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 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 II binding. Binding studies employing the RAP variants K(191)A, K(256)A, K(305)A and K(306)A, however, revealed a modest reduction in cluster II binding for the K(256)A 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.
*Department of Plasma Proteins, Sanquin Research, Amsterdam 1066CX, The Netherlands.
Esther
E
Bloem
*Department of Plasma Proteins, Sanquin Research, Amsterdam 1066CX, The Netherlands.
Eduard H T M
EH
Ebberink
*Department of Plasma Proteins, Sanquin Research, Amsterdam 1066CX, The Netherlands.
Maartje
M
van den Biggelaar
*Department of Plasma Proteins, Sanquin Research, Amsterdam 1066CX, The Netherlands.
Carmen
C
van der Zwaan
*Department of Plasma Proteins, Sanquin Research, Amsterdam 1066CX, The Netherlands.
Koen
K
Mertens
*Department of Plasma Proteins, Sanquin Research, Amsterdam 1066CX, The Netherlands.
Alexander B
AB
Meijer
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D006801:Humans; D026563:LDL-Receptor Related Protein-Associated Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D023302:Protein Footprinting / Q000379:methods; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
D026563:LDL-Receptor Related Protein-Associated Protein; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011994:Recombinant Proteins; D008239:Lysine