Biology subjects

Kiontke, S.

Publications and source records attributed to Kiontke, S..

3 recordsLinked to original sources

bioRxiv2026

NUDT2 loss defines quantitative limits for dinucleoside polyphosphate action on the cGAS-STING-TBK1 axis

NUDT2 is an emerging candidate for therapeutic intervention in cancer, and its inhibition or loss is known to elevate adenosine-containing dinucleoside polyphosphates (ApnNs), including diadenosine tetraphosphate (Ap4A). Ap4A is a stress- and immune-associated nucleotide metabolite proposed to act as a second messenger, raising the possibility that NUDT2 targeting may unintentionally affect important nucleotide-sensitive signaling pathways. One such pathway is cGAS-STING signaling, a central innate immune axis that detects cytosolic DNA, produces the nucleotide second messenger 2'3'-cGAMP, and drives type I interferon responses. Because cGAS-STING also contributes to antitumor immunity and is being pharmacologically targeted in cancer, we asked whether sustained Ap4A accumulation perturbs this pathway. We systematically evaluated Ap4A and related dinucleoside polyphosphates across the cGAS-STING-TBK1 axis using biophysical, enzymatic, structural, and cellular approaches. Contrary to a previous model, STING did not bind Ap4A, Ap3A, or Ap4G, despite robust binding of canonical cyclic dinucleotides. Although cGAS bound these nucleotides with micromolar affinities, DNA-activated cGAMP synthesis was inhibited only at high, supra-substrate ratios. Similarly, TBK1 inhibition required extreme Ap4A ratios beyond physiologically relevant levels. In a THP-1 cell model, NUDT2 knockout caused strong Ap4A accumulation, but the resulting intracellular dinucleoside polyphosphate levels remained below the ratios required to inhibit cGAS or TBK1 in vitro. This study thus distinguishes biochemical possibility from physiological relevance and argues that NUDT2-linked Ap4A accumulation is unlikely to directly compromise cGAS-STING pathway activity.

biochemistry↗

bioRxiv2025

Structural basis of quinone-sensing by the MarR-type repressor MhqR in Staphylococcus aureus

The MarR-family regulator MhqR of Staphylococcus aureus (SaMhqR) was previously characterized as quinone-sensing repressor of the mhqRED operon. Here, we resolved the crystal structures of apo-SaMhqR and the 2-methylbenzoquinone (MBQ)-bound SaMhqR complex. AlphaFold3 modelling was used to predict the structure of the SaMhqR in complex with its operator DNA. In the DNA-bound SaMhqR state, S65 and S66 of an allosteric 3-4 loop adapted a helically wound conformation to elongate helix 4 for optimal DNA binding. Key residues for MBQ interaction were identified as F11, F39, E43, and H111, forming the MBQ-binding pocket. MBQ binding prevented the formation of the extended helix 4 in the allosteric loop, leading to steric clashes with the DNA. Molecular dynamics (MD) simulations revealed an increased intrinsic dynamics within the allosteric loop and the {beta}1/{beta}2-wing regions after MBQ binding, to prevent DNA binding. Using mutational analyses, we validated that F11, F39, and H111 are required for quinone sensing in vivo, whereas S65 and S66 of the allosteric loop and D88, K89, V91 and Y92 of the {beta}1/{beta}2-wing are essential for DNA binding in vitro and in vivo. In conclusion, our structure-guided modelling and mutational analyses identified a quinone-binding pocket of SaMhqR and the mechanism of SaMhqR inactivation, which involves local structural rearrangements of an allosteric loop and a high intrinsic dynamics to prevent DNA interactions. Our results provide novel insights into the redox-mechanism of the conserved SaMhqR repressor, that functions as an important determinant of quinone and antimicrobial resistance in S. aureus. IMPORTANCES. aureus is a major human pathogen, which can cause life-threatening infections in humans. However, treatment options are limited due to the prevalence of antimicrobial resistant isolates in the hospital and the community. The MarR-type repressor SaMhqR was described to control resistance towards quinones and quinone-like antimicrobials. However, the redox-regulatory mechanism of SaMhqR by quinones was unknown. In this work, we explored the DNA-binding and quinone-sensing mechanism of SaMhqR and identified a quinone-binding pocket and an allosteric loop, which facilitates DNA binding activity via a helical wound conformation and adapts an unstructured coiled conformation upon quinone binding to inhibit DNA binding. A similar mechanism has been recently discovered for regulation of uric acid resistance by UrtR family repressors (1). Our results contribute to a better understanding of antimicrobial resistance regulation, which can be exploited for future drug-design to eradicate multidrug-resistant S. aureus.

microbiology↗

bioRxiv2022

Structure of the lysosomal membrane fusion machinery

Lysosomes are of central importance in cellular recycling, nutrient signaling 1,2 and endocytosis, and are tightly connected to autophagy 3 and the invasion of pathogenic bacteria and viruses 1,4. Lysosomal fusion events are fundamental to cell survival and require HOPS, a conserved heterohexameric tethering complex 5,6. HOPS recognizes and binds small membrane-associated GTPases on lysosomes and organelles, and assembles membrane bound SNAREs for fusion 7,8. Through tethering, HOPS brings membranes in close proximity to each other and significantly increases fusion efficacy by catalysing SNARE assembly. Consequently, different HOPS mutations are causative for severe diseases 6. Despite its fundamental cellular duties, it remained speculative how HOPS fulfils its function as high-resolution structural data were unavailable. Here, we used cryo-electron microscopy to reveal the structure of HOPS. In the complex, two central subunits form the backbone and an assembly hub for the functional domains. Two GTPase binding units extend to opposing ends, while the SNARE binding module points to the side, resulting in a triangular shape of the complex. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities. We show that HOPS complex variants with mutations proximal to the backbone can still tether membranes but fail to efficiently promote fusion indicating, that the observed integrity of HOPS is essential to its function. In our model, the core of HOPS acts as a counter bearing between the flexible GTPase binding domains. This positions the SNARE binding module exactly between the GTPase anchored membranes to promote fusion. Our structural and functional analysis reveals the link between the spectacular architecture of HOPS and its mechanism that couples membrane tethering and SNARE assembly, to catalyse lysosomal fusion.

biophysics↗