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

TitleStructure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies.
Publication TypeJournal Article
Year of Publication2014
AuthorsChaikuad, A., T. Keates, C. Vincke, M. Kaufholz, M. Zenn, B. Zimmermann, C. Gutiérrez, R-G. Zhang, C. Hatzos-Skintges, A. Joachimiak, S. Muyldermans, F. W. Herberg, S. Knapp, and S. Müller
JournalBiochem J
Date Published2014 Apr 1
KeywordsAnimals, Apoproteins, Camels, Catalytic Domain, Crystallization, Enzyme Activation, Humans, Intracellular Signaling Peptides and Proteins, Protein Conformation, Protein Multimerization, Protein-Serine-Threonine Kinases, Single-Domain Antibodies

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.

Alternate JournalBiochem. J.
PubMed ID24438162
PubMed Central IDPMC3957475
Grant List092809/Z/10/Z / / Wellcome Trust / United Kingdom
/ / Canadian Institutes of Health Research / Canada
Research group: