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.

Figure 1

Conformational properties of Tip. (A). Prediction of the degree of disorder in the Tip protein as calculated from its primary structure using the program PONDR. A score of >0.5 indicates a high probability of disorder. A schematic of the Tip construct used (residues 1-187 of Tip-C484) is shown above the graph. The location of Lck binding domains LBD1 and LBD2, as well as the transmembrane and extracellular portions (removed in this work), are indicated. (B). Analytical gel filtration results for recombinant, renatured, purified Tip (dotted line) compared with standard proteins. The standards and Tip were analyzed on the same day, under identical conditions. (C). Circular dichroism spectrum of the same protein as shown in (B). The spectrum was recorded with a 0.1 mm path length and thermostatically controlled temperature of 25 °C at a final Tip concentration of 0.6 mg/mL.

Figure 2

Tip:Lck SH3 binding assay. GST-LckSH3 was purified as described in the Materials and Methods and loaded alone (lane 1). For co-incubations, proteins were mixed as indicated (approximate 1:6 ratio for LckSH3:Tip) and incubated for 30 minutes prior to washing. Approximately 5 μg of protein were loaded per lane onto a 10% SDS-PAGE gel, resolved and stained with Coomassie. The mass of Lck SH3 is 7929 Da. Tip normally appears as multiple bands in SDS-PAGE (see explanation in text).

Figure 3

Phosphorylation of Tip by Lck. (A). Coomassie-stained SDS-PAGE of Tip and LckYEEI coincubation. The proteins were allowed to interact for 30 minutes at a 1:10 Lck:Tip ratio (lane 2) in the presence of ATP and magnesium. Tip normally appears as multiple bands in SDS-PAGE (see explanation in text). (B). Electrospray mass spectra of Tip phosphorylation by LckYEEI (as in lanes 1,2 from part A). The vertical dashed lines show the theoretical masses for native Tip and for singly (+1P) and doubly (+2P) phosphorylated Tip. A small peak at +78 Da is due to the formation of a Tip/β-mercaptoethanol adduct. The LckYEEI:Tip molar ratio was 1:5 with the Mg²⁺ and ATP concentrations provided in the Materials and Methods. (C). Recombinant LckYEEI was purified from Sf9 insect cells and assayed for kinase activity with a peptide substrate either alone or in the presence of purified recombinant Tip or Nef in the molar ratios shown. Details of the FRET-based tyrosine kinase assay can be found under Materials and Methods. Each condition was repeated in quadruplicate, and the extent of phosphorylation is expressed as mean percent phosphorylation relative to a control phosphopeptide ± S.D. The overall experiment was repeated twice with comparable results.

Figure 4

Tip is functional in yeast. (A). S. cerevisiae cultures were transformed with combinations of galactose-inducible expression plasmids for Tip (T), HIV-1 Nef (N), wild-type Lck, Lck with a high-affinity SH2-binding tail sequence (LckYEEI), or no insert (-) as indicated. Four-fold dilutions of each culture were spotted on galactose plates to assess growth suppression. Plates were scanned and yeast patches appear as dark circles . (B). Immunoblots from yeast cultures shown in part A. Lck activity was assessed on anti-phosphotyrosine immunoblots (pTyr) while control blots verify expression of Lck, Nef and Tip. In this system, Tip strongly activates both Lck and LckYEEI, suggesting that tail release from SH2 is not required for activation. Tip runs as multiple bands in the presence of Lck due to phosphorylation.

Figure 5

Deuterium incorporation into intact Tip. (A). Mass spectra of the +26 charge state of intact recombinant Tip. The time in D₂O is indicated on the right. A dotted line is provided at m/z 900 for optical guidance. (B). Deuterium levels for intact, recombinant, renatured Tip. Each data point was determined from the mass scale transformation of the raw m/z spectra (see Materials and Methods) and is an average of triplicate measurements. Variation between measurements was within 2 Da so error bars are not shown. The maximum number of exchangeable backbone amide hydrogens in Tip is 191 (indicated by the dotted line). These results have been adjusted for back exchange (see Materials and Methods).

Figure 6

Mass spectra of selected Tip peptic peptides during deuteration. (A) residues (-19)-6, (B) residues 145-177, (C) residues 89-110, (D) residues 109-126. The time spent in D₂O is shown only in panel (D) but was the same for panels (A-C). The charge state of each peptide is indicated. The peptide in panel (A) contains sequence prior to the first official amino acid of Tip and is therefore numbered negatively. The highly deuterated (TD) protein was prepared as described in the Materials and Methods. A complete list of peptic peptides and deuteration is shown in Supplementary Table 1.

Figure 7

Summary of deuterium exchange in Tip peptic peptides. Each bar represents a peptic peptide. Residues are labeled beginning with -19 to account for affinity tag spanning the first 19 residues of recombinant Tip. The percent unprotected after 1 minute of deuterium exchange was calculated by dividing the number of residues that exchanged after one minute (after back-exchange correction) by the total number of possible exchangeable backbone amide hydrogens in each peptide. A schematic of Tip, aligned with the protection data, is shown at the top of the diagram. The number of exchangeable backbone amide hydrogens in each peptide is shown in the upper right of each bar. Some bars were obtained by subtraction of overlapping peptides. A full list of deuterium levels in all peptic peptides can be found in Supplementary Table 1.