Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5

Donghyuk Shin; Sei Young Lee; Seungsoo Han; Shuo Ren; Soyoun Kim; Yoshikatsu Aikawa; Sangho Lee

doi:10.1016/j.bbrc.2012.06.032

Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5

Donghyuk Shin, Sei Young Lee, Seungsoo Han, Shuo Ren, Soyoun Kim, Yoshikatsu Aikawa, Sangho Lee

Division of Life Sciences

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

3 Citations (Scopus)

Abstract

Linkage-specific polyubiquitination regulates many cellular processes. The N-terminal fragment of Rabex-5 (Rabex-5 _9-73) contains tandem ubiquitin binding domains: A20_ZF and MIU. The A20_ZF-MIU of Rabex-5 is known to bind monoubiquitin but molecular details of polyubiquitin binding affinity and linkage selectivity by Rabex-5 _9-73 remain elusive. Here we report that Rabex-5 _9-73 binds linear, K63- and K48-linked tetraubiquitin (Ub ₄) chains with K _d of 0.1-1μM, determined by biolayer interferometry. Mutational analysis of qualitative and quantitative binding data reveals that MIU is more important than A20_ZF in linkage-specific polyubiquitin recognition. MIU prefers binding to linear and K63-linked Ub ₄ with sub μM affinities. However, A20_ZF recognizes the three linkage-specific Ub ₄ with similar affinities with K _d of 3-4μM, unlike ZnF4 of A20. Taken together, our data suggest differential physiological roles of the two ubiquitin binding domains in Rabex-5.

Original language	English
Pages (from-to)	757-762
Number of pages	6
Journal	Biochemical and Biophysical Research Communications
Volume	423
Issue number	4
DOIs	https://doi.org/10.1016/j.bbrc.2012.06.032
Publication status	Published - 2012 Jul 13

Bibliographical note

Funding Information:
We thank Prof. Hongtae Kim for assistance in pull-down assays. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (2008-0058783) and the Agency for Defense Development through Chemical and Biological Defense Research Center to S.L., and the National Junior Research Fellowship (NRF-2011-0031264) to D.S. from the National Research Foundation (NRF) grant funded by the Ministry of Education, Science, and Technology (MEST) of Korea.

All Science Journal Classification (ASJC) codes

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1016/j.bbrc.2012.06.032

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title = "Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5",

abstract = "Linkage-specific polyubiquitination regulates many cellular processes. The N-terminal fragment of Rabex-5 (Rabex-5 9-73) contains tandem ubiquitin binding domains: A20_ZF and MIU. The A20_ZF-MIU of Rabex-5 is known to bind monoubiquitin but molecular details of polyubiquitin binding affinity and linkage selectivity by Rabex-5 9-73 remain elusive. Here we report that Rabex-5 9-73 binds linear, K63- and K48-linked tetraubiquitin (Ub 4) chains with K d of 0.1-1μM, determined by biolayer interferometry. Mutational analysis of qualitative and quantitative binding data reveals that MIU is more important than A20_ZF in linkage-specific polyubiquitin recognition. MIU prefers binding to linear and K63-linked Ub 4 with sub μM affinities. However, A20_ZF recognizes the three linkage-specific Ub 4 with similar affinities with K d of 3-4μM, unlike ZnF4 of A20. Taken together, our data suggest differential physiological roles of the two ubiquitin binding domains in Rabex-5.",

keywords = "A20-type zinc finger, Motif-interacting with ubiquitin, Polyubiquitin, Rabex-5, Ubiquitin binding domain",

author = "Donghyuk Shin and Lee, {Sei Young} and Seungsoo Han and Shuo Ren and Soyoun Kim and Yoshikatsu Aikawa and Sangho Lee",

note = "Funding Information: We thank Prof. Hongtae Kim for assistance in pull-down assays. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (2008-0058783) and the Agency for Defense Development through Chemical and Biological Defense Research Center to S.L., and the National Junior Research Fellowship (NRF-2011-0031264) to D.S. from the National Research Foundation (NRF) grant funded by the Ministry of Education, Science, and Technology (MEST) of Korea. ",

year = "2012",

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doi = "10.1016/j.bbrc.2012.06.032",

language = "English",

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T1 - Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5

AU - Shin, Donghyuk

AU - Lee, Sei Young

AU - Han, Seungsoo

AU - Ren, Shuo

AU - Kim, Soyoun

AU - Aikawa, Yoshikatsu

AU - Lee, Sangho

N1 - Funding Information: We thank Prof. Hongtae Kim for assistance in pull-down assays. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (2008-0058783) and the Agency for Defense Development through Chemical and Biological Defense Research Center to S.L., and the National Junior Research Fellowship (NRF-2011-0031264) to D.S. from the National Research Foundation (NRF) grant funded by the Ministry of Education, Science, and Technology (MEST) of Korea.

PY - 2012/7/13

Y1 - 2012/7/13

N2 - Linkage-specific polyubiquitination regulates many cellular processes. The N-terminal fragment of Rabex-5 (Rabex-5 9-73) contains tandem ubiquitin binding domains: A20_ZF and MIU. The A20_ZF-MIU of Rabex-5 is known to bind monoubiquitin but molecular details of polyubiquitin binding affinity and linkage selectivity by Rabex-5 9-73 remain elusive. Here we report that Rabex-5 9-73 binds linear, K63- and K48-linked tetraubiquitin (Ub 4) chains with K d of 0.1-1μM, determined by biolayer interferometry. Mutational analysis of qualitative and quantitative binding data reveals that MIU is more important than A20_ZF in linkage-specific polyubiquitin recognition. MIU prefers binding to linear and K63-linked Ub 4 with sub μM affinities. However, A20_ZF recognizes the three linkage-specific Ub 4 with similar affinities with K d of 3-4μM, unlike ZnF4 of A20. Taken together, our data suggest differential physiological roles of the two ubiquitin binding domains in Rabex-5.

AB - Linkage-specific polyubiquitination regulates many cellular processes. The N-terminal fragment of Rabex-5 (Rabex-5 9-73) contains tandem ubiquitin binding domains: A20_ZF and MIU. The A20_ZF-MIU of Rabex-5 is known to bind monoubiquitin but molecular details of polyubiquitin binding affinity and linkage selectivity by Rabex-5 9-73 remain elusive. Here we report that Rabex-5 9-73 binds linear, K63- and K48-linked tetraubiquitin (Ub 4) chains with K d of 0.1-1μM, determined by biolayer interferometry. Mutational analysis of qualitative and quantitative binding data reveals that MIU is more important than A20_ZF in linkage-specific polyubiquitin recognition. MIU prefers binding to linear and K63-linked Ub 4 with sub μM affinities. However, A20_ZF recognizes the three linkage-specific Ub 4 with similar affinities with K d of 3-4μM, unlike ZnF4 of A20. Taken together, our data suggest differential physiological roles of the two ubiquitin binding domains in Rabex-5.

KW - A20-type zinc finger

KW - Motif-interacting with ubiquitin

KW - Polyubiquitin

KW - Rabex-5

KW - Ubiquitin binding domain

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JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

IS - 4

ER -

Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5

Abstract

Bibliographical note

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