TY - JOUR
T1 - Structural analysis of the LDL receptor-interacting FERM domain in the E3 ubiquitin ligase IDOL reveals an obscured substrate-binding site
AU - Martinelli, Luca
AU - Adamopoulos, Athanassios
AU - Johansson, Patrik
AU - Wan, Paul T.
AU - Gunnarsson, Jenny
AU - Guo, Hongwei
AU - Boyd, Helen
AU - Zelcer, Noam
AU - Sixma, Titia K.
PY - 2020/9/25
Y1 - 2020/9/25
N2 - Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
AB - Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.
KW - E3 ubiquitin ligase
KW - FERM
KW - FERM domain
KW - IDOL
KW - LDL receptor
KW - LDLR
KW - MYLIP
KW - cholesterol metabolism
KW - crystal structure
KW - enzyme purification
KW - lipoprotein metabolism
KW - low-density lipoprotein (LDL)
KW - protein structure
KW - small-angle X-ray scattering (SAXS)
KW - ubiquitylation (ubiquitination)
UR - http://www.scopus.com/inward/record.url?scp=85091806562&partnerID=8YFLogxK
U2 - https://doi.org/10.1074/jbc.RA120.014349
DO - https://doi.org/10.1074/jbc.RA120.014349
M3 - Article
C2 - 32727844
SN - 0021-9258
VL - 295
SP - 13570
EP - 13583
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 39
ER -