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Axon loss underlies symptom onset and progression in many neurodegenerative disorders. Axon degeneration in injury and disease is promoted by activation of the nicotinamide adenine dinucleotide (NAD)-consuming enzyme SARM1 (sterile alpha and TIR motif-containing protein 1). Here, we report vacor mononucleotide (VMN), a metabolite of the pesticide and neurotoxin vacor, as the most potent yet SARM1 activator. Removal of SARM1 shows complete rescue from vacor-induced neuron and axon death in vitro and in vivo. We present the crystal structure of VMN bound to the Drosophila SARM1 regulatory armadillo-repeat domain, thus facilitating drug development to prevent SARM1 activation in human disease. This study indicates the likely mechanism of action of vacor as a pesticide and lethal neurotoxin in humans, provides important new tools for drug discovery, and further demonstrates that SARM1 removal can permanently block programmed axon death specifically induced by toxicity as well as genetic mutation.

The obligate intracellular pathogen Chlamydia trachomatis is a globally significant cause of sexually transmitted bacterial infections and the leading etiological agent of preventable blindness. The first-row transition metal iron (Fe) plays critical roles in chlamydial cell biology, and acquisition of this nutrient is essential for the survival and virulence of the pathogen. Nevertheless, how C. trachomatis acquires Fe from host cells is not well understood, since it lacks genes encoding known siderophore biosynthetic pathways, receptors for host Fe storage proteins, and the Fe acquisition machinery common to many bacteria. Recent studies have suggested that C. trachomatis directly acquires host Fe via the ATP-binding cassette permease YtgABCD. Here, we characterized YtgA, the periplasmic solute binding protein component of the transport pathway, which has been implicated in scavenging Fe(III) ions. The structure of Fe(III)-bound YtgA was determined at 2.0-A resolution with the bound ion coordinated via a novel geometry (3 Ns, 2 Os [3N2O]). This unusual coordination suggested a highly plastic metal binding site in YtgA capable of interacting with other cations. Biochemical analyses showed that the metal binding site of YtgA was not restricted to interaction with only Fe(III) ions but could bind all transition metal ions examined. However, only Mn(II), Fe(II), and Ni(II) ions bound reversibly to YtgA, with Fe being the most abundant cellular transition metal in C. trachomatis Collectively, these findings show that YtgA is the metal-recruiting component of the YtgABCD permease and is most likely involved in the acquisition of Fe(II) and Mn(II) from host cells.IMPORTANCE Chlamydia trachomatis is the most common bacterial sexually transmitted infection in developed countries, with an estimated global prevalence of 4.2% in the 15- to 49-year age group. Although infection is asymptomatic in more than 80% of infected women, about 10% of cases result in serious disease. Infection by C. trachomatis is dependent on the ability to acquire essential nutrients, such as the transition metal iron, from host cells. In this study, we show that iron is the most abundant transition metal in C. trachomatis and report the structural and biochemical properties of the iron-recruiting protein YtgA. Knowledge of the high-resolution structure of YtgA will provide a platform for future structure-based antimicrobial design approaches.

The bacterium Streptococcus pneumoniae (the pneumococcus) is a major human pathogen that requires Zn2+ for its survival and virulence in the host environment. Polyhistidine triad protein D (PhtD) has a known role in pneumococcal Zn2+ homeostasis. However, the mechanistic basis of PhtD function remains unclear, partly due to a lack of structural information. Here, we determined the crystal structure of the fragment PhtD269-339 , containing the third Zn2+ -binding histidine triad (HT) motif of the protein. Analysis of the structure suggests that Zn2+ binding occurs at the surface of the protein and that all five HT motifs in the protein bind Zn2+ and share similar structures. These new structural insights aid in our understanding of how the Pht proteins facilitate pneumococcal Zn2+ acquisition.

Axon degeneration is a central pathological feature of many neurodegenerative diseases. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+)-cleaving enzyme whose activation triggers axon destruction. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD+, activates SARM1 via an unknown mechanism. Using structural, biochemical, biophysical, and cellular assays, we demonstrate that SARM1 is activated by an increase in the ratio of NMN to NAD+ and show that both metabolites compete for binding to the auto-inhibitory N-terminal armadillo repeat (ARM) domain of SARM1. We report structures of the SARM1 ARM domain bound to NMN and of the homo-octameric SARM1 complex in the absence of ligands. We show that NMN influences the structure of SARM1 and demonstrate via mutagenesis that NMN binding is required for injury-induced SARM1 activation and axon destruction. Hence, SARM1 is a metabolic sensor responding to an increased NMN/NAD+ ratio by cleaving residual NAD+, thereby inducing feedforward metabolic catastrophe and axonal demise.

Streptococcus pneumoniae is a leading cause of life-threatening bacterial infections, especially in young children in developing countries. Pneumococcal infections can be treated with β-lactam antibiotics, but rapid emergence of multidrug-resistant strains of S. pneumoniae over the past two decades has emphasized the need to identify novel drug targets. Pneumococcal surface antigen A (PsaA) is one such target, found on the cell surface of S. pneumoniae. It functions as a high-affinity substrate-binding protein, facilitating acquisition of Mn(2+), which has an important role in protecting S. pneumoniae from reactive oxygen species and, hence, oxidative stress. Consequently, PsaA is essential for bacterial survival and an important virulence factor, which makes it a promising target for antibiotic drug development. To design novel PsaA inhibitors, we used a combination of de novo fragment-based drug discovery and in silico virtual screening methods. We profiled a collection of low molecular weight compounds that were selected based on their structural diversity and ability to bind to apo-PsaA in a virtual docking experiment. The screening resulted in two initial hits that were further optimized by structural variation to improve their potency while maintaining their ligand efficiency and favorable physicochemical properties. The optimized hits were validated using a cell-based assay and molecular dynamics simulations. We found that virtual screening substantially augmented fragment-based drug design approaches, leading to the identification of novel pneumococcal PsaA inhibitors.

Cadmium is a transition metal ion that is highly toxic in biological systems. Although relatively rare in the Earth’s crust, anthropogenic release of cadmium since industrialization has increased biogeochemical cycling and the abundance of the ion in the biosphere. Despite this, the molecular basis of its toxicity remains unclear. Here we combine metal-accumulation assays, high-resolution structural data and biochemical analyses to show that cadmium toxicity, in Streptococcus pneumoniae, occurs via perturbation of first row transition metal ion homeostasis. We show that cadmium uptake reduces the millimolar cellular accumulation of manganese and zinc, and thereby increases sensitivity to oxidative stress. Despite this, high cellular concentrations of cadmium (~17 mM) are tolerated, with negligible impact on growth or sensitivity to oxidative stress, when manganese and glutathione are abundant. Collectively, this work provides insight into the molecular basis of cadmium toxicity in prokaryotes, and the connection between cadmium accumulation and oxidative stress., The molecular basis for the high toxicity of cadmium is unclear. Here, Begg et al. use the bacterium Streptococcus pneumoniae as a model system, and show that cadmium uptake increases sensitivity to oxidative stress by reducing intracellular concentrations of manganese and zinc through different mechanisms.