John R. Engen
Thomas Smithgall, Max Diem, Penny J. Beuning, Paul Vouros
Date of Award
Doctor of Philosophy
Department or Academic Unit
College of Science. Department of Chemistry and Chemical Biology.
chemistry, biochemistry, biophysics, APOBEC3G, Elongin BC, hydrogen exchange, mass spectrometry, protein conformation, Vif
HIV (Viruses), Proteins - Conformation
Amino Acids, Peptides, and Proteins | Biochemistry
Every twelve seconds another person contracts the HIV Virus and every sixteen seconds another person dies from AIDS (UNAIDS, 2010). Despite current treatments for the HIV virus, the AIDS pandemic is still a serious threat to human health. Currently, there are large gaps in our biophysical understanding of the processes that underlie HIV infectivity and the viral components responsible for promoting the disease.
One protein that is essential for the productive infectivity of HIV is Vif (Viral Infectivity Factor). Vif, one of the HIV accessory proteins, acts to promote infection by serving as an adaptor molecule which links the potent cellular antiviral APOBEC3G enzyme with the cellular ubiquitin tagging machinery (ubiquitin ligase) that is necessary for protein degradation (Chiu, 2008). Despite the importance of Vif in HIV biology, the structure and conformation of Vif is unknown and the interactions between Vif, APOBEC3G, and ubiquitin ligase are poorly understood. Due to the instability of Vif at concentrations required for traditional biophysical analysis, it is not amenable to X-ray crystallography or NMR spectroscopy. Any biophysical information that could be obtained for Vif either alone or interacting with its cellular partners would be an invaluable and profound contribution to the field of virology.
Hydrogen Exchange monitored by Mass Spectrometry (HX MS) was employed to analyze the conformation and dynamics of recombinant HIV-1 Vif. HX MS analysis of Vif alone in solution revealed that the N-terminal portion of the molecule that contains the APOBEC3 binding region was protected from amide exchange and likely contained the majority of structural elements present in Vif. The C-terminal portion of Vif, which is responsible for association with the ubiquitin ligase machinery, was easily deuterated and was likely unfolded and solvent exposed in solution. Vif conformation was also analyzed in the absence and presence of the co-factor Zn+2 which is responsible for mediating the interaction with the ubiquitin ligase component Cullin 5. The region responsible for zinc binding was found to undergo conformational exchange in solution in the absence of zinc which could have resulted from conformational transitions of the Vif monomer or an effect of Vif oligomerization. Upon zinc binding, the conformational exchange was slowed suggesting zinc coordination stabilized Vif.
The conformational consequences of the interaction between Vif and the ubiquitin ligase components Elongin BC were then studied. HX MS revealed that conformational changes occurred in Elongin B and C upon Vif binding. The region in Vif responsible for Elongin BC association, the Vif BC box, was unfolded in the absence of the Elongin BC complex and folded upon incubation. HX MS was also used to probe the affinity of a series of Vif variants for the Elongin BC complex and revealed that removal of the Vif BC box abolished Elongin BC binding.
The information obtained from HX MS analysis of the Vif:Elongin BC interaction was then utilized in the design of hydrocarbon stapled Vif peptide inhibitors targeting the Vif:Elongin BC axis. Peptides were produced using sold phase synthesis, hydrocarbon stapled, and purified successfully. HX MS analysis indicated that hydrocarbon stapling stabilized the peptide into an alpha helical conformation and that stapling did not interfere with Elongin BC binding. Vif peptides were capable of inhibiting the formation of Vif:Elongin BC complex and displayed anti HIV activity.
Lastly, the work with Vif peptides and the Elongin BC complex was employed in the development of a fluorous based flow HX platform using the Vif:Elongin BC complex as a model system. A Vif peptide capable of binding Elongin BC was fluorous tagged and immobilized onto a fluorous surface. The fluorous system was capable of capturing Elongin BC from a purified mixture and also from an E. coli lysate. Flow HX was conducted on captured Elongin BC and compared to solution HX experiments which yielded similar results illustrating the feasibility of using fluorous chemistry in HX applications.
Sean R. Marcsisin
Marcsisin, Sean R., "Towards a more complete conformational understanding of the HIV-1 viral infectivity factor" (2011). Chemistry Dissertations. Paper 35. http://hdl.handle.net/2047/d20002111
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