2014
DOI: 10.1042/bj20131399
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Structure of cyclin G-associated kinase (GAK) trapped in different conformations using nanobodies

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 activ… Show more

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Cited by 58 publications
(63 citation statements)
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“…105 By contrast, sdAb paratopes can clearly adopt both flat 106,107 and convex 11 topologies, although possibly only inefficiently adopt concave ones. The CDR1 and CDR2 loops of V H Hs depart from the typical canonical structures of conventional antibodies (Figure 2A), potentially through somatic mutation since germline human V H and camelid V H H repertoires appear to have similar canonical structures.…”
Section: Single-domain Antibody Paratope Structuresmentioning
confidence: 98%
“…105 By contrast, sdAb paratopes can clearly adopt both flat 106,107 and convex 11 topologies, although possibly only inefficiently adopt concave ones. The CDR1 and CDR2 loops of V H Hs depart from the typical canonical structures of conventional antibodies (Figure 2A), potentially through somatic mutation since germline human V H and camelid V H H repertoires appear to have similar canonical structures.…”
Section: Single-domain Antibody Paratope Structuresmentioning
confidence: 98%
“…We have recently solved the structure of GAK in complex with a single chain antibody (nanobody), which resulted in reproducible crystallization conditions for the GAK kinase domain. 22 The nanobody bound distal to the ATP binding site interacting with the lower kinase lobe (Figure 3). The lead compound 12i was co-crystallized and bound as expected to the ATP binding site of GAK.…”
Section: X-ray Crystallographymentioning
confidence: 99%
“…Two kinase domain:nanobody complexes were present in the asymmetric unit that interacted via the extended activation loops of GAK as described previously. 22 The upper lobe of the kinase domain was flexible as indicated by high B-factors in particular in the N-terminal region (Supplemental Figure 1). The helix αC and the DFG motive were in an active conformation compatible with the type I binding mode of the inhibitor (Figure 3).…”
Section: X-ray Crystallographymentioning
confidence: 99%
“…When the epitope is located at the interface of the interacting domains or subunits of a protein, the nanobody can be used to disrupt domain interactions (22). The main advantage of the nanobodies in such conventional applications is that a large panel of nanobodies can be easily screened to identify preferred epitopes or the best co-crystals between a nanobody and the target protein, or to reveal and stabilize unknown target conformers (23).…”
Section: Nanobodies Versus Fragments Of the Conventional Antibodies Amentioning
confidence: 99%