Across all families of enzymes, only a dozen or so distinct classes of non-natural small molecule activators have been characterized, with only four known modes of activation among them. All of these modes of activation rely on naturally evolved binding sites that trigger global conformational changes. Among the enzymes that are of greatest interest for small molecule activation are the seven sirtuin enzymes, nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play a central role in the regulation of healthspan and lifespan in organisms ranging from yeast to mammals. However, there is currently no understanding of how to design sirtuin-activating compounds beyond allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates. Here, we introduce a general mode of sirtuin activation that is distinct from the known modes of enzyme activation. Based on the conserved mechanism of sirtuin-catalyzed deacylation reactions, we establish biophysical properties of small molecule modulators that can in principle result in enzyme activation for diverse sirtuins and substrates. Building upon this framework, we propose strategies for the identification, characterization and evolution of hits for mechanism-based enzyme activating compounds.

Fn1 fibrils have long been viewed as continuous fibers composed of extended, periodically aligned Fn1 molecules. However, our live imaging and single-molecule localization microscopy (SMLM) are inconsistent with this traditional view and show Fn1 fibrils composed of roughly spherical nanodomains containing 6-11 Fn1 dimers. As they move toward the cell center, Fn1 nanodomains become organized into linear arrays, wherein nanodomains are spaced at the average periodicity of 105±17 nm. Periodical Fn1 nanodomain arrays can be visualized between cells in culture and within tissues; they are resistant to deoxycholate treatment and retain nanodomain periodicity in the absence of cells. The nanodomain periodicity in fibrils remained constant when probed with antibodies recognizing distinct Fn1 epitopes or combinations of antibodies recognizing epitopes spanning the length of Fn1. FUD, a peptide that binds Fn1 N-terminus and disrupts Fn1 fibrillogenesis, blocks the organization of Fn1 nanodomains into periodical arrays. These studies establish a new paradigm of Fn1 fibrillogenesis.

Mammalian sirtuins (SIRT1-SIRT7) are a family of nicotinamide adenine dinucleotide (NAD + )-dependent protein deacylases that play critical roles in lifespan and age-related diseases.The physiological importance of sirtuins has stimulated intense interest in designing sirtuinactivating compounds. However, except for allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates, a general framework for the design of sirtuin-activating compounds has been lacking. Recently, we introduced a general mode of sirtuin activation that is distinct from the known modes of enzyme activation, establishing biophysical properties of small molecule modulators that can, in principle, result in enzyme activation for various sirtuins and substrates. Here, we characterize small molecules reported in the literature to activate the SIRT3 enzyme, using a variety of computational, biochemical and biophysical techniques including protein-ligand docking, molecular dynamics simulation, numerical reaction dynamics simulation, kinetic assays and thermodynamic assays with multiple substrates and protocols. In particular, we identify the mechanism of action of the compound honokiol on the human SIRT3 enzyme, modeling its effect on active site conformational degrees of freedom and demonstrating how it nonallosterically activates the human SIRT3 enzyme under physiologically relevant conditions. We show that honokiol constitutes a hit compound for the design of a new generation of nonallosteric activators that can activate SIRT3 through the proposed mechanism-based mode of activation. 4 The search for pharmacological agents that activate sirtuins has become a focus for many anti-aging studies due to sirtuins' lifespan extending effects in mammals [8,9]. Almost all reported sirtuin activators target SIRT1 through allosteric activation [9][10][11][12][13]. These allosteric activators reduce the dissociation constant of the substrate (e.g. the acylated protein dissociation constant , Pr d Ac K  in the case of sirtuins) to activate the enzyme. Allosteric activators of SIRT1 bind outside of the active site to an allosteric domain that is not shared by SIRT2-7 [11]. Moreover, allosteric activators only work with a limited set of SIRT1 substrates [14-18]. Allosteric activation is one of four known modes by which small molecules can activate enzymes [10]. Aside from allosteric activation, enhancement of enzymatic activity via "derepression of inhibition" has been explored theoretically and experimentally. Theoretical models proposed have been limited to inhibitors that are exogenous to the reaction, and experimental studies have considered alleviation of product inhibition through competition with product binding [2]. These approaches can only enhance enzyme activity in the presence of inhibitor or product accumulation and hence are not included among the four known modes of enzyme activation.Small molecules that can activate sirtuins through modes of action other than the reduction of substrate Kd are of particular int...

DNA photolyase can be used to study how a protein with its required cofactor has adapted over a large temperature range. The enzymatic activity and thermodynamics of substrate binding for protein from Sulfolobus solfataricus were directly compared to protein from Escherichia coli. Turnover numbers and catalytic activity were virtually identical, but organic cosolvents may be necessary to maintain activity of the thermophilic protein at higher temperatures. UV-damaged DNA binding to the thermophilic protein is less favorable by ∼2 kJ/mol. The enthalpy of binding is ∼10 kJ/mol less exothermic for the thermophile, but the amount and type of surface area buried upon DNA binding appears to be somewhat similar. The most important finding was observed when ionic strength studies were used to separate binding interactions into electrostatic and nonelectrostatic contributions; DNA binding to the thermophilic protein appears to lack the electrostatic contributions observed with the mesophilic protein.

Fibronectin (Fn1) is an essential ECM glycoprotein important for embryonic development and homeostasis. The functions of Fn1 in regulating cell fate decisions, morphogenesis and cellular responses to injury are intimately linked to the process of Fn1 fibrillogenesis. Therefore, understanding the mechanisms by which Fn1 proteins assemble into fibrils is necessary to gain insights into diverse functions of Fn1. Using CRISPR/Cas9 mutagenesis, we generated mice and cell lines wherein a sequence encoding a fluorescent protein (FP) was knocked into the Fn1 locus replacing the termination codon, resulting in the expression of Fn1-FP proteins subject to endogenous regulation. Live imaging and super-resolution microscopy revealed that Fn1 fibrils are not continuous fibers as was thought before, instead, they are comprised of a discontinuous array of small nanodomains. Live imaging showed that Fn1 nanodomains are mobile and that they become arranged into progressively longer linear arrays as they move toward the nucleus in parallel with the rearward actin flow. The organization of Fn1 nanodomains into linear fibrillar arrays but not the formation of Fn1 nanodomains is regulated by the interactions mediated by the Fn1 N-terminal assembly domain. The nanodomain architecture of Fn1 fibrils is observed in multiple contexts: in three-dimensional ECM in vivo, on substrata of different composition and stiffness, and is retained when the linkage of Fn1 fibrils to cells is disrupted. The modular assembly and structure of Fn1 fibrils bears important implications for mechanisms of ECM remodeling and signal transduction.