Biology subjects

Quade, B.

Publications and source records attributed to Quade, B..

3 recordsLinked to original sources

bioRxiv2026

Cryo-EM structure of detergent-bound Vibrio cholerae MakA captures a candidate intermediate between the soluble form and mature assembly of an α-pore-forming toxin

The tripartite toxin MakA/MakB/MakE from Vibrio cholerae belongs to a family of -pore-forming toxins that undergo extensive conformational rearrangements upon interacting with membrane. While soluble structures of MakA, MakB and MakE have been determined, the structural basis of membrane association and pore assembly remains poorly understood. Here, we report a cryo-electron microscopy structure of a detergent-associated form of MakA at a global resolution of 3.13 Angstrom, with focused refinement of a flexible region improving the local resolution to 2.68 Angstrom. This structure consists of a trimer of MakA asymmetric homodimers, in which each subunit adopts a transmembrane -helical conformation distinct from the previously reported soluble structure. The asymmetric homodimer closely resembles the building block observed in the previously reported helical MakA assembly and is structurally similar to the homodimeric building blocks of the Aeromonas hydrophila AhlB and Serratia marcescens SmhA pores, and the heterodimeric building blocks of the Xenorhabdus nematophila XaxAB and Yersinia enterocolitica YaxAB pores, suggesting a conserved assembly principle among distantly related -pore-forming toxins. Complementary hydropathy analysis, AlphaFold2 modeling, and liposome-based assays reveal distinct roles for the three components, with MakA acting as the primary membrane-inserting subunit, MakE exhibiting low but consistent membrane-association, and MakB alone remaining largely soluble. Together, these results support a model in which the MakA asymmetric homodimer represents a plausible early membrane-insertion unit and building block for pore assembly, while the trimer of dimers represents an early assembly intermediate.

microbiology↗

bioRxiv2024

Structural architecture of TolQ-TolR inner membrane protein complex from opportunistic pathogen Acinetobacter baumannii

Gram-negative bacteria harness the proton motive force (PMF) within their inner membrane (IM) to uphold the integrity of their cell envelope, an indispensable aspect for both division and survival. The IM TolQ-TolR complex is the essential part of the Tol-Pal system, serving as a conduit for PMF energy transfer to the outer membrane. Here we present cryo-EM reconstructions of Acinetobacter baumannii TolQ in apo and TolR- bound forms at atomic resolution. The apo TolQ configuration manifests as a symmetric pentameric pore, featuring a trans-membrane funnel leading towards a cytoplasmic chamber. In contrast, the TolQ-TolR complex assumes a proton non-permeable stance, characterized by the TolQ pentamers flexure to accommodate the TolR dimer, where two protomers undergo a translation-based relationship. Our structure-guided analysis and simulations support the rotor-stator mechanism of action, wherein the rotation of the TolQ pentamer harmonizes with the TolR protomers interplay. These findings broaden our mechanistic comprehension of molecular stator units empowering critical functions within the Gram-negative bacterial cell envelope. TeaserApo TolQ and TolQ-TolR structures depict structural rearrangements required for cell envelope organization in bacterial cell division.

molecular biology↗

bioRxiv2021

Control of neurotransmitter release and presynaptic plasticity by re-orientation of membrane-bound Munc13-1

Munc13-1 plays a central role in neurotransmitter release through its conserved C-terminal region, which includes a diacyglycerol (DAG)-binding C1 domain, a Ca2+/PIP2-binding C2B domain, a MUN domain and a C2C domain. Munc13-1 was proposed to bridge synaptic vesicles to the plasma membrane in two different orientations mediated by distinct interactions of the C1C2B region with the plasma membrane: i) one involving a polybasic face that yields a perpendicular orientation of Munc13-1 and hinders release; and ii) another involving the DAG-Ca2+-PIP2-binding face that induces a slanted orientation and facilitates release. Here we have tested this model and investigated the role of the C1C2B region in neurotransmitter release. We find that K603E or R769E point mutations in the polybasic face severely impair synaptic vesicle priming in primary murine hippocampal cultures, and Ca2+-independent liposome bridging and fusion in in vitro reconstitution assays. A K720E mutation in the polybasic face and a K706E mutation in the C2B domain Ca2+-binding loops have milder effects in reconstitution assays and do not affect vesicle priming, but enhance or impair Ca2+-evoked release, respectively. The phenotypes caused by combining these mutations are dominated by the K603E and R769E mutations. Our results show that the C1-C2B region of Munc13-1 plays a central role in vesicle priming and support the notion that re-orientation of Munc13-1 controls neurotransmitter release and short-term presynaptic plasticity.

neuroscience↗