Differential polyubiquitin recognition by tandem ubiquitin binding domains of Rabex-5.
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 0.1-1 μM, 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 3-4 μM, unlike ZnF4 of A20. Taken together, our data suggest differential physiological roles of the two ubiquitin binding domains in Rabex-5.
Department of Biological Sciences, Sungkyunkwan University, Suwon 440-746, Republic of Korea.
Donghyuk
D
Shin
Sei Young
SY
Lee
Seungsoo
S
Han
Shuo
S
Ren
Soyoun
S
Kim
Yoshikatsu
Y
Aikawa
Sangho
S
Lee
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology
D020662:Guanine Nucleotide Exchange Factors; D025821:Polyubiquitin