Solution structure of RING finger-like domain of retinoblastoma-binding protein-6 (RBBP6) suggests it functions as a U-box

TY - JOUR

T1 - Solution structure of RING finger-like domain of retinoblastoma-binding protein-6 (RBBP6) suggests it functions as a U-box

AU - Kappo, Mautin A

AU - Ab, Eiso

AU - Hassem, Faqeer

AU - Atkinson, R Andrew

AU - Faro, Andrew

AU - Muleya, Victor

AU - Mulaudzi, Takalani

AU - Poole, John O

AU - McKenzie, Jean M

AU - Chibi, Moredreck

AU - Moolman-Smook, Joanna C

AU - Rees, D Jasper G

AU - Pugh, David J R

PY - 2012/3/2

Y1 - 2012/3/2

N2 - Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that the RING finger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control.

AB - Retinoblastoma-binding protein-6 (RBBP6) plays a facilitating role, through its RING finger-like domain, in the ubiquitination of p53 by Hdm2 that is suggestive of E4-like activity. Although the presence of eight conserved cysteine residues makes it highly probable that the RING finger-like domain coordinates two zinc ions, analysis of the primary sequence suggests an alternative classification as a member of the U-box family, the members of which do not bind zinc ions. We show here that despite binding two zinc ions, the domain adopts a homodimeric structure highly similar to those of a number of U-boxes. Zinc ions could be replaced by cadmium ions without significantly disrupting the structure or the stability of the domain, although the rate of substitution was an order of magnitude slower than any previous measurement, suggesting that the structure is particularly stable, a conclusion supported by the high thermal stability of the domain. A hallmark of U-box-containing proteins is their association with chaperones, with which they cooperate in eliminating irretrievably unfolded proteins by tagging them for degradation by the proteasome. Using a yeast two-hybrid screen, we show that RBBP6 interacts with chaperones Hsp70 and Hsp40 through its N-terminal ubiquitin-like domain. Taken together with the structural similarities to U-box-containing proteins, our data suggest that RBBP6 plays a role in chaperone-mediated ubiquitination and possibly in protein quality control.

KW - Cadmium

KW - Carrier Proteins

KW - DNA-Binding Proteins

KW - HSP40 Heat-Shock Proteins

KW - HSP70 Heat-Shock Proteins

KW - Humans

KW - Protein Binding

KW - RING Finger Domains

KW - Saccharomyces cerevisiae

KW - Two-Hybrid System Techniques

KW - Ubiquitination

KW - Zinc

U2 - 10.1074/jbc.M110.217059

DO - 10.1074/jbc.M110.217059

M3 - Article

C2 - 22130672

SN - 0021-9258

VL - 287

SP - 7146

EP - 7158

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 10

M1 - N/A

ER -