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

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 Lys²⁵⁶, Lys²⁷⁰ and Lys³⁰⁵-Lys³⁰⁶ suggesting that these residues contribute to cluster II binding. This agrees with previous findings that Lys²⁵⁶ and Lys²⁷⁰ 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 K²⁵⁶A, K³⁰⁵A and K³⁰⁶A now showed that Lys³⁰⁶ contributes to cluster II binding as well. For full-length RAP, we observed that peptides including Lys⁶⁰, Lys¹⁹¹, Lys²⁵⁶, Lys²⁷⁰ and Lys³⁰⁵-Lys³⁰⁶ exhibited reduced modification with TMT in the RAP–cluster II complex. Notably, Lys⁶⁰ has previously been implicated to mediate D1 domain interaction with cluster II. Our results suggest that also Lys¹⁹¹ of the D2 domain contributes to cluster II binding. Binding studies employing the RAP variants K¹⁹¹A, K²⁵⁶A, K³⁰⁵A and K³⁰⁶A, however, revealed a modest reduction in cluster II binding for the K²⁵⁶A variant only. This suggests that the other lysine residues can compensate for the absence of a single lysine residue for effective complex assembly. Collectively, novel insight has been obtained into the contribution of lysine residues of RAP to cluster II binding. In addition, we propose that TMTs can be utilized to identify lysine residues critical for protein complex formation.