Subtle Dynamic Changes Accompany Hck Activation by HIV-1 Nef and are Reversed by an Antiretroviral Kinase Inhibitor

Abstract

The HIV-1 virulence factor Nef interacts with the macrophage Src-family kinase Hck, resulting in constitutive kinase activation that contributes to viral replication and immune escape. Previous chemical library screens identified the diphenylfuranopyrimdine kinase inhibitor DFP-4AB, which selectively inhibits Nef-dependent Hck activity in biochemical assays and potently blocks HIV replication in vitro. In the present study, hydrogen exchange mass spectrometry (HX MS) was used to study conformational changes in downregulated Hck that result from Nef binding, as well as the impact of DFP-4AB on these changes. Remarkably, interaction with Nef induced only subtle changes in deuterium uptake by Hck, with the most significant changes in the N-lobe of the kinase domain adjacent to the docking site for Nef on the SH3 domain. No changes in hydrogen exchange were observed in the Hck SH2 domain or C-terminal tail, indicating that this regulatory interaction is unaffected by Nef binding. When HX MS was performed in the presence of DFP-4AB, the effect of Nef on Hck N-lobe dynamics was completely reversed. These results show that constitutive activation of Hck by HIV-1 Nef requires only modest changes to the conformational dynamics of the overall kinase structure. DFP-4AB reverses these effects, consistent with its activity against this Nef-induced signaling event in HIV-infected cells.

The Src family kinase Fgr is a transforming oncoprotein that functions independently of SH3-SH2 domain regulation

“…To test this possibility, HX MS was used to compare the rates of deuterium uptake by the Fgr and Src SH3-SH2 dual domains alone and in the context of near-full-length kinases that also include the linker, kinase domain, and phosphorylated tail. The SH3 and SH2 domains of both Fgr and Src were protected from deuterium uptake in the context of the near-full-length kinases, as reported previously for inactive Hck (40). This result suggests that the Fgr core region adopts an assembled conformation similar to inactive Src and Hck, although these intramolecular contacts do not appear to regulate Fgr kinase domain activity.…”

Structure, function, and inhibitor targeting of HIV-1 Nef-effector kinase complexes

“…While these compounds work directly via the Hck active site, they showed enhanced potency for Hck inhibition in the presence of Nef, suggesting that Nef binding may allosterically impact the Hck active site to enhance inhibitor binding. This conclusion is supported by subsequent HX MS studies of the Nef•Hck complex in the presence of a DFP-based compound with antiretroviral activity (74). DFP-based Hck inhibitors also blocked Nef-mediated enhancement of viral replication across a wide range of Nef subtypes, providing additional evidence that Src-family kinase signaling is important to viral replication (101).…”

“…HX MS is particularly powerful when used to compare two states of a given protein -for example, Hck in the presence and absence of Nef. Wales et al performed this comparison using recombinant near-full-length Hck (SH3-SH2-kinase-tail) and full-length Nef (74). The crystal structure of the identical near-full-length Hck protein has been reported (75) and shows that it adopts an assembled, inactive conformation driven by two primary intramolecular interactions: the SH3 domain binds to the SH2-kinase linker (which adopts a PPII helical conformation) while the SH2 domain engages the tyrosine-phosphorylated tail.…”

“…Disruption of either interaction by mutagenesis leads to activation of Hck in cells (76), raising the question of whether Nef selectively disrupts SH3-mediated regulation of Hck or causes a more global disruption of the downregulated state. Comparison HX MS experiments (74) of Hck alone (where the SH2-kinase linker is bound to the SH3 domain as in downregulated Hck) vs. Hck in complex with Nef (where the Nef PxxPxR motif is bound to the SH3 domain as required for Hck activation) demonstrated remarkably few changes to the rest of Hck upon Nef interaction. Very subtle changes in deuterium uptake by Hck were found in only a single peptic peptide that encompasses the C-terminal end of the SH2-kinase linker where it joins the N-lobe of the kinase domain.…”

Selective Proteolysis to Study the Global Conformation and Regulatory Mechanisms of c-Src Kinase

“…Both hypotheses are consistent with increased linker accessibility and faster thermolysin cleavage rate. The latter hypothesis is supported by Smithgall and Engen who have shown that activation of Hck (a Src family kinase) by HIV-1 Nef occurs via SH3 disengagement while the kinase tail remains bound to the SH2 domain of Hck [44]. Thus, further studies are required to elucidate the full mechanism for W121R's ability to overcome Csk inactivation.…”