Aaron T. Smith

Summary: The acquisition of iron is critical for the survival and the virulence of numerous infectious pathogens, and most bacteria acquire ferrous iron (Fe ²⁺ ) by utilizing the ferrous iron transport (Feo) system. FeoB is the main component of this system, and its function is regulated by the soluble cytosolic domain, termed NFeoB. We have recently begun to define the structure and the mechanism of the Feo system from the bacterium Vibrio cholerae , the causative agent of the disease cholera. However, major structural gaps in our understanding of the nucleotide-promiscuous V. cholerae NFeoB still exist. In this work, we have determined several new X-ray crystal structures that reveal distinct snapshots of the Vc NFeoB domain in uncommon and unprecedented states, ultimately illuminating the full catalytic cycle of this NTPase. This work reveals important functional features of Vc NFeoB that may be leveraged and ultimately targeted to prevent the infectivity and the spread of cholera. Graphical

Pseudomonas aeruginosa is a ubiquitous, Gram-negative bacterium that forms biofilms and is responsible for antibiotic-resistant hospital-acquired infections in humans. The P. aeruginosa BqsRS two-component system regulates biofilm formation and dispersal by sensing extracytoplasmic Fe ²⁺ , but the mechanistic details of this process are poorly understood. In this work, we report the crystal and solution structures of the Pa BqsR response regulator receiver domain, comprising a (βα) ₅ response regulator assembly, and the DNA-binding domain, comprising a helix-turn-helix motif. Consistent with its cognate stimulus being Fe ²⁺ , we show that Pa BqsR binds directly to the promoter region of the feo operon that encodes the bacterial Fe ²⁺ transport system FeoABC. Corroborating these in vitro results, transcriptional studies show that Pa BqsR is a global regulator controlling many important genes in PAO1, including the feo operon. Intriguingly, promoter-based assays reveal that Pa BqsR is a dynamic regulator that responds to bioavailable Fe ²⁺ , likely through the ability of Pa BqsR to bind Fe ²⁺ directly via a His-rich motif, independent of the Pa BqsS membrane His kinase. This mode of regulation is unprecedented among OmpR-like response regulators but represents an important level of control over Fe ²⁺ acquisition in P. aeruginosa that could be an attractive therapeutic target to treat hospital-acquired infections.

Prokaryotic two-component signal transduction systems (TCSs) are widely utilized by bacteria to respond to their environment and are typically composed of a transmembrane sensor His kinase (HK) and a cytosolic DNA-binding response regulator (RR) that work together to respond to environmental stimuli. An important TCS that regulates the expression of genes involved in biofilm formation and antibiotic resistance in many pathogens is the BqsRS/CarRS system, originally identified in Pseudomonas aeruginosa . Transcriptomics data suggested that the cognate Pa BqsRS stimulus is Fe ²⁺ , but Pa BqsS has not been characterized at the protein level, and a direct interaction between Fe ²⁺ and Pa BqsS has not been demonstrated. In this work, we biochemically and functionally characterize intact Pa BqsS, an iron-sensing membrane HK, for the first time. Using bioinformatics, protein modeling, metal analyses, site-directed mutagenesis, and X-ray absorption spectroscopy (XAS), we show that Pa BqsS binds a single Fe ²⁺ ion within its periplasmic domain containing an N/O-rich ligation sphere that includes Glu ⁴⁸ as a key metal ligand. Using activity assays, we show that both intact and truncated Pa BqsS have competent ATPase activities, consistent with predicted function. Importantly, we show that the ATP hydrolysis of intact Pa BqsS is stimulated exclusively by Fe ²⁺ , revealing metal-based activation of a functional, intact membrane HK for the first time. Moreover, stimulation assays of Pa BqsS variants demonstrate the importance of Glu ⁴⁵ and Asn ⁴⁹ in the sensing and signal transduction pathway. Taken together, this work uncovers important structural and biochemical properties that could be leveraged to target the BqsRS system for future therapeutic developments.

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important posttranslational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while posttranslational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well those as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR facilitates the formation of a complex between ATE1 and tRNA^Arg, adding a new complexity to the ATE1 structure and providing new insights for future studies of ATE1 functions.

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important post-translational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while post-translational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse ( Mus musculus ) ATE1. Using size-exclusion chromatography (SEC), small angle X-ray scattering (SAXS), and hydrogen deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR likely facilitates the formation of the complex between ATE1 and tRNA ^Arg , adding a new complexity to ATE1 structure and providing new insights for future studies of ATE1 functions. For Table of Contents Use Only: