Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies.
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.
*University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC), Old Road Campus Research Building, Oxford OX3 7DQ, U.K.
Apirat
A
Chaikuad
*University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC), Old Road Campus Research Building, Oxford OX3 7DQ, U.K.
Tracy
T
Keates
†Research Unit of Cellular and Molecular Immunology and Department of Structural Biology, VIB, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
Cécile
C
Vincke
‡Department of Biochemistry, University of Kassel, Heinrich-Plett Strasse 40, 34132 Kassel, Germany.
Melanie
M
Kaufholz
§Biaffin GmbH & CoKG, Heinrich-Plett Strasse 40, 34132 Kassel, Germany.
Michael
M
Zenn
§Biaffin GmbH & CoKG, Heinrich-Plett Strasse 40, 34132 Kassel, Germany.
Bastian
B
Zimmermann
¶Department of Animal Medicine and Surgery, Veterinary Faculty, University of Las Palmas de Gran Canaria, 35416, Arucas, Las Palmas, Spain.
Carlos
C
Gutiérrez
∥Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, U.S.A.
Rong-Guang
RG
Zhang
∥Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, U.S.A.
Catherine
C
Hatzos-Skintges
∥Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, U.S.A.
Andrzej
A
Joachimiak
†Research Unit of Cellular and Molecular Immunology and Department of Structural Biology, VIB, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium.
Serge
S
Muyldermans
Friedrich W
FW
Herberg
*University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC), Old Road Campus Research Building, Oxford OX3 7DQ, U.K.
Stefan
S
Knapp
*University of Oxford, Target Discovery Institute (TDI) and Structural Genomics Consortium (SGC), Old Road Campus Research Building, Oxford OX3 7DQ, U.K.
Susanne
S
Müller
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000502:physiology; D002162:Camelus; D020134:Catalytic Domain / Q000502:physiology; D003460:Crystallization / Q000379:methods; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000502:physiology*; D011487:Protein Conformation; D055503:Protein Multimerization / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000502:physiology*; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000502:physiology*
D001059:Apoproteins; D047908:Intracellular Signaling Peptides and Proteins; D061905:Single-Domain Antibodies; C486325:GAK protein, human; D017346:Protein Serine-Threonine Kinases