Biology subjects

McCormick, J. W.

Publications and source records attributed to McCormick, J. W..

5 recordsLinked to original sources

bioRxiv2026

Protein Surface Site Determines the Evolutionary Accessibility of Allosteric Regulation

Domain recombination is a major source of new allosteric regulation in both evolved and engineered proteins. However, the sequence and structural features that govern where new allostery may emerge remain poorly understood. Here, we test the hypothesis that the evolutionary accessibility of allosteric regulation following domain insertion is constrained by local surface context, specifically association with pre-existing cooperative networks known as protein sectors. We began with two synthetic domain fusions wherein the Avena sativa light-oxygen-voltage (LOV2) domain was inserted into Escherichia coli dihydrofolate reductase (DHFR) at either a sector connected or non-sector connected surface. The insertion sites are only separated by five residues and both DHFR enzymes retain similar catalytic activity, yet the sector connected version exhibits a light-dependent allosteric phenotype, while the non-sector connected version does not. Using deep mutational scanning, we measured the effect of nearly all single point mutations on allostery in each chimera. The sector-connected DL121 was significantly more evolvable, possessing numerous allostery-tuning single mutants. In contrast, DL116 lacked statistically significant mutants that introduce allosteric regulation, suggesting the protein surface used by DL116 may be an evolutionary "dead end" for a regulatory phenotype. Surprisingly, DL116 did not show cooperative unfolding at temperatures up to 80 {degrees}C, suggesting that enhanced protein stability does not promote the evolvability of allosteric regulation as it does with other phenotypes. Together, our findings show that protein surface context influences the mutational pathways available for allosteric regulation, consistent with the view that sector-connected surface sites harbor a latent capacity for allostery while other locations are more evolutionarily inert.

biophysics↗

bioRxiv2024

Local disorder is associated with enhanced catalysis in an engineered photoswitch

Domain insertion is a common strategy for introducing allosteric regulation in both engineered and evolved systems. In this approach, an "input" domain is covalently fused to an "output" domain with the goal of conferring new regulation. In prior work, we found that insertion of the LOV2 domain at evolutionarily conserved allosteric "hot spots" on the metabolic enzyme Dihydrofolate Reductase (DHFR) could confer modest light regulation of enzymatic activity. However, it was not clear if the newly established regulation was achieved by interdomain allosteric conformational coupling, or if it represented a "simpler" mechanism like coupling of LOV2 light activation to global folding stability of DHFR or light-dependent steric occlusion of the DHFR active site. To better understand how these newly formed domain fusions harness light-inducible disorder in LOV2 for allosteric activation, we biochemically characterized a representative synthetic fusion. We observed that LOV2 photoactivation simultaneously: (1) thermally destabilized the fusion and (2) lowered the DHFR catalytic transition free energy of the lit state relative to the dark state. Light-induced NMR chemical shift changes indicated that photochemically-initiated conformational changes propagated from LOV2 to the active site of DHFR. Moreover, ligand binding at DHFR modified LOV2 chemical shifts, demonstrating bidirectional coupling between domains. Examination of select allostery-tuning mutations found a modest negative correlation between the light-induced change in thermal stability and catalytic activity, suggesting an activity-stability tradeoff. Together our data indicate that a domain fusion event can realize localized conformational coupling between active sites even in the absence of extensive evolutionary optimization.

biochemistry↗

bioRxiv2024

Iterative in silico identification of P-glycoprotein inhibitors

Overexpression of the polyspecific efflux transporter, P-glycoprotein (P-gp, MDR1, ABCB1), is a major mechanism by which cancer cells acquire multidrug resistance (MDR), the resistance to diverse chemotherapeutic drugs. Inhibiting drug transport by P-gp can resensitize cancer cells to chemotherapy, but there are no P-gp inhibitors available to patients. Clinically unsuccessful P-gp inhibitors tend to bind at the pumps transmembrane drug binding domains and are often P-gp transport substrates, resulting in lowered intracellular concentration of the drug and altered pharmacokinetics. In prior work, we used computationally accelerated drug discovery to identify novel P-gp inhibitors that target the pumps cytoplasmic nucleotide binding domains. Our first-draft study provided conclusive evidence that the nucleotide binding domains of P-gp are viable targets for drug discovery. Here we develop an enhanced, computationally accelerated drug discovery pipeline that expands upon our prior work by iteratively screening compounds against multiple conformations of P-gp with molecular docking. Targeted molecular dynamics simulations with our homology model of human P-gp were used to generate docking receptors in conformations mimicking a putative drug transport cycle. We offset the increased computational complexity using custom Tanimoto chemical datasets, which maximize the chemical diversity of ligands screened by docking. Using our expanded, virtual-assisted pipeline, we identified nine novel P-gp inhibitors that reverse MDR in two types of P-gp overexpressing human cancer cell lines, reflecting a 13.4% hit rate. Of these inhibitors, all were non-toxic to non-cancerous human cells, and six were not likely to be transport substrates of P-gp. Our novel P-gp inhibitors are chemically diverse and are good candidates for lead optimization. Our results demonstrate that the nucleotide binding domains of P-gp are an underappreciated target in the effort to reverse P-gp-mediated multidrug resistance in cancer.

cancer biology↗

bioRxiv2021

Structurally Distributed Surface Sites Tune Allosteric Regulation

Our ability to rationally optimize allosteric regulation is limited by incomplete knowledge of the mutations that tune allostery. Are these mutations few or abundant, structurally localized or distributed? To examine this, we conducted saturation mutagenesis of a synthetic allosteric switch in which Dihydrofolate reductase (DHFR) is regulated by a blue-light sensitive LOV2 domain. Using a high-throughput assay wherein DHFR catalytic activity is coupled to E. coli growth, we assessed the impact of 1548 viable DHFR single mutations on allostery. Despite most mutations being deleterious to activity, fewer than 5% of mutations had a statistically significant influence on allostery. Most allostery disrupting mutations were proximal to the LOV2 insertion site. In contrast, allostery enhancing mutations were structurally distributed and enriched on the protein surface. Combining several allostery enhancing mutations yielded near-additive improvements to dynamic range. Our results indicate a path towards optimizing allosteric function through variation at surface sites.

biophysics↗

bioRxiv2020

Transport of Alzheimer's Associated Amyloid-β Catalyzed by P-glycoprotein

P-glycoprotein (P-gp) is a critical membrane transporter in the blood brain barrier (BBB) and is implicated in Alzheimers disease (AD). However, previous studies on the ability of P-gp to directly transport the Alzheimers associated amyloid-{beta} (A{beta}) protein have produced contradictory results. Here we use molecular dynamics (MD) simulations, transport substrate accumulation studies in cell culture, and biochemical activity assays to show that P-gp actively transports A{beta}. We observed transport of A{beta}40 and A{beta}42 monomers by P-gp in explicit MD simulations of a putative catalytic cycle. In in vitro assays with P-gp overexpressing cells, we observed enhanced accumulation of fluorescently labeled A{beta}42 in the presence of Tariquidar, a potent P-gp inhibitor. We also showed that A{beta}42 stimulated the ATP hydrolysis activity of isolated P-gp in nanodiscs. Our findings expand the substrate profile of P-gp, and suggest that P-gp may contribute to the onset and progression of AD.

biochemistry↗