التفاصيل البيبلوغرافية
| العنوان: |
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. |
| المؤلفون: |
Bloem, Esther, Ebberink, Eduard H T M, van den Biggelaar, Maartje, van der Zwaan, Carmen, Mertens, Koen, Meijer, Alexander B |
| المساهمون: |
Sub Pharmaceutical proteins, Pharmaceutics, Biomolecular Mass Spectrometry and Proteomics |
| سنة النشر: |
2015 |
| مصطلحات موضوعية: |
Amino Acid Sequence, Amino Acid Substitution, Animals, Binding Sites, Humans, LDL-Receptor Related Protein-Associated Protein, Low Density Lipoprotein Receptor-Related Protein-1, Lysine, Models, Molecular, Mutagenesis, Site-Directed, Protein Binding, Protein Footprinting, Protein Interaction Domains and Motifs, Rats, Recombinant Proteins, Tandem Mass Spectrometry, Taverne |
| الوصف: |
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. |
| نوع الوثيقة: |
article in journal/newspaper |
| وصف الملف: |
application/pdf |
| اللغة: |
English |
| تدمد: |
0264-6021 |
| العلاقة: |
https://dspace.library.uu.nl/handle/1874/329427Test |
| الإتاحة: |
https://dspace.library.uu.nl/handle/1874/329427Test |
| حقوق: |
info:eu-repo/semantics/OpenAccess |
| رقم الانضمام: |
edsbas.88DF1FCD |
| قاعدة البيانات: |
BASE |
