Biology subjects

Simon, M. A.

Publications and source records attributed to Simon, M. A..

5 recordsLinked to original sources

bioRxiv2026

Discovery of the Phosphonate Flavophos Produced by Burkholderia

Phosphonate natural products have proven value to society as antibiotics and herbicides. They inhibit a range of enzyme targets usually by mimicking the enzyme substrates. In this study, we investigate a family of phosphonate biosynthetic gene clusters (BGCs) found in Burkholderia. Heterologous expression in Escherichia coli resulted in production of an antimicrobial compound. Spectroscopic characterization and chemical synthesis assigned its structure as 2,4-dioxopentylphosphonic acid. One of the biosynthetic enzymes is a member of the domain of unknown function (DUF) 849 family with homology to {beta}-keto acid cleavage enzymes (BKACEs). In vitro characterization shows that this enzyme catalyzes chemistry that is divergent from previously characterized BKACEs. The observed catalytic activity is explained by a series of co-crystal structures with substrates and intermediates. The BGC also contains a gene encoding lumazine synthase (LS), an essential enzyme in flavin biosynthesis. Biochemical experiments revealed that 2,4-dioxopentylphosphonic acid inhibits LS. In addition, expression of the LS encoded in the BGC, or LS orthologs from a range of organisms, in E. coli conferred resistance to the new phosphonate, which we therefore name flavophos.

biochemistry↗

bioRxiv2023

Estimating the true stability of the prehydrolytic outward-facing state in an ABC protein

CFTR, the anion channel mutated in cystic fibrosis patients, is a model ABC protein whose ATP-driven conformational cycle is observable at single-molecule level in patch-clamp recordings. Bursts of CFTR pore openings are coupled to tight dimerization of its two nucleotide binding domains (NBDs) and in wild-type (WT) channels are mostly terminated by ATP hydrolysis. The slow rate of non-hydrolytic closure - which determines how tightly bursts and ATP hydrolysis are coupled - is unknown, as burst durations of catalytic site mutants span a range of [~]200-fold. Here we show that Walker A mutation K1250A, Walker B mutation D1370N, and catalytic glutamate mutations E1371S and E1371Q all completely disrupt ATP hydrolysis. True non-hydrolytic closing rate of WT CFTR approximates that of K1250A and E1371S. That rate is slowed [~]15-fold in E1371Q by a non-native inter-NBD H-bond, and accelerated [~]15-fold in D1370N. These findings uncover unique features of the NBD interface in human CFTR.

biophysics↗

bioRxiv2021

Molecular pathology of a cystic fibrosis mutation is explained by loss of a hydrogen bond

The phosphorylation-activated anion channel CFTR is gated by an ATP hydrolysis cycle at its two cytosolic nucleotide binding domains, and is essential for epithelial salt-water transport. A large number of CFTR mutations cause cystic fibrosis. Since recent breakthrough in targeted pharmacotherapy, CFTR mutants with impaired gating are candidates for stimulation by potentiator drugs. Thus, understanding the molecular pathology of individual mutations has become important. The relatively common R117H mutation affects an extracellular loop, but nevertheless causes a strong gating defect. Here we identify a hydrogen bond between the side chain of arginine 117 and the backbone carbonyl group of glutamate 1124 in the cryo-electronmicroscopic structure of phosphorylated, ATP-bound CFTR. We address the functional relevance of that interaction for CFTR gating using macroscopic and microscopic inside-out patch-clamp recordings. Employing thermodynamic double-mutant cycles, we systematically track gating-state dependent changes in the strength of the R117-E1124 interaction. We find that the H-bond is formed only in the open state, but neither in the short-lived "flickery" nor in the long-lived "interburst" closed state. Loss of this H-bond explains the entire gating phenotype of the R117H mutant, including robustly shortened burst durations and strongly reduced intraburst open probability. The findings may help targeted potentiator design.

biophysics↗

bioRxiv2020

Binding Profile Mapping of the S100 Protein Family Using a High-throughput Local Surface Mimetic Holdup Assay

S100 proteins are small, typically homodimeric, vertebrate-specific EF-hand proteins that establish Ca2+-dependent protein-protein interactions in the intra- and extracellular environment and are overexpressed in various pathologies. There are about 20 distinct human S100 proteins with numerous potential partner proteins. Here, we used a quantitative holdup assay to measure affinity profiles of most members of the S100 protein family against a library of chemically synthetized foldamers. The profiles allowed us to quantitatively map the binding promiscuity of each member towards the foldamer library. Since the library was designed to systematically contain most binary natural amino acid side chain combinations, the data also provide insight into the promiscuity of each S100 protein towards all potential naturally-occurring S100 partners in the human proteome. Such information will be precious for future drug design of modulators of S100 pathological activities.

biochemistry↗

bioRxiv2020

Effects of Healthy Aging on Right Ventricular Structure and Biomechanical Properties

Healthy aging has been associated with alterations in pulmonary vasculature and right ventricular (RV) hemodynamics, potentially leading to RV remodeling. Despite the current evidence suggesting an association between aging and alterations in RV function and higher prevalence of pulmonary hypertension in the elderly, limited data exist on age-related differences in RV structure and biomechanics. In this work we report our preliminary findings on the effects of healthy aging on RV structure, function, and biomechanical properties. Hemodynamic measurements, biaxial mechanical testing, constitutive modeling, and quantitative histological analysis were employed to study two groups of Sprague-Dawley rats: control (11 weeks) and aging (80 weeks). Aging was associated with increases in RV peak pressures ({approx}{uparrow}17%, p=0.017), RV contractility ({approx}{uparrow}52%, p= 0.004), and RV wall thickness ({approx}{uparrow}34%, p=0.002). Longitudinal realignment of RV collagen (16.4{degrees}, p=0.013) and myofibers (14.6{degrees}, p=0.017) were observed with aging, accompanied by transmural cardiomyocyte loss and fibrosis. A bimodal alteration in biomechanical properties was noted, resulting in increased myofiber stiffness ({approx}{uparrow}158%, p=0.0006) and decreased effective collagen fiber stiffness ({approx}{downarrow}67%, p=0.031). Our results demonstrate the potential of healthy aging to modulate RV remodeling via increased peak pressures, cardiomyocyte loss, fiber reorientation, and altered collagen/myofiber stiffness. Some similarities were observed between aging-induced remodeling patterns and those of RV remodeling in pressure overload.

bioengineering↗