Abstract
We present a new method for the rapid identification of amino acid residues that contribute to protein-protein interfaces. Tail-interacting protein of 47 kDa (TIP47) binds Rab9 GTPase and the cytoplasmic domains of mannose 6-phosphate receptors and is required for their transport from endosomes to the Golgi apparatus. Cysteine mutations were incorporated randomly into TIP47 by expression in Escherichia coli cells harboring specific misincorporator tRNAs. We made use of the ability of the native TIP47 protein to protect 48 cysteine probes from chemical modification by iodoacetamide as a means to obtain a surface map of TIP47, revealing the identity of surface-localized, hydrophobic residues that are likely to participate in protein-protein interactions. Direct mutation of predicted interface residues confirmed that the protein had altered binding affinity for the mannose 6-phosphate receptor. TIP47 mutants with enhanced or diminished affinities were also selected by affinity chromatography. These methods were validated in comparison with the protein's crystal structure, and provide a powerful means to predict protein-protein interaction interfaces.
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Silverman, J.A. & Harbury, P.B. Rapid mapping of protein structure, interactions, and ligand binding by misincorporation proton-alkyl exchange. J. Biol. Chem. 277, 30968–30975 (2002).
Diaz, E. & Pfeffer, S.R. TIP47: a cargo selection device for mannose 6-phosphate receptor trafficking. Cell 93, 433–443 (1998).
Krise, J.P., Sincock, P.M., Orsel, J.G. & Pfeffer, S.R. Quantitative analysis of TIP47-receptor cytoplasmic domain interactions: implications for endosome-to-trans Golgi network trafficking. J. Biol. Chem. 275, 25188–25193 (2000).
Orsel, J.G., Sincock, P.M., Krise, J.P. & Pfeffer, S.R. Recognition of the 300-kDa mannose 6-phosphate receptor cytoplasmic domain by 47-kDa tail-interacting protein. Proc. Natl. Acad. Sci. USA 97, 9047–9051 (2000).
Carroll, K.S. et al. Role of Rab9 GTPase in facilitating receptor recruitment by TIP47. Science 292, 1373–1377 (2001).
Hanna, J., Carroll, K. & Pfeffer, S.R. Identification of residues in TIP47 essential for Rab9 binding. Proc. Natl. Acad. Sci. USA 99, 7450–7454 (2002).
Hickenbottom, S.J., Kimmel, A.R., Londos, C. & Hurley, J.H. Structure of a lipid-droplet protein: the PAT family member TIP47. Structure 12, 1199–1207 (2004).
Jacobson, G.R., Schaffer, M.H., Stark, G.R. & Vanaman, T.C. Specific chemical cleavage in high yield at the amino peptide bonds of cysteine and cystine residues. J. Biol. Chem. 248, 6583–6591 (1973).
Wu, J. & Watson, J.T. Optimization of the cleavage reaction for cyanylated cysteinyl proteins for efficient and simplified mass mapping. Anal. Biochem. 258, 268–276 (1998).
Janin, J. Surface and inside volumes in globular proteins. Nature 277, 491–492 (1979).
Rost, B. PHD: predicting one-dimensional protein structure by profile based neural networks. Methods Enzymol. 266, 525–539 (1996).
Lo Conte, L., Chothia, C. & Janin, J. The atomic structure of protein-protein recognition sites. J. Mol. Biol. 285, 2177–2198 (1999).
Stemmer, W.P., Crameri, A., Ha, K.D., Brennan, T.M. & Heyneker, H.L. Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxy-ribonucleotides. Gene 164, 49–53 (1995).
Janin, J. & Wodak, S. Conformation of amino acid side-chains in proteins. J. Mol. Biol. 125, 357–386 (1978).
Fraczkiewicz, R. & Braun, W. Exact and efficient analytical calculation of the accessible surface areas and their gradients for macromolecules. J. Comp. Chem. 19, 319–333 (1998).
Acknowledgements
We are grateful to J. Silverman for his help in setting up the MPAX method and J. Hurley for sharing structural data before publication. This research was supported by a research grant from the US National Institutes of Health to S.R.P. (DK37332).
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Burguete, A., Harbury, P. & Pfeffer, S. In vitro selection and prediction of TIP47 protein-interaction interfaces. Nat Methods 1, 55–60 (2004). https://doi.org/10.1038/nmeth702
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DOI: https://doi.org/10.1038/nmeth702
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