Application of linker technique to trap transiently interacting protein complexes for structural studies
Main Article Content
Keywords
Protein-protein interactions, transient interactions, Gly-rich linkers
Abstract
Protein-protein interactions are key events controlling several biological processes. We have developed and employed a method to trap transiently interacting protein complexes for structural studies using Gly-rich linkers to fuse interacting partners, one of which is unstructured. Initial steps involve isothermal titration calorimetry to identify the minimum binding region of the unstructured protein in its interaction with its stable binding partner. This is followed by computational analysis to identify the approximate site of the interaction and to design an appropriate linker length. Subsequently, fused constructs are generated and characterized using size exclusion chromatography and dynamic light scattering experiments. The structure of the chimeric protein is then solved by crystallization, and validated both in vitro and in vivo by substituting key interacting residues of the full length, unlinked proteins with alanine. This protocol offers the opportunity to study crucial and currently unattainable transient protein interactions involved in various biological processes
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References
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35. Cui Y ea (2003) Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy. Anal Biochem 315: 175-182.
36. Zhang M, Vogel HJ, Zwiers H (1994) Nuclear magnetic resonance studies of the structure of B50/neuromodulin and its interaction with calmodulin. Biochem Cell Biol 72: 109-116.
37. Kingston RL, Hamel DJ, Gay LS, Dahlquist FW, Matthews BW (2004) Structural basis for the attachment of a paramyxoviral polymerase to its template. Proc Natl Acad Sci U S A 101: 8301-8306.
2. Zimmerman SB, Trach SO (1991) Estimation of macromolecule concentrations and excluded volume effects for the cytoplasm of Escherichia coli. J Mol Biol 222: 599-620.
3. Surin A, Pshenichkin S, Grajkowska E, Surina E, Wroblewski JT (2007) Cyclothiazide selectively inhibits mGluR1 receptors interacting with a common allosteric site for non-competitive antagonists. Neuropharmacology 52: 744-754.
4. Frang H, Cockcroft V, Karskela T, Scheinin M, Marjamaki A (2001) Phenoxybenzamine binding reveals the helical orientation of the third transmembrane domain of adrenergic receptors. J Biol Chem 276: 31279-31284.
5. Vauquelin G, Van Liefde I, Birzbier BB, Vanderheyden PM (2002) New insights in insurmountable antagonism. Fundam Clin Pharmacol 16: 263-272.
6. Kondo H, Shiroishi M, Matsushima M, Tsumoto K, Kumagai I (1999) Crystal structure of anti-Hen egg white lysozyme antibody (HyHEL-10) Fv-antigen complex. Local structural changes in the protein antigen and water-mediated interactions of Fv-antigen and light chain-heavy chain interfaces. J Biol Chem 274: 27623-27631.
7. Pascal SM, Singer AU, Gish G, Yamazaki T, Shoelson SE, et al. (1994) Nuclear magnetic resonance structure of an SH2 domain of phospholipase C-gamma 1 complexed with a high affinity binding peptide. Cell 77: 461-472.
8. Dunker AK, Brown CJ, Lawson JD, Iakoucheva LM, Obradovic Z (2002) Intrinsic disorder and protein function. Biochemistry 41: 6573-6582.
9. Dyson HJ, Wright PE (2005) Intrinsically unstructured proteins and their functions. Nat Rev Mol Cell Biol 6: 197-208.
10. Vucetic S, Xie H, Iakoucheva LM, Oldfield CJ, Dunker AK, et al. (2007) Functional anthology of intrinsic disorder. 2. Cellular components, domains, technical terms, developmental processes, and coding sequence diversities correlated with long disordered regions. J Proteome Res 6: 1899-1916.
11. Xie H, Vucetic S, Iakoucheva LM, Oldfield CJ, Dunker AK, et al. (2007) Functional anthology of intrinsic disorder. 3. Ligands, post-translational modifications, and diseases associated with intrinsically disordered proteins. J Proteome Res 6: 1917-1932.
12. Xie H, Vucetic S, Iakoucheva LM, Oldfield CJ, Dunker AK, et al. (2007) Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J Proteome Res 6: 1882-1898.
13. Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF, Jones DT (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J Mol Biol 337: 635-645.
14. Radivojac P, Iakoucheva LM, Oldfield CJ, Obradovic Z, Uversky VN, et al. (2007) Intrinsic disorder and functional proteomics. Biophys J 92: 1439-1456.
15. Draper DE, Reynaldo LP (1999) RNA binding strategies of ribosomal proteins. Nucleic Acids Res 27: 381-388.
16. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447: 1021-1025.
17. Meszaros B, Simon I, Dosztanyi Z (2011) The expanding view of protein-protein interactions: complexes involving intrinsically disordered proteins. Phys Biol 8: 035003.
18. Reddy Chichili VP, Kumar V, Sivaraman J (2013) Linkers in the structural biology of protein-protein interactions. Protein Sci 22: 153-167.
19. Kumar V, Chichili VP, Zhong L, Tang X, Velazquez-Campoy A, et al. (2013) Structural basis for the interaction of unstructured neuron specific substrates neuromodulin and neurogranin with calmodulin. Sci Rep 3: 1392.
20. Andreasen TJ, Luetje CW, Heideman W, Storm DR (1983) Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes. Biochemistry 22: 4615-4618.
21. Neuner-Jehle M, Denizot JP, Mallet J (1996) Neurogranin is locally concentrated in rat cortical and hippocampal neurons. Brain Res 733: 149-154.
22. Iwakura M, Nakamura T (1998) Effects of the length of a glycine linker connecting the N-and C-termini of a circularly permuted dihydrofolate reductase. Protein Eng 11: 707-713.
23. Hendrickson WA, Horton JR, LeMaster DM (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J 9: 1665-1672.
24. Wiseman T, Williston S, Brandts JF, Lin LN (1989) Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal Biochem 179: 131-137.
25. Jelesarov I, Bosshard HR (1999) Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetics of biomolecular recognition. J Mol Recognit 12: 3-18.
26. Benvenuti M, Mangani S (2007) Crystallization of soluble proteins in vapor diffusion for x-ray crystallography. Nat Protoc 2: 1633-1651.
27. Zbyszek Otwinowski WM (1997) Processing of X-ray diffraction data collected in oscillation mode Methods in Enzymology 276: 307-326.
28. Sheldrick GM (2008) A short history of SHELX. Acta Crystallogr A 64: 112-122.
29. Cowtan K (2006) The Buccaneer software for automated model building. 1. Tracing protein chains. Acta Crystallogr D Biol Crystallogr 62: 1002-1011.
30. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126-2132.
31. Vagin AA, Steiner RA, Lebedev AA, Potterton L, McNicholas S, et al. (2004) REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr D Biol Crystallogr 60: 2184-2195.
32. Laskowski RA, Macarthur, M. W., Moss, D. S. & Thornton, J. M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures. Journal of Applied Crystallography 26: 283-291.
33. Dominy CN, Andrews DW (2003) Site-directed mutagenesis by inverse PCR. Methods Mol Biol 235: 209-223.
34. Ran X, Miao HH, Sheu FS, Yang D (2003) Structural and dynamic characterization of a neuron-specific protein kinase C substrate, neurogranin. Biochemistry 42: 5143-5150.
35. Cui Y ea (2003) Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy. Anal Biochem 315: 175-182.
36. Zhang M, Vogel HJ, Zwiers H (1994) Nuclear magnetic resonance studies of the structure of B50/neuromodulin and its interaction with calmodulin. Biochem Cell Biol 72: 109-116.
37. Kingston RL, Hamel DJ, Gay LS, Dahlquist FW, Matthews BW (2004) Structural basis for the attachment of a paramyxoviral polymerase to its template. Proc Natl Acad Sci U S A 101: 8301-8306.
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Jan 28, 2016
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Chichili VPR, Kumar V, Sivaraman J. Application of linker technique to trap transiently interacting protein complexes for structural studies. J Biol Methods [Internet]. 2016 Jan. 28 [cited 2024 Apr. 23];3(1):e34. Available from: https://jbmethods.org/index.php/jbm/article/view/81
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