Biology subjects

Rawski, M.

Publications and source records attributed to Rawski, M..

6 recordsLinked to original sources

bioRxiv2026

A 13-subunit c-ring in the Chlamydomonas chloroplast ATP synthase lowers the H⁺/ATP cost of carbon fixation

The chloroplast F1Fo ATP synthase is a rotary motor that converts the light-driven proton-motive force into the chemical energy of ATP. The number of c-subunits in its rotor fixes the number of protons translocated per ATP formed, a fundamental parameter of bioenergetic systems. The reference spinach enzyme possesses fourteen c-subunits and a H+/ATP ratio of 4.67. Green algae additionally operate a carbon-concentrating mechanism that sustains CO2 fixation in water at a substantial cost in ATP, yet the structure of the algal motor, and whether its bioenergetic parameters differ from those of vascular plants, remains unresolved. Here, a 2.2 [A] structure of the ATP synthase of Chlamydomonas reinhardtii reveals that the enzyme carries a thirteen-membered c-ring, the first departure from c14 in a chloroplast, and with a lower predicted H+/ATP ratio of 4.33. Ordered waters trace a Grotthuss proton relay through the membrane, where an insulating triad separates the proton loading and unloading sites and couples flux to rotation. A single substitution in the redox switching {gamma}-subunit abolishes the contact with the catalytic {beta}-subunit that idles the enzyme in darkness in vascular plants. These unique features of the algal ATP synthase lower the H+/ATP cost of carbon fixation in the light and facilitate acetate metabolism in the dark.

biochemistry↗

bioRxiv2026

Filament Formation by ChlI Challenges the Current View of Magnesium Chelatase Architecture

Magnesium chelatase (MgCh) catalyzes the first committed step in chlorophyll biosynthesis by inserting Mg2+ into the tetrapyrrole ring. The enzyme comprises three core subunits: ChlI, ChlD, and ChlH, and requires the auxiliary factor GUN4 for full activity. Despite extensive investigation, the structural organization of the active holoenzyme and the mechanistic coupling between ATP hydrolysis and Mg2+ insertion remain poorly defined. Here, we used cryo-electron microscopy to investigate MgCh architecture. We show that both cyanobacterial and plant ChlI homologs assemble into filamentous helical structures in the presence of Mg2+ and either ATP or ADP. However, only ATP-induced oligomers are susceptible to disassembly by ChlD. Structural analysis of the ATP-driven assemblies reveals compact inter-subunit packing, with ADP and Mg2+ coordinated at the interfacial regions. These findings suggest that ATP hydrolysis promotes subunit compaction and may facilitate partial dehydration of the Mg2+ hydration shell. Low-resolution reconstructions further provide a structural framework for ChlD engagement with ChlI filaments. Finally, we demonstrate that phosphatidylglycerol enhances catalytic activity, supporting a role for membrane lipids in modulating MgCh function. Significance statementMagnesium chelatase catalyzes the first committed step of chlorophyll biosynthesis, yet the structural basis of its activation and coupling to ATP hydrolysis remains unclear. Using cryo-electron microscopy, we show that the ATPase subunit ChlI forms filamentous helical oligomers in the presence of Mg2+ and nucleotide, a property conserved across cyanobacterial and plant homologs but not previously recognized. Only oligomers generated through ATP hydrolysis interact efficiently with the ChlD subunit, indicating that hydrolysis produces a distinct, recognition-competent conformation. Structural analysis further suggests that ATP hydrolysis promotes subunit compaction and partial dehydration of Mg2+. Together, these findings reveal a previously unrecognized oligomeric state of ChlI and provide a structural framework for understanding how ATP hydrolysis regulates magnesium chelatase activity.

biochemistry↗

bioRxiv2025

Structures of the LPOR-Chlide Complexes Imply the Basis of Membrane Remodeling and a Photocatalytic Mechanism

Flowering plants rely on the photocatalytic enzyme light-dependent protochlorophyllide oxidoreductase (LPOR) to synthesize chlorophyllide (Chlide), a chlorophyll intermediate, and at the same time to remodel lipid membranes into the cubic phase required for chloroplast development. Yet neither the mechanism of this light-driven catalysis nor the structural basis of its membrane remodeling activity is well understood, largely due to the lack of high-resolution structural information. To address these questions, we analyzed Chlide:LPOR:NADPH oligomeric assemblies by cryo-electron microscopy. Eleven maps were obtained, enabling the reconstruction of nine distinct LPOR oligomer models. Most assemblies adopt helical or stacked-ring forms, whereas one displays a segmented string-of-dimers architecture, representing a previously unobserved structural organization. Strings of LPOR dimers induce distinct periodic deformations of the lipid bilayer that stabilize multiple complex architectures. These architectures are maintained by three different inter-string interfaces occurring in various combinations. The plasticity of these interactions suggest that the filaments are capable of twisting and sliding relative to each other. Our highest-resolution map (2.55 [A]) provided a detailed view of the pigment-binding site, revealing a water channel that connects the central magnesium ion of the pigment to the bulk solvent. Unexpectedly, the propionate group of the pigment protrudes out of the porphyrin plane and arches back toward NADPH, ideally positioned for hydride transfer. These structural insights allowed us to propose a novel reaction mechanism for LPOR photocatalysis. Together, our data highlight the critical role of Chlide:LPOR:NADPH complexes in delaying prolamellar body disassembly and point to their possible regulatory function in mature leaves.

biochemistry↗

bioRxiv2025

Revealing the unexpected interplay between the Proteasome Activator PA200 and the immunoproteasome

The proteasome activator PA200 binds to the catalytic core of the proteasome, the 20S, and activates its proteolytic activities. The cellular function of PA200 is poorly understood and appears to be cell type and differentiation specific. Recent evidence suggests that PA200 not only binds to the standard 20S (s20S) proteasome but also to the specialized immunoproteasome (i20S) which plays a key role in anti-viral and anti-tumor immunity. We here investigated the interaction of PA200 and the immunoproteasome in detail. We show the very first cryo-EM structures of the singly- and doubly-capped i20S-PA200 complexes that revealed no major difference regarding the first binding event of PA200 to the i20S vs. the s20S. However, first PA200 binding triggered a subtle and long range allosteric bending of the i20S barrel which was not seen in the s20S-PA200 complexes. This resulted in major structural rearrangements in the opposite unbound ring - the displacement of atoms up to 5.4 [A] and the increase in its outer diameter - thereby increasing the occupancy of the second PA200 binding site. Mass photometry confirmed higher occupancy of PA200 to the i20S versus the s20S. Binding of PA200 to the i20S enhanced proteasomal activation compared to the s20S. Co-expression of PA200 and the i20S in cells and tissues, however, is restricted but their interaction is favored upon co-expression. The expression of PA200 and the catalytic subunits of the i20S is differentially regulated depending on the cellular context. Our data also suggest that PA200 has the potential to regulate i20S gene expression whereas the i20S has no effects on PA200 expression. Overall, this work sheds new light on the interaction of PA200 with the i20S from a structural, mechanistic and cellular point of view. Importantly, we identify PA200 as a key regulator of the i20S whenever PA200 and the catalytic subunits of the i20S are co-expressed in the same cell.

biochemistry↗

bioRxiv2023

miR-125b-5p impacts extracellular vesicle biogenesis, trafficking, and EV subpopulation release in the porcine trophoblast by regulating ESCRT-dependent pathway

1.Intercellular communication is a critical process that ensures cooperation between distinct cell types at the embryo-maternal interface. Extracellular vesicles (EVs) are considered to be potent mediators of this communication by transferring biological information in their cargo (e.g. miRNAs) to the recipient cells. miRNAs are small non-coding RNAs that affect the function and fate of neighboring and distant cells by regulating gene expression. Focusing on the maternal side of the dialog, we recently revealed the impact of embryonic signals, including miRNAs, on EV-mediated cell-to-cell communication. In this study, we show the regulatory mechanism of the miR-125b-5p ESCRT-mediated EV biogenesis pathway and the further secretion of EVs by trophoblasts at the time when the crucial steps of implantation are taking place. To test the ability of miR-125b-5p to influence the expression of genes involved in the generation and release of EV subpopulations in porcine conceptuses, we used an ex vivo approach. Next, in silico and in vitro analyses were performed to confirm miRNA-mRNA interactions. Finally, EV trafficking and release were assessed using several imaging and particle analysis tools. Our results indicated that conceptus development and implantation are accompanied by changes in the abundance of EV biogenesis and trafficking machinery. ESCRT-dependent EV biogenesis and the further secretion of EVs were modulated by miR-125b-5p, specifically impacting the ESCRT-II complex (via VPS36) and EV trafficking in primary porcine trophoblast cells. The identified miRNA-ESCRT interplay led to the generation and secretion of specific subpopulations of EVs. miRNA present at the embryo-maternal interface governs EV-mediated communication between the mother and the developing conceptus, leading to the generation, trafficking, and release of characteristic subpopulations of EVs.

physiology↗

bioRxiv2022

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses protect their single-stranded RNA genome with a methylated cap during replication. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16 where nsp10 acts as a co-factor to both. Aditionally, nsp14 carries an exonuclease domain, which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins should be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex. This interaction is expected to encourage formation of mature capped viral mRNA, modulating the nsp14s exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

microbiology↗