Functional characterization and conformational analysis of the Herpesvirus saimiri Tip-C484 protein.

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.

Altered dynamics in Lck SH3 upon binding to the LBD1 domain of Herpesvirus saimiri Tip.

The Tip protein from Herpesvirus saimiri interacts with the SH3 domain from the Src-family kinase Lck via a proline-containing sequence termed LBD1. Src-family kinase SH3 domains related to Lck have been shown to be dynamic in solution and partially unfold under physiological conditions. The rate of such partial unfolding is reduced by viral protein binding. To determine if the Lck SH3 domain displayed similar behavior, the domain was investigated with hydrogen exchange and mass spectrometry. Lck SH3 was found to be highly dynamic in solution. While other SH3 domains require as much as 10,000 sec to become totally deuterated, Lck SH3 became almost completely labeled within 200 sec. A partial unfolding event involving 8-10 residues was observed with a half-life of approximately 10 sec. Tip LBD1 binding did not cause gross structural changes in Lck SH3 but globally stabilized the domain and reduced the rate of partial unfolding by a factor of five. The region of partial unfolding in Lck SH3 was found to be similar to that identified for other SH3 domains that partially unfold. Although the sequence conservation between Lck SH3 and other closely related SH3 domains is high, the dynamics do not appear to be conserved.

Relative contributions of desolvation, inter- and intramolecular interactions to binding affinity in protein kinase systems.

In several previous studies, we performed sensitivity analysis to gauge the relative importance of different atomic partial charges in determining protein-ligand binding. In this work, we gain further insights by decomposing these results into three contributions: desolvation, intramolecular interactions, and intermolecular interactions, again based on a Poisson continuum electrostatics model. Three protein kinase-inhibitor systems have been analyzed: CDK2-deschloroflavopiridol, PKA-PKI, and LCK-PP2. Although our results point out the importance of specific intermolecular interactions to the binding affinity, they also reveal the remarkable contributions from the solvent-mediated intramolecular interactions in some cases. Thus, it is necessary to look beyond analyzing protein-ligand interactions to understand protein-ligand recognition or to gain insights into designing ligands and proteins. In analyzing the contributions of the three components to the overall binding free energy, the PKA-PKI system with a much larger ligand was found to behave differently from the other two systems with smaller ligands. In the former case, the intermolecular interactions are very favorable, and together with the favorable solvent-mediated intramolecular interactions, they overcome the large desolvation penalties to give a favorable electrostatics contribution to the overall binding affinity. On the other hand, the other two systems with smaller ligands only present modest intermolecular interactions and they are not or are only barely sufficient to overcome the desolvation penalty even with the aid of the favorable intramolecular contributions. As a result, the binding affinity of these two systems do not or only barely benefit from electrostatics contributions.

Inhibition of the activity of SRC and Abl tyrosine protein kinases by the binding of the Wiskott-Aldrich syndrome protein.

The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible.

The herpesvirus saimiri tip484 and tip488 proteins both stimulate lck tyrosine protein kinase activity in vivo and in vitro.

Herpesvirus saimiri (HVS) of subgroup C efficiently induces leukemia in New World primates and transforms human lymphocytes. The viral tyrosine kinase interacting protein (Tip) binds to the tyrosine protein kinase Lck and is essential for transformation. Understanding how Tip modulates Lck activity is important for elucidating the mechanism of herpesvirus saimiri leukemogenesis. However, there are reports suggesting that whereas the Tip protein of HVS strain 484 stimulates the activity of Lck, the Tip protein of HVS strain 488 inhibits Lck. To determine whether these two divergent Tip proteins have opposite effects on Lck activity, we compared them in parallel. We found that both Tip proteins stimulated Lck kinase activity in vivo and in vitro and that both stimulated NF-AT- and STAT3-dependent transcription in T cells. Our data support the model that HVS infection increases the activity of Lck through the action of Tip.