Crystal structure of TBC1D15 GTPase-activating protein (GAP) domain and its activity on Rab GTPases.
TBC1D15 belongs to the TBC (Tre-2/Bub2/Cdc16) domain family and functions as a GTPase-activating protein (GAP) for Rab GTPases. So far, the structure of TBC1D15 or the TBC1D15·Rab complex has not been determined, thus, its catalytic mechanism on Rab GTPases is still unclear. In this study, we solved the crystal structures of the Shark and Sus TBC1D15 GAP domains, to 2.8 Å and 2.5 Å resolution, respectively. Shark-TBC1D15 and Sus-TBC1D15 belong to the same subfamily of TBC domain-containing proteins, and their GAP-domain structures are highly similar. This demonstrates the evolutionary conservation of the TBC1D15 protein family. Meanwhile, the newly determined crystal structures display new variations compared to the structures of yeast Gyp1p Rab GAP domain and TBC1D1. GAP assays show that Shark and Sus GAPs both have higher catalytic activity on Rab11a·GTP than Rab7a·GTP, which differs from the previous study. We also demonstrated the importance of arginine and glutamine on the catalytic sites of Shark GAP and Sus GAP. When arginine and glutamine are changed to alanine or lysine, the activities of Shark GAP and Sus GAP are lost.
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou, 310018, China.
Yan-Na
YN
Chen
Laboratory of Structural Science, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, Michigan, 49503, USA.
Xin
X
Gu
Laboratory of Structural Science, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, Michigan, 49503, USA.
X Edward
XE
Zhou
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou, 310018, China.
Weidong
W
Wang
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou, 310018, China.
Dandan
D
Cheng
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou, 310018, China.
Yinghua
Y
Ge
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou, 310018, China.
Fei
F
Ye
VARI-SIMM Center for Structure and Function of Drug Targets and the CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Science, Shanghai, 201203, China.|Laboratory of Structural Science, Center for Structural Biology and Drug Discovery, Van Andel Research Institute, Grand Rapids, Michigan, 49503, USA.
H Eric
HE
Xu
College of Life Science, Zhejiang Sci-Tech University, Hangzhou, 310018, China.|Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Hangzhou, 310018, China.
Zhengbing
Z
Lv
D000818:Animals; D018360:Crystallography, X-Ray; D029941:Fish Proteins / Q000737:chemistry* / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D012754:Sharks; D013552:Swine; D020691:rab GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism
D029941:Fish Proteins; D020690:GTPase-Activating Proteins; D046912:Multiprotein Complexes; D006160:Guanosine Triphosphate; D020691:rab GTP-Binding Proteins
GAP; GTPase activity assay; Sus; TBC1D15; crystal structure; shark