A novel chemical footprinting approach identifies critical lysine residues involved in the binding of receptor-associated protein to cluster II of LDL receptor-related protein

The Biochemical Journal
Esther BloemAlexander B Meijer

Abstract

Tandem mass tags (TMTs) were utilized in a novel chemical footprinting approach to identify lysine residues that mediate the interaction of receptor-associated protein (RAP) with cluster II of LDL (low-density lipoprotein) receptor (LDLR)-related protein (LRP). The isolated RAP D3 domain was modified with TMT-126 and the D3 domain-cluster II complex with TMT-127. Nano-LC-MS analysis revealed reduced modification with TMT-127 of peptides including Lys(256), Lys(270) and Lys(305)-Lys(306) suggesting that these residues contribute to cluster II binding. This agrees with previous findings that Lys(256) and Lys(270) are critical for binding cluster II sub-domains [Fisher, Beglova and Blacklow (2006) Mol. Cell 22, 277-283]. Cluster II-binding studies utilizing D3 domain variants K(256)A, K(305)A and K(306)A now showed that Lys(306) contributes to cluster II binding as well. For full-length RAP, we observed that peptides including Lys(60), Lys(191), Lys(256), Lys(270) and Lys(305)-Lys(306) exhibited reduced modification with TMT in the RAP-cluster II complex. Notably, Lys(60) has previously been implicated to mediate D1 domain interaction with cluster II. Our results suggest that also Lys(191) of the D2 domain contributes to cluster I...Continue Reading

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Citations

Aug 17, 2018·The Biochemical Journal·Małgorzata A PrzeradzkaAlexander B Meijer
Sep 28, 2018·The Protein Journal·Juan GuevaraNatalia Valentinova Guevara
Jun 30, 2019·Analytical and Bioanalytical Chemistry·Khanh K NguyenJoseph C Genereux
Mar 10, 2021·Journal of Thrombosis and Haemostasis : JTH·Nadia FreatoEduard H T M Ebberink
Oct 2, 2019·ACS Central Science·Barbara SteigenbergerRichard A Scheltema

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