3ZUV

Crystal structure of a designed selected Ankyrin Repeat protein in complex with the phosphorylated MAP kinase ERK2

Summary for 3ZUV

• Entry DOI: 10.2210/pdb3zuv/pdb
• Related: 1ERK 1GOL 2ERK 2GPH 3ERK 3ZU7 4ERK
• Descriptor: MITOGEN-ACTIVATED PROTEIN KINASE 1, DESIGNED ANKYRIN REPEAT PROTEIN, SULFATE ION, ... (4 entities in total)
• Functional Keywords: de novo protein-transferase complex, ankyrin repeat protein, selected binder, protein design, de novo protein/transferase
• Biological source: RATTUS NORVEGICUS (NORWAY RAT); More
• Cellular location: Nucleus (By similarity): P63086
• Total number of polymer chains: 4
• Total formula weight: 114329.96

Authors

Kummer, L.,Mittl, P.R.,Pluckthun, A. (deposition date: 2011-07-20, release date: 2012-06-27, Last modification date: 2024-11-13)

Primary citation

Kummer, L.,Parizek, P.,Rube, P.,Millgramm, B.,Prinz, A.,Mittl, P.R.,Kaufholz, M.,Zimmermann, B.,Herberg, F.W.,Pluckthun, A.
Structural and Functional Analysis of Phosphorylation-Specific Binders of the Kinase Erk from Designed Ankyrin Repeat Protein Libraries.
Proc.Natl.Acad.Sci.USA, 109:E2248-, 2012

PubMed Abstract: We have selected designed ankyrin repeat proteins (DARPins) from a synthetic library by using ribosome display that selectively bind to the mitogen-activated protein kinase ERK2 (extracellular signal-regulated kinase 2) in either its nonphosphorylated (inactive) or doubly phosphorylated (active) form. They do not bind to other kinases tested. Crystal structures of complexes with two DARPins, each specific for one of the kinase forms, were obtained. The two DARPins bind to essentially the same region of the kinase, but recognize the conformational change within the activation loop and an adjacent area, which is the key structural difference that occurs upon activation. Whereas the rigid phosphorylated activation loop remains in the same form when bound by the DARPin, the more mobile unphosphorylated loop is pushed to a new position. The DARPins can be used to selectively precipitate the cognate form of the kinases from cell lysates. They can also specifically recognize the modification status of the kinase inside the cell. By fusing the kinase with Renilla luciferase and the DARPin to GFP, an energy transfer from luciferase to GFP can be observed in COS-7 cells upon intracellular complex formation. Phosphorylated ERK2 is seen to increase by incubation of the COS-7 cells with FBS and to decrease upon adding the ERK pathway inhibitor PD98509. Furthermore, the anti-ERK2 DARPin is seen to inhibit ERK phosphorylation as it blocks the target inside the cell. This strategy of creating activation-state-specific sensors and kinase-specific inhibitors may add to the repertoire to investigate intracellular signaling in real time.

PubMed: 22843676
DOI: 10.1073/PNAS.1205399109

Experimental method

X-RAY DIFFRACTION (2.72 Å)