Biology subjects

Brown, S. H. J.

Publications and source records attributed to Brown, S. H. J..

9 recordsLinked to original sources

bioRxiv2026

The Lower γ Region Ensures Unidirectional Rotation and Torque Generation in the Latter Half of the 80° Substep of F1-ATPase

F1-ATPase achieves unidirectional rotation of its {gamma} shaft through coordinated conformational cycling of the 3{beta}3 ring, yet how the shaft itself enforces directionality remains unclear. Here we analyzed an axle-less TF1 lacking the lower half of the rotor shaft by combining single-molecule rotation assays with cryo-EM structural analysis under catalysis conditions. Under ATP-saturated conditions, wild-type TF1 exhibited only three pauses per turn corresponding to the catalytic dwells, whereas axle-less TF1 exhibited six pauses per turn, indicating the presence of an additional intermediate during rotation. This previously unreported intermediate dwell was observed at 40{degrees} between the binding and catalytic dwells. High-speed recordings revealed frequent backsteps confined to the 40{degrees} transition between the intermediate and catalytic dwells. Analysis of the dwell-time distributions indicated an approximately zero free energy bias between these two states, consistent with thermally driven interconversion. Cryo-EM resolved three corresponding intermediates--binding, intermediate, and catalytic dwells--showing a major {beta} conformational change from 0{degrees} to 40{degrees}, but minimal {beta} rearrangement between 40{degrees} and 80{degrees}, suggesting that the {beta} power stroke is not operative in this interval. Analysis of the {gamma} rotational scheme indicates that the driving force changes across the 0-80{degrees} step: the 0-40{degrees} advance proceeds via a power stroke to an intermediate dwell, whereas the subsequent 40{degrees} transition to the catalytic dwell lacks a power stroke and is dominated by thermal fluctuations. Taken together, these findings suggest that the lower {gamma} region contributes to unidirectional rotation and torque generation specifically in the latter half of the 0-80{degrees} step, while the initial half can proceed even without contributions from the lower {gamma} region.

biophysics↗

bioRxiv2025

The structure and composition of native human nucleosomes

Since the first high-resolution structures of recombinantly assembled nucleosomes, efforts have shifted towards understanding chromatin structure in a native context. Most of these efforts have focused on native-like, yet still recombinantly assembled, nucleosomes that contain native DNA sequences. To date, no high-resolution structures of native human nucleosomes have been reported. Here we report the high-resolution cryo-EM structure of native human nucleosomes isolated from HEK293 cells. The HEK293-NCP structure reveals that native human nucleosomes store 145 bp of DNA. Despite the DNA sequence diversity of native nucleosomes, we observe conserved nucleotides that support the idea of a nucleosome positioning code. In addition to these striking features of nucleosomal DNA, we note alternate conformations of several DNA contacting histone residues that hint at dynamics in the HEK293-NCP. To complement the HEK293-NCP structure, we provide a mass spectrometry analysis of histone modifications and variants present in the sample, which demonstrates that a typical HEK293-NCP is composed of canonical histones with N-terminal tails that are methylated at K9, K27 and/or K36 of histone H3. Altogether these findings have implications for biological processes such as chromatin remodelling and transcription and improve our understanding of nucleosome and chromatin structure in a native context.

biochemistry↗

bioRxiv2025

Functional and Structural Characterization of F1-ATPase with common ancestral core domains in stator ring

Extant F1-ATPases exhibit diverse rotational stepping behaviors--3-, 6-, or 9-step cycles--yet the evolutionary origin of these patterns remains unclear. Here, we used ancestral sequence reconstruction to infer the catalytic {beta} and non-catalytic subunits of a putative ancestral F1-ATPase. We then fused their functionally critical domains into the thermostable F1 from Bacillus PS3, yielding a stable chimeric enzyme. Cryo-EM revealed two distinct conformational states--binding and catalytic dwell states--separated by a [~]34{degrees} rotation of the {gamma} subunit, suggesting a fundamental six-step mechanism akin to that of extant 6-stepping F1-ATPases. Single-molecule rotation assays with ATP and the slowly hydrolyzed ATP analog ATP{gamma}S demonstrated that the chimeric motor is intrinsically a 6-stepper, pausing at binding and catalytic dwell positions separated by 32.1{degrees}, although the binding dwell is significantly prolonged by an unknown mechanism. These findings indicate that F1-ATPase was originally a 6-stepper and diversified into 3-, 6- and 9-step forms in evolutional adaptation. Based on these results, we discuss plausible features of the entire FoF1 complex, along with potential physiological contexts in last universal common ancestor and related lineages.

biophysics↗

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↗

bioRxiv2024

The molecular structure of an axle-less F1-ATPase

F1Fo ATP synthase is a molecular rotary motor that can generate ATP using a transmembrane proton motive force. Isolated F1-ATPase catalytic cores can hydrolyse ATP, passing through a series of conformational states involving rotation of the central {gamma} rotor subunit and the opening and closing of the catalytic {beta} subunits. Cooperativity in F1-ATPase has long thought to be conferred through the {gamma} subunit, with three key interaction sites between the {gamma} and {beta} subunits being identified. Single molecule studies have demonstrated that the F1 complexes lacking the {gamma} axle still "rotate" and hydrolyse ATP, but with less efficiency. We solved the cryogenic electron microscopy structure of an axle-less Bacillus sp. PS3 F1-ATPase. The unexpected binding-dwell conformation of the structure in combination with the observed lack of interactions between the axle-less {gamma} and the open {beta} subunit suggests that the complete {gamma} subunit is important for coordinating efficient ATP binding of F1-ATPase.

biochemistry↗

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↗

bioRxiv2022

Broadly neutralizing SARS-CoV-2 antibodies through epitope-based selection from convalescent patients

Emerging variants of concern (VOCs) are threatening to limit the effectiveness of SARS-CoV-2 monoclonal antibodies and vaccines currently used in clinical practice; broadly neutralizing antibodies and strategies for their identification are therefore urgently required. Here we demonstrate that broadly neutralizing antibodies can be isolated from peripheral blood mononuclear cells (PBMCs) of convalescent patients using SARS-CoV-2 receptor binding domains (RBDs) carrying epitope-specific mutations. This is exemplified by two human antibodies, GAR05, binding to epitope class 1, and GAR12, binding to a new epitope class 6 (located between class 3 and class 5). Both antibodies broadly neutralize VOCs, exceeding the potency of the clinical monoclonal sotrovimab (mAb S309) by orders of magnitude. They also provide potent prophylactic and therapeutic in vivo protection of hACE2 mice against viral challenge. Our results indicate that exposure to Wuhan SARS-CoV-2 induces antibodies that maintain potent and broad neutralization against emerging VOCs using two unique strategies: either by targeting the divergent class 1 epitope in a manner resistant to VOCs (ACE2 mimicry, as illustrated by GAR05 and mAbs P2C-1F11/S2K14); or alternatively, by targeting rare and highly conserved epitopes, such as the new class 6 epitope identified here (as illustrated by GAR12). Our results provide guidance for next generation monoclonal antibody development and vaccine design.

immunology↗

bioRxiv2021

Analysis of sex-specific lipid metabolism in P. falciparum gametocytes points to importance of sphingomyelin for gametocytogenesis

Male and female Plasmodium falciparum gametocytes are the parasite lifecycle stage responsible for transmission of malaria from the human host to mosquito vector. Not only are gametocytes able to survive in radically different host environments, but they are also precursors for male and female gametes that reproduce sexually soon after ingestion by the mosquito. Here we investigate the sex-specific lipid metabolism of gametocytes within their host red blood cell and poised for ingestion by the mosquito vector and subsequent sexual reproduction. Comparison of the male and female lipidome identifies cholesteryl esters and dihydrosphingomyelin enrichment in female gametocytes. Chemical inhibition of each of these lipid types in mature gametocytes suggests dihydrosphingomyelin synthesis but not cholesteryl ester synthesis is important for sex-specific gametocyte viability. Genetic disruption of each of the two sphingomyelin synthase gene points towards sphingomyelin synthesis contributing to gametocytogenesis. This study shows that gametocytes are not only distinct from asexual stages, but that the lipid composition is also vastly different between male and female gametocytes, reflecting the different cellular roles these stages play. Together our results highlight the sex-specific nature of gametocyte lipid metabolism that has the potential to be targeted to block malaria transmission.

microbiology↗

bioRxiv2018

Identification of a novel tetrameric structure for human apolipoprotein-D

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded {beta}-barrel with a single adjacent -helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimers disease, where apolipoprotein-D overexpression reduces the amyloid-{beta} burden in Alzheimers disease mouse models.\n\nOligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids.\n\nHere, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a {bsim}95 to {bsim}100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

biochemistry↗