Biology subjects

Dinan, J. C.

Publications and source records attributed to Dinan, J. C..

3 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↗

bioRxiv2026

Global Proteomics Investigation of SAMT-247 Targets: An Antiviral Thioester that Acetylates Zinc Finger Proteins

Covalent modification of target proteins is a well-established mechanism of action for small molecule inhibitors. Cysteine residues in particular have been exploited for their reactivity toward electrophilic molecules. SAMT-247 is a mercaptobenzamide thioester that covalently acetylates cysteines in the zinc-coordinating domains of the HIV nucleocapsid protein. This SAMT-247-promoted reaction leads to loss of zinc binding by the protein, with concomitant loss of protein structure and function. Although it has low cytotoxicity in animal models, recent studies have indicated that it affects other protein targets in uninfected cells, for example leading to increased immune cell functions. In this study, global proteomics approaches have been used to better understand other protein targets of SAMT-247. Minimal effects are observed when unstimulated THP-1 monocyte cells were treated with SAMT-247. In contrast, thermal proteome profiling identified 170 proteins with altered thermal stability when THP-1 cells were stimulated with phorbol 12-myristate 13-acetate/Ionomycin (PMA/Iono) before SAMT-247 treatment. Among the affected proteins, 81 contain a zinc-coordinating domain and/or have been shown to have a reactive cysteine residue. Among these, several play a role in cellular metabolism, and Seahorse assays demonstrated that SAMT-247 significantly increased the anti-metabolic and pro-glycolytic effect of PMA/Iono in THP-1 cells. Two of the most-affected proteins were ZC3H7A, a microRNA-binding protein with four zinc finger domains, and MGMT, a DNA damage repair protein with a reactive cysteine. Both proteins were modified by SAMT-247 when tested alone or in the presence of THP-1 cell lysate, indicating that they are bona fide targets of the inhibitor. The low activity of SAMT-247 in unstimulated THP-1 cells is consistent with its low cytotoxicity. The increased effects of SAMT-247 in stimulated immune cells suggests that this molecule could be developed to target diseases other than HIV.

cell biology↗

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↗