The NMR structure determination of the immunoglobulin-alpha ITAM peptide substrate bound to Lyn tyrosine kinase
Abstract
The purpose of this study was to investigate the structural interactions between Lyn tyrosine kinase and a known substrate by NMR spectroscopy. An eighteen-residue peptide (ITP-18) and a twelve-residue peptide (ITP-12) of the immunoreceptor tyrosine activation motif (ITAM) of Ig-α were used in this study along with a glutathione-S-transferase (GST) fused portion of Lyn tyrosine kinase. Chapter 1 presents the background and biological motivations that are the basis for this work. Chapter 2 describes the expression, purification, and characterization of the GST-Lyn protein product. The activity of GST-Lyn for the ITP-12 substrate is also described. The structure determination of the peptide substrate bound to the Lyn tyrosine kinase by exchange-transferred nuclear Overhauser effect (ET-NOE) is explained in Chapter 3. The peptide structure has an irregular helical nature. The acidic residues on the opposing side of the helix to the tyrosine side chain may be important to the binding; glutamtes at positions 2 and 6 are conserved in the ITAM and in the concensus substrate sequences. The presence of NOEs across the entire peptide may indicate that the peptide is binding to a large surface in the kinase active site. In Chapter 4, the peptide structure is docked into the active site of a known src-type tyrosine kinase structure, Lck kinase domain. The docked structure shows the two glutamates contacting the enzyme as well as the ITAM conserved leucine residues at positions 4 and 8. The docked structure shows that the binding is dependent on electrostatic interactions of the peptide's acidic residues and hydrophobic contacts from the leucines. The docked structure has been shown to be more similar to the peptide inhibitor bound to cAMP-dependent protein kinase than to the peptide substrate bound into insulin receptor kinase. The docked structure also helps demonstrate how the autophosphorylation of src-kinases aids the binding of substrate in the active site. The docked structure of ITP-12 represents the first structural model of a peptide substrate bound to a Src-type kinase.
Degree
Ph.D.
Advisors
Post, Purdue University.
Subject Area
Biophysics|Biochemistry|Immunology
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