protein-protein interactions; controlling these interactions in cells has important applications for reengineering biological functions. As many regulatory proteins are modular, considerable progress in engineering signaling circuits has been made by recombining commonly occurring domains. Our ability to predictably engineer cellular functions, however, is constrained by complex crosstalk observed in naturally occurring domains. Here we demonstrate a strategy for improving and simplifying protein network engineering: using computational design to create orthogonal (non-crossreacting) protein-protein interfaces. We validated the design of the interface between a key signaling protein, the GTPase Cdc42, and its activator, Intersectin, biochemically and by solving the crystal structure of the engineered complex. The designed GTPase (orthoCdc42) is activated exclusively by its engineered cognate partner (orthoIntersectin), but maintains the ability to interface with other GTPase signaling circuit components in vitro. In mammalian cells, orthoCdc42 activity can be regulated by orthoIntersectin, but not wild-type Intersectin, showing that the designed interaction can trigger complex processes. Computational design of protein interfaces thus promises to provide specific components that facilitate the predictable engineering of cellular functions.
authors.affiliation: Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, CA 94158, USA.
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Kapp
authors.forename: Sen
authors.initials: S
authors.lastname: Liu
authors.forename: Amelie
authors.initials: A
authors.lastname: Stein
authors.forename: Derek T
authors.initials: DT
authors.lastname: Wong
authors.forename: Attila
authors.initials: A
authors.lastname: Reményi
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Yeh
authors.forename: James S
authors.initials: JS
authors.lastname: Fraser
authors.forename: Jack
authors.initials: J
authors.lastname: Taunton
authors.forename: Wendell A
authors.initials: WA
authors.lastname: Lim
authors.forename: Tanja
authors.initials: T
authors.lastname: Kortemme
mesh_terms: D000818:Animals; D003461:Crystallography; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D011506:Proteins / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D011506:Proteins; D020558:GTP Phosphohydrolases