Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.
Centrioles and cilia are microtubule-based structures, whose precise formation requires controlled cytoplasmic tubulin incorporation. How cytoplasmic tubulin is recognized for centriolar/ciliary-microtubule construction remains poorly understood. Centrosomal-P4.1-associated-protein (CPAP) binds tubulin via its PN2-3 domain. Here, we show that a C-terminal loop-helix in PN2-3 targets β-tubulin at the microtubule outer surface, while an N-terminal helical motif caps microtubule's α-β surface of β-tubulin. Through this, PN2-3 forms a high-affinity complex with GTP-tubulin, crucial for defining numbers and lengths of centriolar/ciliary-microtubules. Surprisingly, two distinct mutations in PN2-3 exhibit opposite effects on centriolar/ciliary-microtubule lengths. CPAP(F375A), with strongly reduced tubulin interaction, causes shorter centrioles and cilia exhibiting doublet- instead of triplet-microtubules. CPAP(EE343RR) that unmasks the β-tubulin polymerization surface displays slightly reduced tubulin-binding affinity inducing over-elongation of newly forming centriolar/ciliary-microtubules by enhanced dynamic release of its bound tubulin. Thus CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures using a 'clutch-like' mechanism.
Beijing Advanced Innovation Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China.|MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.|Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Xiangdong
X
Zheng
Institute for Biochemistry I and Center for Molecular Medicine of the University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany.
Anand
A
Ramani
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, Neuherberg 85764, Germany.|Biomolecular NMR at Center for Integrated Protein Science Munich and Department Chemie, Technische Universität München, Lichtenbergstr. 4, Garching 85747, Germany.
Komal
K
Soni
Department of Life Sciences, University of Siena, Siena 53100, Italy.
Marco
M
Gottardo
Beijing Advanced Innovation Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China.|MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Shuangping
S
Zheng
Institute for Biochemistry I and Center for Molecular Medicine of the University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany.
Li
L
Ming Gooi
MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.|Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Wenjing
W
Li
MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.|Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Shan
S
Feng
Institute for Biochemistry I and Center for Molecular Medicine of the University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany.
Aruljothi
A
Mariappan
Institute for Biochemistry I and Center for Molecular Medicine of the University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany.
Arpit
A
Wason
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Str. 108, Dresden 01307, Germany.
Per
P
Widlund
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Str. 108, Dresden 01307, Germany.
Andrei
A
Pozniakovsky
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Str. 108, Dresden 01307, Germany.
Ina
I
Poser
MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.
Haiteng
H
Deng
MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.|Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing 100084, China.
Guangshuo
G
Ou
Department of Life Sciences, University of Siena, Siena 53100, Italy.
Maria
M
Riparbelli
Department of Life Sciences, University of Siena, Siena 53100, Italy.
Callaini
C
Giuliano
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer Str. 108, Dresden 01307, Germany.
Anthony A
AA
Hyman
Institute of Structural Biology, Helmholtz Zentrum München, Ingolstädter Landstr. 1, Neuherberg 85764, Germany.|Biomolecular NMR at Center for Integrated Protein Science Munich and Department Chemie, Technische Universität München, Lichtenbergstr. 4, Garching 85747, Germany.
Michael
0000-0002-1594-0527
M
Sattler
Institute for Biochemistry I and Center for Molecular Medicine of the University of Cologne, Robert-Koch-Str. 21, Cologne 50931, Germany.
Jay
J
Gopalakrishnan
Beijing Advanced Innovation Center for Structural Biology, Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing 100084, China.|MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China.|Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Haitao
0000-0002-6686-1677
H
Li
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002502:Centrioles / Q000378:metabolism* / Q000648:ultrastructure; D002923:Cilia / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D054730:Protein Interaction Domains and Motifs; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C414735:CENPJ protein, human; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014404:Tubulin