Exploring the Allosteric Territory of Protein Function

Abstract

While the pervasiveness of allostery in proteins is commonly accepted, we further show the generic nature of allosteric mechanisms by analyzing here transmembrane ion-channel viroporin 3a and RNA-dependent RNA polymerase (RdRp) from SARS-CoV-2 along with metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and fumarate hydratase (FH) implicated in cancers. Using the previously developed structure-based statistical mechanical model of allostery (SBSMMA), we share our experience in analyzing the allosteric signaling, predicting latent allosteric sites, inducing and tuning targeted allosteric response, and exploring the allosteric effects of mutations. This, yet incomplete list of phenomenology, forms a complex and unique allosteric territory of protein function, which should be thoroughly explored. We propose a generic computational framework, which not only allows one to obtain a comprehensive allosteric control over proteins but also provides an opportunity to approach the fragment-based design of allosteric effectors and drug candidates. The advantages of allosteric drugs over traditional orthosteric compounds, complemented by the emerging role of the allosteric effects of mutations in the expansion of the cancer mutational landscape and in the increased mutability of viral proteins, leave no choice besides further extensive studies of allosteric mechanisms and their biomedical implications.

Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19

“…An in silico docking report suggested that RIFs, FDX, and other small molecules including a prodrug form of RDV-TP and ivermectin, may bind to SARS-CoV-2 nsp12 [26] at a site that could be involved in allosteric signaling [24]. This hypothetical hotspot lies at the interface between the NiRAN and RdRp domains (Figure 4A), and our results support the existence of allosteric communications between the two nsp12 active sites [23].…”

“…For orthosteric ligands that bind to an active site the relevance is clear, but for allosteric ligands the effect cannot be predicted from a simple free energy of binding [82]. The available data argue that SARS-CoV-2 RdRp [23,24,42] and ExoN [83] are subject to allosteric control. Furthermore, while virtual screening is necessary to evaluate millions of compounds as potential novel antivirals, its value for drug repurposing now appears questionable.…”

“…Plasmids used in this study are listed in Table S1. Expression vectors for wild-type SARS-CoV-2 nsp7/8/9/12 proteins were described previously [24], and the nsp12 mutant plasmids were constructed by standard molecular biology approaches with restriction and modification enzymes from New England Biolabs (Ipswich, MA, USA), taking advantage of the existing or silent restriction sites engineered into the nsp12 coding sequence. DNA oligonucleotides for vector construction and sequencing were obtained from Millipore Sigma (Burlington, VT, USA).…”

“…The expression and purification of nsp7/8/9 were performed as described previously [24]. Nsp12 variants were overexpressed in E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany, Cat#69450).…”

“…Both nsp12 active sites are essential for viral growth [19] and targeting either site is expected to inhibit SARS-CoV-2. Fourth, computational and functional studies suggest that, like multi-subunit bacterial RNAPs, SARS-CoV-2 RdRp may be controlled allosterically [17,23] and several hypothetical allosteric pockets have been identified in nsp12 [24][25][26]. Finally, the transcribing RdRp associates with other nsps to form a large replication-transcription complex [16], which presents multiple hypothetical targets for small-molecule inhibitors.…”