title: Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein.
abstract: Tyrosine kinase interacting protein (Tip) of Herpesvirus saimiri (HVS) activates the lymphoid-specific member of the Src family kinase Lck. The Tip:Lck interaction is essential for transformation and oncogenesis in HVS-infected cells. As there are no structural data for Tip, hydrogen-exchange mass spectrometry was used to investigate the conformation of a nearly full-length form (residues 1-187) of Tip from HVS strain C484. Disorder predictions suggested that Tip would be mostly unstructured, so great care was taken to ascertain whether recombinant Tip was functional. Circular dichroism and gel-filtration analysis indicated an extended, unstructured protein. In vitro and in vivo binding and kinase assays confirmed that purified, recombinant Tip interacted with Lck, was capable of activating Lck kinase activity strongly and was multiply phosphorylated by Lck. Hydrogen-exchange mass spectrometry of Tip then showed that the majority of backbone amide hydrogen atoms became deuterated after only 10 s of labeling. Such a result suggested that Tip was almost totally unstructured in solution. Digestion of deuterium-labeled Tip revealed some regions with minor protection from exchange. Overall, it was found that, although recombinant Tip is still functional and capable of binding and activating its target Lck, it is largely unstructured.
authors.affiliation: Department of Chemistry, University of New Mexico, Albuquerque, NM 87131, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Mitchell
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Trible
authors.forename: Lori A
authors.initials: LA
authors.lastname: Emert-Sedlak
authors.forename: David D
authors.initials: DD
authors.lastname: Weis
authors.forename: Edwina C
authors.initials: EC
authors.lastname: Lerner
authors.forename: Jeremy J
authors.initials: JJ
authors.lastname: Applen
authors.forename: Bartholomew M
authors.initials: BM
authors.lastname: Sefton
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Smithgall
authors.forename: John R
authors.initials: JR
authors.lastname: Engen
mesh_terms: D006567:Herpesvirus 2, Saimiriine / Q000201:enzymology*; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011522:Protons; D011994:Recombinant Proteins / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010455:Peptides; D010750:Phosphoproteins; D011522:Protons; D011994:Recombinant Proteins; D014764:Viral Proteins; C092384:tyrosine kinase interacting protein, Saimiriine herpesvirus 2