Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies

DOI: 10.1042/BJ20131399
PMID: 24438162

Authors: Apirat Chaikuad (University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC)) , Tracy Keates (University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC)) , Cécile Vincke (Research Unit of Cellular and Molecular Immunology and Department of Structural Biology, VIB, Vrije Universiteit Brussel) , Melanie Kaufholz (Department of Biochemistry, University of Kassel) , Michael Zenn (Biaffin GmbH & CoKG) , Bastian Zimmermann (Biaffin GmbH & CoKG) , Carlos Gutiérrez (Department of Animal Medicine and Surgery, Veterinary Faculty, University of Las Palmas de Gran Canaria) , Rong‑guang Zhang (Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory) , Catherine Hatzos‑skintges (Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory) , Andrzej Joachimiak (Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory) , Serge Muyldermans (Research Unit of Cellular and Molecular Immunology and Department of Structural Biology, VIB, Vrije Universiteit Brussel) , Friedrich w. Herberg (Department of Biochemistry, University of Kassel) , Stefan Knapp (University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC)) , Susanne Müller (University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC))
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical Journal , VOL 459 , PAGES 59 - 69

State: Published (Approved)
Published: April 2014
Diamond Proposal Number(s): 8421

Abstract: GAK (cyclin G-associated kinase) is a key regulator of clathrin-coated vesicle trafficking and plays a central role during development. Additionally, due to the unusually high plasticity of its catalytic domain, it is a frequent ‘off-target’ of clinical kinase inhibitors associated with respiratory side effects of these drugs. In the present paper, we determined the crystal structure of the GAK catalytic domain alone and in complex with specific single-chain antibodies (nanobodies). GAK is constitutively active and weakly associates in solution. The GAK apo structure revealed a dimeric inactive state of the catalytic domain mediated by an unusual activation segment interaction. Co-crystallization with the nanobody NbGAK_4 trapped GAK in a dimeric arrangement similar to the one observed in the apo structure, whereas NbGAK_1 captured the activation segment of monomeric GAK in a well-ordered conformation, representing features of the active kinase. The presented structural and biochemical data provide insight into the domain plasticity of GAK and demonstrate the utility of nanobodies to gain insight into conformational changes of dynamic molecules. In addition, we present structural data on the binding mode of ATP mimetic inhibitors and enzyme kinetic data, which will support rational inhibitor design of inhibitors to reduce the off-target effect on GAK.

Journal Keywords: Apoproteins; Camels; Catalytic; Crystallization; Enzyme; Humans; Intracellular; Protein; Protein-Serine-Threonine; Single-Domain Antibodies

Subject Areas: Biology and Bio-materials


Instruments: I02-Macromolecular Crystallography , I04-Macromolecular Crystallography

Other Facilities: Advanced Photon Source (19-ID)