Biology subjects

Bouwer, J. C.

Publications and source records attributed to Bouwer, J. C..

6 recordsLinked to original sources

bioRxiv2025

Electron tomography reveals mitochondrial network and cristae remodelling during cell differentiation in the human placenta

Mitochondria adapt their structure through fusion and fission, yet how their morphology and cristae architecture alter as cells differentiate remains unclear. The human placenta is an ideal model for studying mitochondrial diversity within a single tissue. As epithelial trophoblast cells of the placenta differentiate, their accompanying mitochondria morphologically and functionally transform. We used array tomography to characterise mitochondrial volume and network complexity. Cryo-electron tomography revealed two distinct subpopulations of mitochondria within progenitor cytotrophoblasts, and a singular, homogeneous population in the differentiated syncytiotrophoblast. We showcased the 3D topology of individual cristae through a standardised metric of "curvedness". Proteomic analysis of isolated mitochondria identified reduced dynamics proteins and cristae organisation complexes, accompanied by subunit variations within the electron transport chain, ATP synthase, and altered supercomplex abundance. This study highlights the advantages of combining multimodal imaging with mechanistic insights, to elucidate the process of mitochondrial morphology and cristae architecture remodelling as cells differentiate.

cell biology↗

bioRxiv2025

Structural Studies of an Anti-necroptosis Viral:Human Functional Hetero-amyloid M45:RIPK3 using SSNMR

The formation of RIP-homotypic interaction motif (RHIM)-based heteromeric amyloid assemblies between effector proteins such as RIPK1, ZBP1, or TRIF and the kinase RIPK3 serve as regulating signals for the necroptosis process, a key element of innate immune defense. Murine cytomegalovirus (MCMV) expresses the M45-encoded viral inhibitor of RIP activation (vIRA) which inhibits necroptosis in a RHIM-dependent manner. A pivotal question is how viral M45 forms hetero-amyloids with RIPK3 to effectively create an inhibitory assembly. We report a novel high-resolution structure of the M45:RIPK3 complex where M45 and RIPK3 alternately stack in an amyloid-state structure. Mutagenesis of the residues flanking the IQIG tetrad in M45 results in specific impacts on co-assembly with RIPK3, indicating an extended interface in the heteromeric fibrils. Other key interactions support the formation of stable viral:host fibrils. The M45: RIPK3 hetero-amyloid is likely to act as an anti-necroptotic signal by competing with formation of other pro-necroptotic species and introducing a barrier to RIPK3 autophosphorylation. Significance StatementThis study investigates the structural biology of the necroptotic pathway, an understudied programmed cell death mechanism that plays a crucial role in innate immunity and has implications for infectious diseases, cell cycle regulation, and cancer. We present the high-resolution structure of a cross-species hetero-amyloid in which M45, a murine cytomegalovirus (MCMV) protein, co-assembles with human RIPK3 to inhibit necroptosis by competing with pro-necroptotic amyloids. Using solid-state NMR, cryo-EM, mutagenesis, and biophysical analyses, we uncover a novel structural paradigm for cross-species hetero-amyloids, shedding light on viral strategies to manipulate host immunity and protein interactions.

biophysics↗

bioRxiv2023

Potassium dependent structural changes in the selectivity filter of HERG potassium channels

The fine tuning of biological electrical signaling is mediated by variations in the rates of opening and closing of gates that control ion flux through different ion channels. Human ether-a-go-go related gene (HERG) potassium channels have uniquely rapid inactivation kinetics which are critical to the role they play in regulating cardiac electrical activity. Here, we have exploited the K+ sensitivity of HERG inactivation to determine structures of both a conductive and non-conductive selectivity filter structure of HERG. We propose that inactivation is the result of a high propensity for flipping of the selectivity filter valine carbonyl oxygens. Molecular dynamics simulations point to a low energy barrier, and hence rapid kinetics, for flipping of the valine 625 carbonyl oxygens facilitated by a previously unrecognized interaction between S620 and Y616 that stabilizes the transition state between conducting and non-conducting structures. Our model represents a new mechanism by which ion channels fine tune their activity that explains the uniquely rapid inactivation kinetics of HERG. HighlightsStructures of a conductive and non-conductive HERG selectivity filter have been determined. Reduced potassium causes flipping of selectivity filter valine carbonyl oxygens. The sidechain of S620 on the pore helix coordinates distinct sets of interactions between conductive, non-conductive, and transition states.

biophysics↗

bioRxiv2023

Highly amine-reactive graphene-oxide EM grids for biochemical surface modification in aqueous buffer

Graphene oxide (GO), an oxidized derivative of graphene, has found application in cryo-electron microscopy (cryo-EM) as a hydrophilic and transparent solid support on which to adsorb biological macromolecules, providing an alternative to traditional aqueous films. Current applications generally adsorb the macromolecule directly onto unmodified GO or modify the GO surface with polyethylene glycol-amine reagents. This nucleophilic amine reaction must be performed in an aprotic organic solvent and therefore precludes the use of biological samples such as nucleic acids and peptides. The utility of GO could be expanded by the ability to covalently modify its surface with biochemical affinity reagents such as small- molecule metabolites, peptides, or nucleic acids, in aqueous buffer at neutral pH. Presented here is a chemical procedure that converts all oxygen functionalities of GO to highly amine- reactive glycidyl epoxide groups, achieved without the need of specialized laboratory equipment. We show that single sheets of glycidyl epoxide-modified GO react on the EM grid with primary amines at micromolar concentrations in minutes at room temperature in aqueous buffer. Given the ease of derivatizing biochemical reagents with amines, the chemistry described here will enable imaging of macromolecules immobilized on GO through specific biochemical and biologically relevant binding interactions.

molecular biology↗

bioRxiv2023

Structural characterisation of the complete cycle of sliding clamp loading in Escherichia coli

Ring-shaped DNA sliding clamps are essential for DNA replication and genome maintenance. Clamps need to be opened or trapped open and chaperoned onto DNA by clamp loader complexes (CLCs). Detailed understanding of the mechanisms by which CLCs open and place clamps around DNA remains limited. Here, we present a series of six structures of the Escherichia coli CLC bound to an open or closed clamp on and off a primer-template DNA that represent all intermediates in the clamp loading process. We show that the ATP-bound CLC first binds to a clamp, then constricts to hold onto it. The CLC then expands to open the clamp with a gap large enough for double-stranded DNA to enter. Upon binding to DNA, the CLC constricts slightly, allowing ATP hydrolysis and clamp closing around DNA. Although both yeast and E. coli CLCs open clamps by crab claw-like motions, they do it by the CLC expanding in opposite directions. These structures provide critical high-resolution snapshots of clamp loading by the E. coli CLC, revealing how the molecular machine works.

biochemistry↗

bioRxiv2021

Mechanism of transcription modulation by the transcription-repair coupling factor

Elongation by RNA polymerase is dynamically modulated by accessory factors. The transcription-repair coupling factor (TRCF) recognizes distressed RNAPs and either rescues transcription or initiates transcription termination. Precisely how TRCFs choose to execute either outcome remains unclear. With Escherichia coli as a model, we used single-molecule assays to study dynamic modulation of elongation by Mfd, the bacterial TRCF. We found that nucleotide-bound Mfd converts the elongation complex (EC) into a catalytically poised state, presenting the EC with an opportunity to restart transcription. After long-lived residence in this catalytically poised state, ATP hydrolysis by Mfd remodels the EC through an irreversible process leading to loss of the RNA transcript. Further, biophysical studies revealed that the motor domain of Mfd binds and partially melts DNA containing a template strand overhang. The results explain pathway choice determining the fate of the EC and provide a molecular mechanism for transcription modulation by TRCF.

biophysics↗