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BMC Chem Biol. 2012; 12: 1.
Published online Mar 15, 2012. doi:  10.1186/1472-6769-12-1
PMCID: PMC3328272
HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck
Teodora Pene-Dumitrescu,1 Sherry T Shu,1 Thomas E Wales,2 John J Alvarado,3 Haibin Shi,3 Purushottam Narute,1 Jamie A Moroco,1 Joanne I Yeh,3 John R Engen,2 and Thomas E Smithgallcorresponding author1
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219, USA
2Department of Chemistry and Chemical Biology and Barnett Institute of Chemical & Biological Analysis, Northeastern University, Boston, MA 02115, USA
3Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA
corresponding authorCorresponding author.
Teodora Pene-Dumitrescu: Teodora.Dumitrescu/at/quintiles.com; Sherry T Shu: shu1027/at/gmail.com; Thomas E Wales: T.Wales/at/neu.edu; John J Alvarado: jja24/at/pitt.edu; Haibin Shi: has52/at/pitt.edu; Purushottam Narute: pun1/at/pitt.edu; Jamie A Moroco: jam177/at/pitt.edu; Joanne I Yeh: jiyeh/at/pitt.edu; John R Engen: J.Engen/at/neu.edu; Thomas E Smithgall: tsmithga/at/pitt.edu
Received September 23, 2011; Accepted March 15, 2012.
Abstract
Background
Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association.
Results
To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the Km for ATP as well as enhanced inhibitor potency.
Conclusions
These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting.
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