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

Apirat Chaikuad,Andrzej Joachimiak,Stefan Knapp,Michael Zenn,Rong-Guang Zhang,Bastian Zimmermann,Catherine Hatzos-Skintges,Cécile Vincke,Melanie Kaufholz,Carlos Gutiérrez,Friedrich W Herberg,Serge Muyldermans,Susanne Müller,Tracy Keates

doi:10.1042/bj20131399

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.

Highlights

Kinases are highly dynamic enzymes and key regulators of signalling pathways
This critical role for cyclin G-associated kinase (GAK) in the maintenance of respiratory function is thought to be the cause of the side effects observed with the epidermal growth factor receptor (EGFR) kinase inhibitor gefitinib, used in the treatment of patients with NSCLC, as GAK is potently inhibited by this drug [7]
Our previous work on myristoylated and palmitoylated serine/threonine kinase 1 (MPSK1) revealed several molecular features predicted to be unique for the numb-associated kinase (NAK) family including an atypical activation segment [8]

Summary

INTRODUCTION

Kinases are highly dynamic enzymes and key regulators of signalling pathways. The proper timely and spatial activation of these pathways requires fine tuning and a tight regulation of the proteins involved. GAK (cyclin G-associated kinase) is a member of the NAK (numb-associated kinase) family, which in humans includes STK16 (serine/threonine kinase 16)/MPSK1 (myristoylated and palmitoylated serine/threonine kinase 1), AAK1 (adaptorassociated kinase 1) and BIKE [BMP2 (bone morphogenetic protein 2)-inducible kinase] This small subfamily of kinases is located in the centre of the kinase phylogenetic tree and shows a large sequence and structure diversity to other kinases as well as within the NAK subfamily [1]. Death is caused by respiratory dysfunction due to altered distribution of surfactant protein [7] This critical role for GAK in the maintenance of respiratory function is thought to be the cause of the side effects observed with the EGFR (epidermal growth factor receptor) kinase inhibitor gefitinib, used in the treatment of patients with NSCLC (non-small-cell lung cancer), as GAK is potently inhibited by this drug [7].

Chaikuad and others

RESULTS

DISCUSSION