SH3 Binding Project
SH3 domains are the most common protein interaction domain in animals and are found across all forms of life. There are over 400 SH3 domain family members in humans which are involved in diverse cellular processes, such as signal transduction and cytoskeleton regulation, yet it is unknown how these repeated domains encode specific information for each family. The SH3 domain, and other interaction domains, interact with long flexible peptides (>12 residues) that are intrinsically disordered, meaning they do not behave like folded proteins. Most SH3 domains contain a highly conserved shallow hydrophobic surface that interacts with a proline region in their target peptides. Therefore, it is highly likely that many of these SH3 domains share some common binding features, but little is known about the molecular basis of these extended interactions.
In this project, we are exploring how the intrinsically disordered protein, ArkA, interacts and binds to the SH3 domain, Abp1, through a multi-step process. We use molecular dynamic simulations and advanced sampling techniques to simulate each stage of the binding process, from the disordered unbound ArkA to the fully bound ArkA-Abp1 complex. We also compare our simulation data to experimental data collected from collaborators in the Stollar lab at Eastern New Mexico University.
HIV virion infectivity factor (Vif) is a viral protein that defeats host defenses by ubiquitinating antiviral proteins, which causes the host cell to degrade these protective proteins, making it easier for the virus to take over and replicate. To ubiquitinate the antiviral proteins, the virus must hijack the host cell’s own ubiquitination machinery, repurposing it to add a ubiquitin tag to a protective protein rather than the normal ubiquitination target. Vif mimics host cellular substrate receptors, binding the host E3 ligase ubiquitination complex, consisting of the scaffold protein cullin5 (Cul5), RBX2, and the substrate adaptors elongin B (EloB) and elongin C (EloC). Recently, it was found that Vif also binds the co-transcription core binding factor subunit beta (CBF β) in the early stage of complex formation. The four proteins, Vif/EloB/EloC/CBF β, form the basic HIV hijacked ubiquitination complex before going on to bind Cul5 and the antiviral proteins that HIV degrades. Protein exchange factors can then accelerate the dissociation of the cullin scaffold protein (Cul5) from the substrate receptor (Vif) allowing the cullin to be used by other substrate receptors. This means that the Vif/EloB/EloC/CBF β complex is likely recycled as well. The main targets of ubiquitination by Vif that are currently known are the apolipoprotein B mRNA editing enzymes, catalytic polypeptide like (APOBEC), particularly APOBEC3F and APOBEC3G. The APOBEC proteins destroy viral RNA, and they would restrict the replication of HIV if Vif were not present. Understanding how Vif recruits and interacts with the ubiquitination complex is crucial for developing therapeutics to prevent this interaction.
In this project, we are studying the conformational ensemble of the Vif-host protein complex using molecular dynamics simulations. Using simulations we can compare the dynamics and alternate states occupied by the complex when different proteins are removed or added to the complex, or when the sequence of any of the proteins are modified. Through this computational method we hope to improve understanding of how the intrinsically disordered protein Vif mediates flexibility of the entire complex that may be important for function. This project is a collaboration with experimentalists in the Gross lab at the University of California San Francisco.