Catalytic Mechanism of Bacteriophage T4 Rad50 ATP Hydrolysis

Spontaneous double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage, and their improper repair can lead to cellular dysfunction. The Mre11 and Rad50 proteins, a nuclease and an ATPase, respectively, form a well-conserved complex that is involved in the initial processing of DSBs. Here we examine the kinetic and catalytic mechanism of ATP hydrolysis by T4 Rad50 (gp46) in the presence and absence of Mre11 (gp47) and DNA. Single-turnover and pre-steady state kinetics on the wild-type protein indicate that the rate-limiting step for Rad50, the MR complex, and the MR-DNA complex is either chemistry or a conformational change prior to catalysis. Pre-steady state product release kinetics, coupled with viscosity steady state kinetics, also supports that the binding of DNA to the MR complex does not alter the rate-limiting step. The lack of a positive deuterium solvent isotope effect for the wild type and several active site mutants, combined with pH–rate profiles, implies that chemistry is rate-limiting and the ATPase mechanism proceeds via an asymmetric, dissociative-like transition state. Mutation of the Walker A/B and H-loop residues also affects the allosteric communication between Rad50 active sites, suggesting possible routes for cooperativity between the ATP active sites

Fish Assemblages, Connectivity, and Habitat Rehabilitation in a Diked Great Lakes Coastal Wetland Complex

Fish and plant assemblages in the highly modified Crane Creek coastal wetland complex of Lake Erie were sampled to characterize their spatial and seasonal patterns and to examine the implications of the hydrologic connection of diked wetland units to Lake Erie. Fyke netting captured 52 species and an abundance of fish in the Lake Erie–connected wetlands, but fewer than half of those species and much lower numbers and total masses of fish were captured in diked wetland units. Although all wetland units were immediately adjacent to Lake Erie, there were also pronounced differences in water quality and wetland vegetation between the hydrologically isolated and lake‐connected wetlands. Large seasonal variations in fish assemblage composition and biomass were observed in connected wetland units but not in disconnected units. Reestablishment of hydrologic connectivity in diked wetland units would allow coastal Lake Erie fish to use these vegetated habitats seasonally, although connectivity does appear to pose some risks, such as the expansion of invasive plants and localized reductions in water quality. Periodic isolation and drawdown of the diked units could still be used to mimic intermediate levels of disturbance and manage invasive wetland vegetation.Received October 1, 2013; accepted March 5, 2014Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/141054/1/tafs1130-sup-0001.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/141054/2/tafs1130.pd

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Aquifex aeolicus PilT, Homologue of a Surface Motility Protein, Is a Thermostable Oligomeric NTPase

Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a ≈5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed