| Title: |
Prediction of a New Ligand-Binding Site for Type 2 Motif based on the Crystal Structure of ALG-2 by Dry and Wet Approaches |
| Authors: |
Masatoshi Maki; Takeshi Takahashi; Hironori Suzuki; Hideki Shibata; Tatsutoshi Inuzuka |
| Source: |
International Journal of Molecular Sciences, Vol 13, Iss 6, Pp 7532-7549 (2012) |
| Publisher Information: |
MDPI AG |
| Publication Year: |
2012 |
| Collection: |
Directory of Open Access Journals: DOAJ Articles |
| Subject Terms: |
ALG-2; calcium-binding protein; computational prediction; protein-protein interaction; proline-rich motif; Biology (General); QH301-705.5; Chemistry; QD1-999 |
| Description: |
ALG-2 is a penta-EF-hand Ca 2+ -binding protein and interacts with a variety of intracellular proteins. Two types of ALG-2-binding motifs have been determined: type 1, P X YP X nYP ( X , variable; n = 4), in ALIX and PLSCR3; type 2, P X PGF, in Sec31A and PLSCR3. The previously solved X-ray crystal structure of the complex between ALG-2 and an ALIX peptide containing type 1 motif showed that the peptide binds to Pocket 1 and Pocket 2. Co-crystallization of ALG-2 and type 2 motif-containing peptides has not been successful. To gain insights into the molecular basis of type 2 motif recognition, we searched for a new hydrophobic cavity by computational algorithms using MetaPocket 2.0 based on 3D structures of ALG-2. The predicted hydrophobic pocket designated Pocket 3 fits with N -acetyl-ProAlaProGlyPhe-amide, a virtual penta-peptide derived from one of the two types of ALG-2-binding sites in PLSCR3 (type 2 motif), using the molecular docking software AutoDock Vina. We investigated effects of amino acid substitutions of the predicted binding sites on binding abilities by pulldown assays using glutathione- S -transferase -fused ALG-2 of wild-type and mutant proteins and lysates of cells expressing green fluorescent protein -fused PLSCR3 of wild-type and mutants. Substitution of either L52 with Ala or F148 with Ser of ALG-2 caused loss of binding abilities to PLSCR3 lacking type 1 motif but retained those to PLSCR3 lacking type 2 motif, strongly supporting the hypothesis that Pocket 3 is the binding site for type 2 motif. |
| Document Type: |
article in journal/newspaper |
| Language: |
English |
| Relation: |
http://www.mdpi.com/1422-0067/13/6/7532; https://doaj.org/toc/1422-0067; https://doaj.org/article/dd2be9a6344c4666ae6a01956fe05346 |
| DOI: |
10.3390/ijms13067532 |
| Availability: |
https://doi.org/10.3390/ijms13067532; https://doaj.org/article/dd2be9a6344c4666ae6a01956fe05346 |
| Accession Number: |
edsbas.CC09D080 |
| Database: |
BASE |