Taking up about a third of cellular proteins, oligomeric proteins may confer several advantages, such as functional control, allosteric regulation, and establishment of higher order complexity (Ali et al., Bioorg Med Chem 2005;13:5013–5020). A number of techniques, including biophysical and fluorescent methods, can be used to detect in vitro/in vivo protein association, and/or provide affinity and stoichiometry determination of the interaction (for example, see Gell et al., Adv Exp Med Biol 2012;747:19–41). Among these different approaches, fluorescence polarization, for instance, not only can measure association constants, but is also high-throughput amenable for potential screening of small molecular modulators of protein stability.
In their article, Gray and co-workers* report an alternative plate based assay to study regulation of protein self-assembly and its effect on protein interactions. An emerging cancer drug target, pro-oncogenic protein anterior gradient-2 (AGR2), was applied as a model protein for this study due to the existence of a previously reported dimeric species. Using a fluorescently conjugated monoclonal antibody (MAB) and a far infrared emission as the readout, the assay design was based on the MAB recognition of separate epitopes that occurred only when AGR2 was in an oligomeric format (see Figure 1).
The assay, termed 2SMTA, was further demonstrated as being capable of quantitative measurement of protein oligomerization. More interestingly, the AGR2 protein contains an intrinsically disordered N-terminal region, and testing (de)stabilization effects induced by small molecule modulators or self-peptides generated based on dimer interface or this intrinsically disordered region would be a relevant next step. The authors subsequently demonstrated the assay's ability to differentiate such self-peptides into categories that either acted as stabilizers or disruptors of protein oligomer equilibrium.
A third relevant utility of the assay was to examine oligomerization effect on protein biochemical activity, such as interaction with other protein substrates. Between two well-characterized AGR2 substrates, a pentapeptide motif and Reptin, no substantial binding activity was observed among wild-type and mutant AGR2 for the former client, while both stabilization and attenuation effects were recognized for the latter (Figure 2). Overall, this work presents a high-throughput friendly assay that can be used to quantitatively measure protein oligomerization, screen for protein stability modulators, and further correlate protein subunit structure with its biochemical activity.
*Abstract from Protein Science 2013 Jun 18 [Epub ahead of print]; DOI: 10.1002/pro.2299
Many regulatory proteins are homo-oligomeric and designing assays that measure self-assembly will provide novel approaches to study protein allostery and screen for novel small molecule modulators of protein interactions. We present an assay to begin to define the biochemical determinants that regulate dimerization of the cancer-associated oncoprotein AGR2.
A two site-sandwich microtiter assay (2SMTA) was designed using a DyLight800-labeled monoclonal antibody that binds to an epitope in AGR2 to screen for synthetic self-peptides that might regulate dimer stability. Peptides derived from the intrinsically disordered N-terminal region of AGR2 increase in trans oligomer stability as defined using the 2SMTA assay. A DSS crosslinking assay that traps the AGR2 dimer through K95-K95 adducts confirmed that Δ45-AGR2 was a more stable dimer using denaturing gel electrophoresis. A titration of wt-AGR2, Δ45-AGR2 (a more stable dimer), and monomeric AGR2E60A revealed that Δ45-AGR2 was more active in binding to Reptin than either wt-AGR2 or the AGR2E60A mutant.
Our data have defined a functional role for the AGR2 dimer in the binding to its most well characterized interacting protein, Reptin. The ability to regulate AGR2 oligomerization in trans opens the possibility for developing small molecules that regulate its biochemical activity as potential cancer therapeutics. The data also highlight the utility of this oligomerization assay to screen chemical libraries for ligands that could regulate AGR2 dimer stability and its oncogenic potential.