The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation

Tal Oppenheim; Meytal Radzinski; Merav Braitbard; Esther S. Brielle; Ohad Yogev; Eliya Goldberger; Yarden Yesharim; Tommer Ravid; Dina Schneidman-Duhovny; Dana Reichmann

doi:10.1016/j.str.2023.05.014

The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation

Tal Oppenheim, Meytal Radzinski, Merav Braitbard, Esther S. Brielle, Ohad Yogev, Eliya Goldberger, Yarden Yesharim, Tommer Ravid, Dina Schneidman-Duhovny^*, Dana Reichmann^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.

Original language	American English
Pages (from-to)	764-779.e8
Journal	Structure
Volume	31
Issue number	7
DOIs	https://doi.org/10.1016/j.str.2023.05.014
State	Published - 6 Jul 2023

Bibliographical note

Funding Information:
We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program ( CDA00064/2014 ), the US- Israel Binational Science Foundation (grant number: 2015056 ), Israel Ministry of Science and Technology (grant number: 80802 ) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation.

Funding Information:
We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program (CDA00064/2014), the US-Israel Binational Science Foundation (grant number: 2015056), Israel Ministry of Science and Technology (grant number: 80802) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation. Conceptualization: T. O. M. R. M. B. D. S-D. and D. R.; Data curation: T. O. M. R. E. S. B. and D. R.; Formal Analysis: T. O. M. R. M. B. E. S. B. D. S-D. and D. R.; Funding acquisition: D. S-D. and D. R.; Investigation: T. O. M. R. E. S. B. O. Y. E. G. and Y. Y.; Methodology: T. O. M. R. M. B. T. R. D. S-D. and D. R.; Resources: T. R. D. S-D. and D. R.; Software: M. B. E. S. B. and D. S-D.; Supervision: D. S-D. and D. R.; Writing - original draft: T. O. M. R. D. S-D. and D. R.; Writing - review & editing: T. O. M. R. M. B. D. S-D. and D. R. The authors state that no competing financial interests exist. We support inclusive, diverse, and equitable conduct of research.

Publisher Copyright:
© 2023 Elsevier Ltd

Keywords

CDC48
CL-MS
NPL4
UFD1
crosslinking coupled with MS (XL-MS)
integrative modeling
protein quality control
structural mass spectrometry

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10.1016/j.str.2023.05.014

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title = "The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation",

abstract = "Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.",

keywords = "CDC48, CL-MS, NPL4, UFD1, crosslinking coupled with MS (XL-MS), integrative modeling, protein quality control, structural mass spectrometry",

author = "Tal Oppenheim and Meytal Radzinski and Merav Braitbard and Brielle, {Esther S.} and Ohad Yogev and Eliya Goldberger and Yarden Yesharim and Tommer Ravid and Dina Schneidman-Duhovny and Dana Reichmann",

note = "Funding Information: We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program ( CDA00064/2014 ), the US- Israel Binational Science Foundation (grant number: 2015056 ), Israel Ministry of Science and Technology (grant number: 80802 ) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation. Funding Information: We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program (CDA00064/2014), the US-Israel Binational Science Foundation (grant number: 2015056), Israel Ministry of Science and Technology (grant number: 80802) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation. Conceptualization: T. O. M. R. M. B. D. S-D. and D. R.; Data curation: T. O. M. R. E. S. B. and D. R.; Formal Analysis: T. O. M. R. M. B. E. S. B. D. S-D. and D. R.; Funding acquisition: D. S-D. and D. R.; Investigation: T. O. M. R. E. S. B. O. Y. E. G. and Y. Y.; Methodology: T. O. M. R. M. B. T. R. D. S-D. and D. R.; Resources: T. R. D. S-D. and D. R.; Software: M. B. E. S. B. and D. S-D.; Supervision: D. S-D. and D. R.; Writing - original draft: T. O. M. R. D. S-D. and D. R.; Writing - review & editing: T. O. M. R. M. B. D. S-D. and D. R. The authors state that no competing financial interests exist. We support inclusive, diverse, and equitable conduct of research. Publisher Copyright: {\textcopyright} 2023 Elsevier Ltd",

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doi = "10.1016/j.str.2023.05.014",

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T1 - The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation

AU - Oppenheim, Tal

AU - Radzinski, Meytal

AU - Braitbard, Merav

AU - Brielle, Esther S.

AU - Yogev, Ohad

AU - Goldberger, Eliya

AU - Yesharim, Yarden

AU - Ravid, Tommer

AU - Schneidman-Duhovny, Dina

AU - Reichmann, Dana

N1 - Funding Information: We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program ( CDA00064/2014 ), the US- Israel Binational Science Foundation (grant number: 2015056 ), Israel Ministry of Science and Technology (grant number: 80802 ) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation. Funding Information: We are grateful for the financial support from the Israel Science Foundation (grant number: 1537/18 for DR and 1466/18 for DS), Human Frontier Science program (CDA00064/2014), the US-Israel Binational Science Foundation (grant number: 2015056), Israel Ministry of Science and Technology (grant number: 80802) and the Azrieli Foundation for supporting MR. We are tremendously thankful to Prof. Maya Schuldiner for providing us with the UPRE-GFP plasmid and for stimulating discussions, to Adi Levy for her assistance with the cell imaging, to Dr. Hadar Amartely for the assistance with ITC and protein purification, and to Yael Sidkiyahu for her assistance with figure preparation. Conceptualization: T. O. M. R. M. B. D. S-D. and D. R.; Data curation: T. O. M. R. E. S. B. and D. R.; Formal Analysis: T. O. M. R. M. B. E. S. B. D. S-D. and D. R.; Funding acquisition: D. S-D. and D. R.; Investigation: T. O. M. R. E. S. B. O. Y. E. G. and Y. Y.; Methodology: T. O. M. R. M. B. T. R. D. S-D. and D. R.; Resources: T. R. D. S-D. and D. R.; Software: M. B. E. S. B. and D. S-D.; Supervision: D. S-D. and D. R.; Writing - original draft: T. O. M. R. D. S-D. and D. R.; Writing - review & editing: T. O. M. R. M. B. D. S-D. and D. R. The authors state that no competing financial interests exist. We support inclusive, diverse, and equitable conduct of research. Publisher Copyright: © 2023 Elsevier Ltd

PY - 2023/7/6

Y1 - 2023/7/6

N2 - Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.

AB - Cdc48 (VCP/p97) is a major AAA-ATPase involved in protein quality control, along with its canonical cofactors Ufd1 and Npl4 (UN). Here, we present novel structural insights into the interactions within the Cdc48-Npl4-Ufd1 ternary complex. Using integrative modeling, we combine subunit structures with crosslinking mass spectrometry (XL-MS) to map the interaction between Npl4 and Ufd1, alone and in complex with Cdc48. We describe the stabilization of the UN assembly upon binding with the N-terminal-domain (NTD) of Cdc48 and identify a highly conserved cysteine, C115, at the Cdc48-Npl4-binding interface which is central to the stability of the Cdc48-Npl4-Ufd1 complex. Mutation of Cys115 to serine disrupts the interaction between Cdc48-NTD and Npl4-Ufd1 and leads to a moderate decrease in cellular growth and protein quality control in yeast. Our results provide structural insight into the architecture of the Cdc48-Npl4-Ufd1 complex as well as its in vivo implications.

KW - CDC48

KW - CL-MS

KW - NPL4

KW - UFD1

KW - crosslinking coupled with MS (XL-MS)

KW - integrative modeling

KW - protein quality control

KW - structural mass spectrometry

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U2 - 10.1016/j.str.2023.05.014

DO - 10.1016/j.str.2023.05.014

M3 - Article

C2 - 37311459

AN - SCOPUS:85163932903

SN - 0969-2126

VL - 31

SP - 764-779.e8

JO - Structure

JF - Structure

IS - 7

ER -

The Cdc48 N-terminal domain has a molecular switch that mediates the Npl4-Ufd1-Cdc48 complex formation

Abstract

Bibliographical note

Keywords

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