Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2
Abstract
:1. Introduction
2. Results
2.1. Co-Crystallization
2.2. Overall Structure of the Complex between ALG-2 and Sec31A Peptide
2.3. Structure of the Sec31A Peptide
2.4. Binding of the Sec31A Peptide to Pocket 3
2.4.1. Analysis of the Ca2+-Dependent Interaction of Sec31A by GST-Pulldown
2.4.2. Identification of Critical Pocket 3 Residues for Interaction
2.5. Co-Immunoprecipitation Assays of Sec31A Mutants
2.6. Identification of Critical Residues in Sec31A for Binding to ALG-2
2.7. Comparison of ALG-2-Binding Motif Type 2 Sequences
3. Discussion
4. Experimental Section
4.1. Expression Plasmids
4.2. Purification of Recombinant Proteins
4.3. Crystallization
4.4. Data Collection, Structure Determination and Refinement
4.5. Cell Culture and DNA Transfection
4.6. GST-ALG-2 Pulldown
4.7. Far-Western
4.8. Co-Immunoprecipitation Assays
4.9. Miscellaneous
Supplementary Materials
Acknowledgments
Author Contributions
Abbreviation
aa | amino acids |
Anx | annexin |
bio-ALG-2 | biotin-labeled ALG-2 |
COPII | coat protein complex II |
ESCRT | endosomal sorting complex required for transport |
FW | far-Western |
GFP | green fluorescent protein |
GST | glutathione-S-transferase |
IP | immunoprecipitation |
PEF | penta-EF-hand |
PPII | polyproline helix type II |
WB | Western blotting |
Conflicts of Interest
References
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Takahashi, T.; Kojima, K.; Zhang, W.; Sasaki, K.; Ito, M.; Suzuki, H.; Kawasaki, M.; Wakatsuki, S.; Takahara, T.; Shibata, H.; et al. Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2. Int. J. Mol. Sci. 2015, 16, 3677-3699. https://doi.org/10.3390/ijms16023677
Takahashi T, Kojima K, Zhang W, Sasaki K, Ito M, Suzuki H, Kawasaki M, Wakatsuki S, Takahara T, Shibata H, et al. Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2. International Journal of Molecular Sciences. 2015; 16(2):3677-3699. https://doi.org/10.3390/ijms16023677
Chicago/Turabian StyleTakahashi, Takeshi, Kyosuke Kojima, Wei Zhang, Kanae Sasaki, Masaru Ito, Hironori Suzuki, Masato Kawasaki, Soichi Wakatsuki, Terunao Takahara, Hideki Shibata, and et al. 2015. "Structural Analysis of the Complex between Penta-EF-Hand ALG-2 Protein and Sec31A Peptide Reveals a Novel Target Recognition Mechanism of ALG-2" International Journal of Molecular Sciences 16, no. 2: 3677-3699. https://doi.org/10.3390/ijms16023677