Abstract
Structural flexibility plays a crucial role in protein function. To assess whether specific structural changes are associated with the binding of an immunoreceptor tyrosine-based activation motif (ITAM) to the tandem Src homology-2 domains (tSH2) of the spleen tyrosine kinase [EC 2.7.7.112] (Syk), we used an approach based on protein hydrogen/deuterium exchange in the presence and absence of the diphosphorylated ITAM peptide. The protein deuterium uptake by the intact Syk protein was monitored in time by electrospray mass spectrometry, which revealed a dramatic relative decrease in deuterium uptake when the protein was bound to the ITAM peptide, suggesting an overall change in protein dynamics. Subsequently, the deuterium incorporation of individual segments of the protein was investigated using proteolysis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass-analysis, which revealed that several regions of Syk tSH2 are significantly more protected from exchange in the presence of the ITAM peptide. Four protected regions encompass the phosphotyrosine and hydrophobic binding sites on the SH2 domains, whereas two other protected regions are located in the inter-SH2 linker motif and do not make any direct contacts with the peptide. Interestingly, our data suggest that binding of the ITAM peptide to Syk tSH2 induces distal structural effects on the protein that stabilize the inter-SH2 linker region, possibly by raising the degree of helical structure upon binding.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Turner, M.; Schweighoffer, E.; Colucci, F.; Di Santo, J. P.; Tybulewicz, V. L. Tyrosine kinase SYK: Essential Functions for Immunoreceptor Signaling. Immunol. Today 2000, 21, 148–154.
Yanagi, S.; Inatome, R.; Takano, T.; Yamamura, H. Syk Expression and Novel Function in a Wide Variety of Tissues. Biochem. Biophys. Res. Commun. 2001, 288, 495–498.
Reth, M. Antigen Receptor Tail Clue. Nature 1989, 338, 383–384.
Pribluda, V. S.; Pribluda, C.; Metzger, H. Transphosphorylation as the Mechanism by Which the High-Affinity Receptor for IgE is Phosphorylated Upon Aggregation. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 11246–11250.
Cambier, J. C.; Pleiman, C. M.; Clark, M. R. Signal Transduction by the B Cell Antigen Receptor and Its Coreceptors. Annu. Rev. Immunol. 1994, 12, 457–486.
Chan, A. C.; Desai, D. M.; Weiss, A. The Role of Protein Tyrosine Kinases and Protein Tyrosine Phosphatases in T Cell Antigen Receptor Signal Transduction. Annu. Rev. Immunol. 1994, 12, 555–592.
Kimura, T.; Sakamoto, H.; Appella, E.; Siraganian, R. Conformational Changes Induced in the Protein Tyrosine Kinase p72syk by Tyrosine Phosphorylation or by Binding of Phosphorylated Immunoreceptor Tyrosine-Based Activation Motif Peptides. Mol. Cell. Biol. 1996, 16, 1471–1478.
Shiue, L.; Zoller, M. J.; Brugge, J. S. Syk is Activated by Phosphotyrosine-Containing Peptides Representing the Tyrosine-Based Activation Motifs of the High Affinity Receptor for IgE. J. Biol. Chem. 1995, 270, 10498–10502.
Hatada, M. H.; Lu, X. D.; Laird, E. R.; Green, J. P.; et al. Molecular Basis for Interaction of the Protein Tyrosine Kinase ZAP-70 with the T-Cell Receptor. Nature 1995, 377, 32–38.
Sada, K.; Zhang, J.; Siraganian, R. P. SH2 Domain-Mediated Targeting, but not Localization, of Syk in the Plasma Membrane is Critical for Fc{epsilon}RI Signaling. Blood 2001, 97, 1352–1359.
Sicheri, F.; Moarefi, I.; Kuriyan, J. Crystal Structure of the Src Family Tyrosine Kinase Hck. Nature 1997, 385, 602–609.
Arold, S. T.; Ulmer, T. S.; Mulhern, T. D.; Werner, J. M.; Ladbury, J. E.; Campbell, I. D.; Noble, M. E. M. The Role of the Src Homology 3-Src Homology 2 Interface in the Regulation of Src Kinases. J. Biol. Chem. 2001, 276, 17199–17205.
Engh, R. A.; Bossemeyer, D. Structural Aspects of Protein Kinase Control—Role of Conformational Flexibility. Pharmacol. Ther. 2002, 93, 99–111.
Grucza, R.; Bradshaw, J.; Mitaxov, V.; Waksman, G. Role of Electrostatic Interactions in SH2 Domain Recognition: Salt Dependence of Tyrosyl-Phosphorylated Peptide Binding to the Tandem SH2 Domain of the Syk Kinase and the Single SH2 Domain of the Src Kinase. Biochemistry 2000, 39, 10072–10081.
Dekker, F. J.; de Mol, N. J.; van Ameijde, J.; Fischer, M. J. E.; Ruijtenbeek, R.; Redegeld, F. A. M.; Liskamp, R. M. J. Replacement of the Intervening Amino Acid Sequence of a Syk-Binding Diphosphopeptide by a Nonpeptide Spacer with Preservation of High Affinity. Chem. Biochem. 2002, 3, 238–242.
Dekker, F. J.; de Mol, N. J.; Fischer, M. J.; Liskamp, R. M. Amino Propynyl Benzoic Acid Building Block in Rigid Spacers of Divalent Ligands Binding to the Syk SH2 Domains with Equally High Affinity as the Natural Ligand. Bioorg. Med. Chem. Lett. 2003, 13, 1241–1234.
Fütterer, K.; Wong, J.; Grucza, R. A.; Chan, A. C.; Waksman, G. Structural Basis for Syk Tyrosine Kinase Ubiquity in Signal Transduction Pathways Revealed by the Crystal Structure of Its Regulatory SH2 Domains Bound to a Dually Phosphorylated ITAM Peptide 1. J. Mol. Biol. 1998, 281, 523–537.
Folmer, R. H. A.; Geschwindner, S.; Yafeng Xue, Y. Crystal Structure and NMR Studies of the Apo SH2 Domains of ZAP-70: Two Bikes Rather than a Tandem. Biochemistry 2002, 41, 14176–14184.
Grucza, R. A.; Fütterer, K.; Chan, A. C.; Waksman, G. Thermodynamic Study of the Binding of the Tandem-SH2 Domain of the Syk Kinase to a Dually Phosphorylated ITAM Peptide: Evidence for Two Conformers. Biochemistry 1999, 38, 5024–5033.
Kumaran, S.; Grucza, R. A.; Waksman, G. The Tandem Src Homology 2 Domain of the Syk Kinase: A Molecular Device that Adapts to Interphosphotyrosine Fistances. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 14828–14833.
Anderegg, R.; Wagner, D. Mass Spectrometric Characterization of Protein-Ligand Interaction. J. Am. Chem. Soc. 1995, 117, 1374–1377.
Guy, P.; Remigy, H.; Jaquinod, M.; Bersch, B.; Blanchard, L.; Dolla, A.; Forest, E. Study of the New Stability Properties Induced by Amino Acid Replacement of Tyrosine 64 in Cytochrome c553 from Desulfovibrio vulgaris Hildenborough Using Electrospray Ionization Mass Spectrometry. Biochem. Biophys. Res. Commun. 1996, 218, 97–103.
Zhang, Z.; Smith, D. L. Determination of Amide Hydrogen Exchange by Mass Spectrometry: A New Tool for Protein Structure Elucidation. Protein Sci. 1993, 2, 522–531.
Mandell, J. G.; Falick, A. M.; Komives, E. A. Measurement of Amide Hydrogen Exchange by MALDI-TOF Mass Spectrometry. Anal. Chem. 1998, 70, 3987–3995.
Catalina, M. I.; Dekker, F. J.; Liskamp, R. M. J.; Versluis, C.; Maier, C. S.; Heck, A. J. R. Structural Analysis of High Affinity Divalent Phosphopeptide Hybrids of Spleen Tyrosine Kinase. Int. J. Mass Spectrom. 2003, 228, 879–890.
de Mol, N. J.; Catalina, M. I.; Fischer, M. J. E.; Broutin, I.; Maier, C. S.; Heck, A. J. R. Changes in Structural Dynamics of the Grb2 Adaptor Protein Upon Binding of Phosphotyrosine Ligand to Its SH2 Domain. Biochem. Biophys. Acta 2004, 1700, 53–64.
Kipping, M.; Schierhorn, A. Improving Hydrogen/Deuterium Exchange Mass Spectrometry by Reduction of the Back-Exchange Effect. J. Mass Spectrom. 2003, 38, 271–276.
Thevenon-Emeric, G.; Kozlowski, J.; Zhang, Z.; Smith, D. L. Determination of Amide Hydrogen Exchange Rates in Peptides by Mass Spectrometry. Anal. Chem. 1992, 64, 2456–2458.
Jensen, O. A.; Podtelejnikov, A.; Mann, M. Delayed Extraction Improves Specificity in Database Searches by Matrix-Assisted Laser Desorption/Ionization Peptide Maps. Rapid Commun. Mass Spectrom. 1996, 10, 1371–1378.
Zhang, Z.; Smith, D. L. Determination of Amide Hydrogen Exchange by Mass Spectrometry: A New Tool for Protein Structure Elucidation. Protein Sci. 1993, 2, 522–531.
Dharmasiri, K.; Smith, D. L. Mass Spectrometric Determination of Isotopic Exchange Rates of Amide Hydrogens Located on the Surfaces of Proteins. Anal. Chem. 1996, 68, 2340–2344.
Yan, X.; Broderick, D.; Leid, M.; Schimerlik, M.; Deinzer, M. Dynamics and Ligand-Induced Solvent Accessibility Changes in Human Retinoid X Receptor Homodimer Determined by Hydrogen Deuterium Exchange and Mass Spectrometry. Biochemistry 2004, 43, 909–917.
Yan, X.; Watson, J.; Ho, P. S.; Deinzer, M. L. Mass Spectrometric Approaches Using Electrospray Ionization Charge States and Hydrogen-Deuterium Exchange for Determining Protein Structures and Their Conformational Changes. Mol. Cell. Proteom. 2004, 3, 10–23.
Miranker, A.; Robinson, C. V.; Radford, S. E.; Aplin, R. T.; Dobson, C. M. Detection of Transient Protein Folding Populations by Mass Spectrometry. Science 1993, 262, 896–900.
Wagner, D. S.; Melton, L. G.; Yan, Y.; Erickson, B. W.; Anderegg, R. J. Deuterium Exchange of α-Helices and β-Sheets as Monitored by Electrospray Ionization Mass Spectrometry. Protein Sci. 1994, 3, 1305–1314.
Mandell, J. G.; Baerga-Ortiz, A.; Akashi, S.; Takio, K.; Komives, E. A. Solvent Accessibility of the Thrombin-Thrombomodulin Interface. J. Mol. Biol. 2001, 306, 575–589.
Wintrode, P.; Friedrich, K.; Vierling, E.; Smith, J.; Smith, D. Solution Structure and Dynamics of a Heat Shock Protein Assembly Probed by Hydrogen Exchange and Mass Spectrometry. Biochemistry 2003, 42, 10667–10673.
Croy, C.; Koeppe, J.; Bergqvist, S.; Komives, E. Allosteric Changes in Solvent Accessibility Observed in Thrombin Upon Active Site Occupation. Biochemistry 2004, 43, 5246–5255.
Andersen, M. D.; Shaffer, J.; Jennings, P. A.; Adams, J. A. Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding. Hydrogen-Deuterium Exchange Studies Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Detection. J. Biol. Chem. 2001, 276, 14204–14211.
Dekker, F. J.; de Mol, N. J.; Fischer, M. J. E.; Liskamp, R. M. J. Amino Propynyl Benzoic Acid Building Block in Rigid Spacers of Divalent Ligands Binding to the Syk SH2 Domains with Equally High Affinity as the Natural Ligand. Bioorg. Med. Chem. Lett. 2003, 13, 1241–1244.
Hvidt, A.; Linderstrom-Lang, K. Exchange of Hydrogen Atoms in Insulin with Deuterium Atoms in Aqueous Solutions. Biochem. Biophys. Acta 1954, 14, 575–575.
Hoofnagle, A. N.; Resing, K. A.; Ahn, N. G. Protein Analysis by Hydrogen Exchange Mass Spectrometry. Annu. Rev. Biophys. Biomol. Struct. 2003, 32, 1–25.
Engen, J. R.; Gmeiner, W. H.; Smithgall, T. E.; Smith, D. L. Hydrogen Exchange Shows Peptide Binding Stabilizes Motions in Hck SH2. Biochemistry 1999, 38, 8926–8935.
Farrow, N. A.; Muhandiram, R.; Singer, A. U.; Pascal, S. M.; Kay, C. M.; Gish, G.; Shoelson, S. E.; Pawson, T.; Forman-Kay, J. D.; Kay, L. E. Backbone Dynamics of a Free and Phosphopeptide-Complexed Src Homology 2 Domain Studied by 15N NMR Relaxation. Biochemistry 1994, 33, 5984–6003.
Shoelson, S. E.; Sivaraja, M.; Williams, K. P.; Hu, P.; Schlessinger, J.; Weiss, M. A. Specific Phosphopeptide Binding Regulates a Conformational Change in the PI 3-Kinase SH2 Domain Associated with Enzyme Activation. Embo J. 1993, 12, 795–802.
Breeze, A. L.; Kara, B. V.; Barratt, D. G.; Anderson, M.; Smith, J. C.; Luke, R. W.; Best, J. R.; Cartlidge, S. A. Structure of a Specific Peptide Complex of the Carboxy-Terminal Sh2 Domain from the P85-α Subunit of Phosphatidylinositol 3-Kinase. Embo J. 1996, 15, 3579–3589.
Van den Bremer, E. T. J.; Jiskoot, W.; James, R.; Moore, G. R.; Kleanthous, C.; Heck, A. J. R.; Maier, C. S. Probing Metal Ion Binding and Conformational Properties of the Colicin E9 Endonuclease by Electrospray Ionization Time-of-Flight Mass Spectrometry. Protein Sci. 2002, 11, 1738–1752.
Mohimen, A.; Dobo, A.; Hoerner, J. K.; Kaltashov, I. A. A Chemometric Approach to Detection and Characterization of Multiple Protein Conformers in Solution Using Electrospray Ionization Mass Spectrometry. Anal. Chem. 2003, 75, 4139–4147.
Konermann, L.; Douglas, D. J. Equilibrium Unfolding of Proteins Monitored by Electrospray Ionization Mass Spectrometry: Distinguishing Two-State from Multi-State Transitions. Rapid Commun. Mass Spectrom. 1998, 12, 435–442.
Waksman, G.; Shoelson, S. E.; Pant, N.; Cowburn, D.; Kuriyan, J. Binding of a High Affinity Phosphotyrosyl Peptide to the Src SH2 Domain: Crystal Structures of the Complexed and Peptide-Free Forms. Cell 1993, 72, 779–790.
Narula, S.; Yuan, R.; Adams, S.; Green, O.; Green, J.; Philips, T.; Zydowsky, L.; Botfield, M.; Hatada, M.; Laird, E.; Zoller, M.; Karas, J.; Dalgarno, D. Solution Structure of the C-Terminal SH2 Domain of the Human Tyrosine Kinase Syk Complexed with a Phosphotyrosine Pentapeptide. Structure 1995, 3, 1061–1073.
Author information
Authors and Affiliations
Corresponding author
Additional information
Published online May 23, 2005
Rights and permissions
About this article
Cite this article
Catalina, M.I., Fischer, M.J.E., Dekker, F.J. et al. Binding of a Diphosphorylated-ITAM peptide to spleen tyrosine kinase (Syk) induces distal conformational changes: A hydrogen exchange mass spectrometry study. J Am Soc Mass Spectrom 16, 1039–1051 (2005). https://doi.org/10.1016/j.jasms.2005.02.011
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1016/j.jasms.2005.02.011