Biology subjects

Klos, P.

Publications and source records attributed to Klos, P..

3 recordsLinked to original sources

bioRxiv2025

Molecular insights into the dual activation of PomZ, a ParA/MinD P-loop ATPase, by the two ATPase activating proteins PomX and PomY

Positioning of the division site is precisely regulated. In Myxococcus xanthus, the PomX/Y/Z assembly associates with and translocates across the nucleoid in a PomZ ATPase-dependent manner to position the cytokinetic FtsZ-ring at midcell. Unlike other systems incorporating a ParA/MinD ATPase, the PomX/Y/Z system incorporates two ATPase Activating Proteins (AAPs), PomX and PomY, that stimulate PomZ activity. While PomX AAP activity, similarly to other characterized AAPs of ParA/MinD ATPases, resides in a short positively charged N-terminal peptide (PomXNPEP), the mechanism of PomY-mediated activation of PomZ remained unknown. We map PomY AAP activity to its C-terminal residues 496-682 (PomY496-682) and show that this activity is important for division site positioning and the initiation of cell division. Structural modeling suggests a bipartite PomY496-682 architecture, comprising a globular domain and an intrinsically disordered region containing the short 5 helix. However, modeling supported an interaction only between 5 and the PomZ dimer. The 5 binding site partially overlaps with that of the prototype AAP ParB on the ParA dimer but 5 lacks a positively charged residue critical for ParB AAP activity. Modeling supported that PomXNPEP binds the PomZ dimer at a site distinct from that of 5 and which overlaps with that of the prototype AAP MinE on the MinD dimer. These findings reveal a non-canonical AAP mechanism of PomY, while PomXNPEP likely functions analogously to MinE, indicating that PomX and PomY act in concert through distinct but complementary mechanisms to stimulate PomZ ATPase activity, thereby efficiently driving the positioning of the cytokinetic FtsZ-ring. ImportanceIn bacteria, the regulatory systems that position the division site often incorporate a ParA/MinD ATPase. These ATPases also function in the positioning of numerous other macromolecular complexes in bacteria. Characteristically, these ATPases depend on stimulation of their ATPase activity by a single cognate ATPase activating protein (AAP). However, the Myxococcus xanthus cell division regulatory system PomX/Y/Z system stands out by incorporating two AAPs, PomX and PomY, that activate the PomZ ATPase. Similar to other AAPs, PomX uses a short, positively charged N-terminal peptide (PomXNPEP) to stimulate PomZ activity. We map PomY AAP activity to a C-terminal region spanning residues 496-682 (PomY496-682). Structural modeling using AlphaFold 3 supports that the AAP activity of PomY496-682 and PomXNPEP involves two distinct, non-overlapping binding sites at the dimer interface of ATP-bound PomZ. Altogether, these results provide insights into the molecular mechanism of the activation by two AAPs of a ParA/MinD ATPase.

microbiology↗

bioRxiv2022

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social, predatory bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single large megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we studied the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization, suggesting a novel cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. PomY-nucleated FtsZ polymerization shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanisms ancient origin.

cell biology↗

bioRxiv2021

Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

In bacteria, the nucleotide-based second messenger bis-(3-5)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in the formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP receptors. We confirm that PlpA, a stand-alone PilZ-domain protein, is specifically important for motility and that Pkn1, which is composed of a Ser/Thr domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility upstream of the Frz chemosensory system as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility downstream of the Frz chemosensory system by setting up the polarity of the two motility systems. Our results support a model whereby the three proteins PlpA, PixA and PixB act in parallel pathways and have distinct functions to regulation of motility. Importancec-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts upstream of the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts downstream of the Frz system to regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple parallel pathways to regulate motility and development in M. xanthus.

microbiology↗