The cardiac troponin (cTn) complex is an important regulatory protein in heart contraction. Upon binding of Ca2+, cTn undergoes a conformational shift that allows the troponin I switch peptide (cTnISP) to be released from the actin filament and bind to the troponin C hydrophobic patch (cTnCHP). Mutations and modifications to this complex can change its sensitivity to Ca2+ and alter the energetics of the transition from the Ca2+-unbound, cTnISP-unbound form to the Ca2+-bound, cTnISP-bound form. We utilized targeted molecular dynamics (TMD) to obtain a trajectory of this transition pathway, followed by umbrella sampling to estimate the free energy associated with the cTnISP–cTnCHP binding and the cTnCHP opening events for wild-type (WT) cTn. We were able to reproduce experimental values for the cTnISP–cTnCHP binding event and obtain cTnCHP opening free energies in agreement with previous computational measurements of smaller cTnC systems. This excellent agreement for WT cTn demonstrated the strength of computational methods in studying the dynamics and energetics of the cTn complex. We then introduced mutations to the cTn complex that cause cardiomyopathy or alter its Ca2+ sensitivity and observed a general decrease in the free energy of opening the cTnCHP. For these same mutations, we observed no general trend in the effect on the cTnISP–cTnCHP binding event. Our method sets the stage for future computational studies on this system that predict the consequences of yet uncharacterized mutations on cTn dynamics and energetics.

Ca 2+ binding to cardiac troponin C (cTnC) causes a conformational shift that exposes a hydrophobic patch (cTnC HP ) for binding of the cTnI switch peptide (cTnI SP ), ultimately resulting in contraction of the heart. The inhibitory peptide (cTnI IP ), attached at the N-terminal end of the cTnI SP , serves as a tether for the cTnI SP to the rest of the troponin complex. Due to this tethered nature, the cTnI SP remains within proximity of the hydrophobic patch region, resulting in the cTnC HP experiencing an elevated "effective concentration" of the cTnI SP . Mutations to the cTnI IP region have been hypothesized to cause disease by affecting the ability of the cTnI SP to "find" the hydrophobic patch, resulting in alterations to the heart's ability to contract normally. We tested this hypothesis using molecular dynamics (MD) simulations of the troponin complex using a model that contained all three subunits of troponin: C, I, and T. We developed methods that allowed us to quantitatively measure the effective concentration of the cTnI SP from the simulations. A significant reduction in the cTnI SP effective concentration was observed when the cTnI IP was removed from the system, showcasing the importance of a tethered cTnI SP . Through accelerated MD methods, we proposed the minimum effective concentration of a tethered cTnI SP to be approximately 21 mM. Modification of the cTnI IP via PKC-mediated phosphorylation of T143 was shown to significantly increase the estimated effective concentration of cTnI SP , help the cTnI SP find the cTnC HP more effectively, and maintain the relative shape of the cTnI IP when compared to the native model. All of these data indicate that pT143 may be able to help promote binding of cTnI SP to the cTnC HP . We then tested six mutations within the cTnI IP region that are known cTnC Ca 2+ -sensitizing mutations and have been linked with cardiomyopathy. We did not observe a significant reduction in the effective concentration upon the introduction of these mutations; however, we did observe increased variability in the flexibility and dynamics of the cTnI IP region when compared to native. Our observations led us to hypothesize that the mechanism by which these cardiomyopathic mutations affect Ca 2+ sensitivity is by altering the off rate of cTnI SP from the hydrophobic patch.

Although salmonellosis, an infectious disease, is a significant global healthcare burden, there are no Salmonella-specific vaccines or therapeutics for humans. Motivated by our finding that FraB, a Salmonella deglycase responsible for fructose-asparagine catabolism, is a viable drug target, we initiated experimental and computational efforts to identify inhibitors of FraB. To this end, our recent high-throughput screening initiative yielded almost exclusively uncompetitive inhibitors of FraB. In parallel with this advance, we report here how a separate structural and computational biology investigation of FrlB, a FraB paralog, led to the serendipitous discovery that 2-deoxy-6-phosphogluconate is a competitive inhibitor of FraB (KI ~ 3 μM). However, this compound was ineffective in inhibiting the growth of Salmonella in a liquid culture. In addition to poor uptake, cellular metabolic transformations by a Salmonella dehydrogenase and different phosphatases likely undermined the efficacy of 2-deoxy-6-phosphogluconate in live-cell assays. These insights inform our ongoing efforts to synthesize non-hydrolyzable/-metabolizable analogs of 2-deoxy-6-phosphogluconate. We showcase our findings largely to (re)emphasize the role of serendipity and the importance of multi-pronged approaches in drug discovery.

Amadori rearrangement products are stable sugar-amino acid conjugates that are formed nonenzymatically during preparation, dehydration, and storage of foods. Because Amadori compounds such as fructose-lysine (F-Lys), an abundant constituent in processed foods, shape the animal gut microbiome, it is important to understand bacterial utilization of these fructosamines. In bacteria, F-Lys is first phosphorylated, either during or after uptake to the cytoplasm, to form 6-phosphofructose-lysine (6-P-F-Lys). FrlB, a deglycase, then converts 6-P-F-Lys to L-lysine and glucose-6-phosphate. Here, to elucidate the catalytic mechanism of this deglycase, we first obtained a 1.8-Å crystal structure of Salmonella FrlB (without substrate) and then used computational approaches to dock 6-P-F-Lys on this structure. We also took advantage of the structural similarity between FrlB and the sugar isomerase domain of Escherichia coli glucosamine-6-phosphate synthase (GlmS), a related enzyme for which a structure with substrate has been determined. An overlay of FrlB-6-P-F-Lys on GlmS-fructose-6-phosphate structures revealed parallels in their active-site arrangement and guided our selection of seven putative active-site residues in FrlB for site-directed mutagenesis. Activity assays with eight recombinant single-substitution mutants identified residues postulated to serve as the general acid and general base in the FrlB active site and indicated unexpectedly significant contributions from their proximal residues. By exploiting native mass spectrometry (MS) coupled to surface-induced dissociation, we distinguished mutations that impaired substrate binding versus cleavage. As demonstrated with FrlB, an integrated approach involving x-ray crystallography, in silico approaches, biochemical assays, and native MS can synergistically aid structure-function and mechanistic studies of enzymes.

The cardiac thin filament comprises F-actin, tropomyosin, and troponin (cTn). cTn is composed of three subunits: troponin C (cTnC), troponin I (cTnI), and troponin T (cTnT). To computationally study the effect of the thin filament on cTn activation events, we employed targeted molecular dynamics followed by umbrella sampling using a model of the thin filament to measure the thermodynamics of cTn transition events. Our simulations revealed that the thin filament causes an increase in the free energy required to open the cTnC hydrophobic patch and causes a more favorable interaction between this region and the cTnI switch peptide. Mutations to the cTn complex can lead to cardiomyopathy, a collection of diseases that present clinically with symptoms of hypertrophy or dilation of the cardiac muscle, leading to impairment of the heart's ability to function normally and ultimately myocardial infarction or heart failure. Upon introduction of cardiomyopathic mutations to R145 of cTnI, we observed a general decrease in the free energy of opening the cTnC hydrophobic patch, which is on par with previous experimental results. These mutations also exhibited a decrease in electrostatic interactions between cTnI-R145 and actin-E334. After introduction of a small molecule to the wild-type cTnI−actin interface to intentionally disrupt intersubunit contacts, we successfully observed similar thermodynamic consequences and disruptions to the same protein−protein contacts as observed with the cardiomyopathic mutations. Computational studies utilizing the cTn complex in isolation would have been unable to observe these effects, highlighting the importance of using a more physiologically relevant thin-filament model to investigate the global consequences of cardiomyopathic mutations to the cTn complex.