Biology subjects

Szalai, V. A.

Publications and source records attributed to Szalai, V. A..

4 recordsLinked to original sources

bioRxiv2025

Copper acquisition in Bacillus subtilis involves Cu(II) exchange between YcnI and YcnJ

The transition metal copper is biologically essential across all three domains of life. Several copper-dependent proteins and enzymes produced by the Gram-positive bacterium Bacillus subtilis have been characterized. However, many questions remain about how copper is recognized and trafficked to metalate cuproproteins. The ycnKJI operon in B. subtilis encodes a suite of proteins implicated in copper uptake and regulation, including the copper-binding protein YcnI and the putative copper importer YcnJ. Here, we demonstrate that one of the extracellular domains within YcnJ (YcnJCopC) binds Cu(II) in 1:1 stoichiometry with high affinity using a histidine brace motif. Biochemical results reveal that YcnJCopC and YcnI can exchange Cu(II). Genetic studies reveal that loss of either YcnI or YcnJ, or mutation of the key residues required for Cu(II)-binding, leads to a growth defect under conditions of copper limitation. Together, these data suggest that the Cu(II)-binding sites in both YcnI and YcnJ contribute to efficient import under Cu limited conditions. Our results support a model in which YcnI may sequester Cu(II) from YcnJ, serving a regulatory role to limit the amount of copper that enters the cytoplasm and allowing Cu(II) to be stored for later import in the outer face of the membrane. This transfer of Cu(II) between extracellular domains of membrane-bound proteins represents a potential new paradigm in bacterial copper usage.

biochemistry↗

bioRxiv2021

The YcnI protein from Bacillus subtilis contains a copper-binding domain

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through its ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized DUF1775 domain-containing YcnI. DUF1775 domains are found across bacterial phylogeny and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance (EPR) and inductively-coupled plasma-mass spectrometry (ICP-MS) find that it can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper binding site featuring a unique mono-histidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.

biophysics↗

bioRxiv2021

Iron-sulfur clusters are involved in post-translational arginylation

Eukaryotic arginylation is an essential post-translational modification that both modulates protein stability and regulates protein half-life through the N-degron pathway. Arginylation is catalyzed by a family of enzymes known as the arginyl-tRNA transferases (ATE1s), which are conserved across the eukaryotic domain. Despite its conservation and importance, little is known regarding the structure, mechanism, and regulation of ATE1s. In this work, we have discovered that ATE1s bind a previously unknown iron-sulfur cluster that is conserved across evolution. We have extensively characterized the nature of this iron-sulfur cluster, and we show that the presence of the iron-sulfur cluster is linked to alterations in arginylation efficacy. Finally, we demonstrate that the ATE1 iron-sulfur cluster is oxygen sensitive, which could be a molecular mechanism of the N-degron pathway to sense oxidative stress. Thus, our data provide the framework of a cluster-based paradigm of ATE1 regulatory control.

biochemistry↗

bioRxiv2020

The C-terminus and Third Cytoplasmic Loop Cooperatively Activate Mouse Melanopsin Phototransduction

Melanopsin, an atypical vertebrate visual pigment, mediates non-image forming light responses including circadian photoentrainment and pupillary light reflexes, and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsins deactivation have been characterized (i.e. C-terminal phosphorylation and {beta}-arrestin binding), but a detailed analysis of melanopsins activation is lacking. We propose that an extended 3rd cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation which is stabilized by ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the 3rd cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in 3-dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsins activation rate. Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo melanopsin--which has substitutions of various potential phosphorylation sites and a substitution of the conserved tyrosine--indicates that substitution of these potential phosphorylation sites and the tyrosine residue result in dramatically slower activation kinetics, a finding that also supports the role of phosphorylation in signaling activation. We therefore propose that melanopsins C-terminus is proximal to intracellular loop 3 and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling. STATEMENT OF SIGNIFICANCEMelanopsin is an important visual pigment in the mammalian retina that mediates non-image forming responses such as circadian photoentrainment and pupil constriction, and supports contrast detection for image formation. In this study, we detail two critical structural features of mouse melanopsin--its 3rd cytoplasmic loop and C-terminus--that are important in the activation of melanopsins light responses. Furthermore, we propose that these two regions directly participate in coupling mouse melanopsin to its G-protein. These findings contribute to further understanding of GPCR-G-protein coupling, and given recent findings suggesting flexibility of melanopsin signal transduction in the retina (possibly by coupling more than one G-protein type), these findings provide insight into the molecular basis of melanopsin function in the retina.

neuroscience↗