Biology subjects

Shvarev, D.

Publications and source records attributed to Shvarev, D..

11 recordsLinked to original sources

bioRxiv2026

Assembly and activation of the AAA+ motor of the multi-subunit cobaltochelatase

Cobalamin is a complex yet essential cobalt-containing cofactor synthesized exclusively by prokaryotes and archaea. Aerobic cobalamin biosynthesis requires the CobNST multi-subunit class I cobaltochelatase for cobalt incorporation. Cobalt chelation is dependent on the energy transfer from the CobST AAA+/VWA motor unit to the substrate-binding catalytic unit (CobN). However, the mechanism for assembly and function of CobNST cobaltochelatase remains poorly understood. Here, we used cryo-EM to solve the structures of the AAA+ motor CobS, in the absence and presence of ATP and its VWA-partner protein CobT. Our data reveal that the AAA+ CobS assembles into various conformations, including hexameric planar rings and different spiral left-handed assemblies: hexamers, double rings and filaments. In the absence of nucleotide, the CobT N-terminal domain interacts with CobS left-handed oligomers, remaining in a half-assembled, nonfunctional motor unit. In the CobST complex, ATP induces conformational rearrangement of CobS from left-handed helical oligomers to a right-handed closed hexamer. The simultaneous repositioning of the CobT VWA domain to the AAA+ central pore shifts the motor unit to a functional state, ready-to-drive CobN-catalyzed chelation reaction. These findings define the mechanism for assembly and activation of the class I cobaltochelatase CobST motor.

biochemistry↗

bioRxiv2026

Cholesterol transport cycle of human ABCA2

Cholesterol is a key component of cellular membranes and is critical for brain function, particularly axon myelination. Among the 48 human ATP-binding cassette (ABC) transporters, ABCA2 exhibits the highest expression in the brain and is involved in cholesterol metabolism, primarily in oligodendrocytes. Notably, ABCA2 has been associated with myelin sheath integrity and maintenance, as well as Alzheimer's disease. Here, we report cryo-EM structures of human ABCA2 that reveal critical endogenous lipid-binding sites unique to ABCA2. Our five distinct conformations include a previously uncharacterized intermediate between the closed and apo states of ABCA subfamily transporters. Most importantly, we elucidated the cholesterol transport mechanism of ABCA2, which involves novel interdependent rotations of the exocytoplasmic domains (ECDs) and regulatory domains (RDs). Our structural findings provide a new perspective on ABCA transporter function and highlight the role of ABCA2 in facilitating efficient cholesterol recycling and transport in the brain.

molecular biology↗

bioRxiv2026

Assembly of N-acetyl-L-glutamate kinase complex with the PII signal transduction protein follows a two-step mechanism

PII signal transduction proteins control central carbon/nitrogen metabolism via interacting with and modulating many cellular targets. In photoautotrophs, N-acetyl-L-glutamate kinase (NAGK), the rate-limiting enzyme of arginine biosynthesis, is the primary target of PII. Here, we focused on how PII initiates the interaction with NAGK. Through biochemical and biophysical analyses, we show that the PII variant lacking the T-loop (PII{Delta}T-loop) is blocked in the first association step with NAGK, but is still able to form a stable complex. However, the NAGK interaction with PII{Delta}T-loop is weaker than with the wild-type PII as indicated by a decrease in the affinity. Using single particle cryo-EM, we resolved the structure of the encounter PII{Delta}T-loop-NAGK complex, revealing that the PII{Delta}T-loop-NAGK interface is established by the B-loop residues of PII. Thus, our data indicate a two-step process of PII-NAGK complex formation with B-loop initiating the contact with NAGK followed by the insertion of the PII T-loop deeply into the NAGK cavity to fully activate the enzyme.

microbiology↗

bioRxiv2026

Structure of chlorophyll synthase in complex with the LHC-like protein HliD

Chlorophyll is the central cofactor in oxygenic photosynthesis, responsible for both light capture in antenna complexes and light-driven charge separation in photosystems. The tetraprenyl tail of chlorophyll is attached to the chlorophyllide macrocycle by the transmembrane enzyme chlorophyll synthase (ChlG). In the cyanobacterium Synechocystis sp. PCC 6803, ChlG forms a complex with the LHC-like high-light-inducible protein HliD. To understand the substrate specificity and catalytic mechanism of ChlG, and how it is photoprotected by HliD, we determined the structure of a ChlG2HliD2 complex in both substrate-free and geranylgeranyl pyrophosphate (GGPP)-bound states using cryogenic electron microscopy (cryo-EM). A homodimer of HliD, which binds four chlorophylls and two zeaxanthin molecules in a quenched state, is flanked by two ChlG monomers. AlphaFold modelling placed chlorophyllide adjacent to the structurally resolved GGPP bound to the active site of ChlG. Cryo-EM data, site-directed mutagenesis and molecular dynamics were used to formulate a molecular mechanism for ChlG catalysis. The structure of the ChlG2HliD2 complex shows how Hlips bind to the synthase, reveals the arrangements of carotenoids and chlorophylls that mediate energy dissipation, and sheds light on the evolution of eukaryotic LHC antennae from their cyanobacterial ancestors.

plant biology↗

bioRxiv2026

A dual sensor regulates P-glycoproteins structural plasticity

P-glycoprotein is an efflux pump with an exceptionally broad substrate profile which drives its profound clinical impact. Despite its biological importance, it remains obscure how P-glycoprotein achieves its polyspecificity and how substrate binding and the lipid environment stimulate its activity. Structural data highlight the importance of transmembrane helices 4 and 10, which surround the binding pocket, and identify them as key players in substrate recognition. Here, we used cryo-EM to study P-glycoprotein in detergent and nanodiscs to strategically leverage environment- and substrate-dependent phenotypes. This approach allowed us to decipher unexpected and distinct roles of transmembrane helices 4 and 10, which structurally explain differences in ATPase activity. Our data highlights helix 4 as an environment sensor and helix 10 as the key player in substrate recognition constituting a dual regulation mechanism for the functional plasticity of P-glycoprotein, and visualizes the intricate interplay between a membrane protein and its environment.

molecular biology↗

bioRxiv2024

The ABC transporter MsbA in a dozen environments

High-resolution structure determination of membrane proteins typically requires isolation from the native lipid bilayer and reconstitution into artificial membrane mimics. For this purpose, numerous detergents, amphipols, polymers and membrane scaffold proteins are available. The choice of the specific membrane substitute can strongly affect the proteins specific activity, stability and conformational spectrum, potentially leading to errors or misinterpretation during analysis. The bacterial ATP-binding cassette transporter MsbA is a prominent example of such environment-specific bias, resulting in apparent conformational and activity responses. Here, we present a systematic analysis of the conformational spectrum of MsbA, stabilized in a dozen environments, using cryo-EM. Our data show pronounced structural feedback of the ABC transporter to the respective membrane mimetics. Detergents generally favour a conformation with wide separation of the nucleotide-binding domains, while nanodiscs induce the narrow conformation. Notably, only three of the dozen tested environments allow MsbA to sample the functional conformational spectrum, enabling full movement of the nucleotide-binding domains between narrow and wide inward-facing conformations. We expect this study to serve as a blueprint for other membrane proteins, even where the structural reaction to the hydrophobic environment is not directly visible but still critical for the proteins function.

molecular biology↗

bioRxiv2024

Structural basis of the allosteric regulation of cyanobacterial glucose-6-phosphate dehydrogenase by the redox sensor OpcA

The oxidative pentose phosphate (OPP) pathway is a fundamental carbon catabolic route for generating reducing power and metabolic intermediates for biosynthetic processes. In addition, its first two reactions form the OPP shunt, which replenishes the Calvin-Bassham cycle under certain conditions. Glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first and rate-limiting reaction of this metabolic route. In photosynthetic organisms, G6PDH is redox-regulated to allow fine-tuning and to prevent futile cycles while carbon is being fixed. In cyanobacteria, regulation of G6PDH requires the redox protein OpcA, but the underlying molecular mechanisms behind this allosteric activation remain elusive. Here, we used enzymatic assays and in vivo interaction analyses to show that OpcA binds G6PDH under different environmental conditions. However, complex formation enhances G6PDH activity when OpcA is oxidized and inhibits it when OpcA is reduced. To understand the molecular basis of this regulation, we used cryogenic electron microscopy to determine the structure of cyanobacterial G6PDH and the G6PDH-OpcA complex. OpcA binds the G6PDH tetramer and induces conformational changes in the active site of G6PDH. The redox sensitivity of OpcA is achieved by intramolecular disulfide bridge formation, which influences the allosteric regulation of G6PDH. In vitro assays reveal that the level of G6PDH activation depends on the number of bound OpcA molecules, which implies that this mechanism allows delicate fine-tuning. Our findings unveil a novel and unique molecular mechanism governing the regulation of the OPP pathway in cyanobacteria.

molecular biology↗

bioRxiv2023

Structure of the endosomal CORVET tethering complex

Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solved the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discovered that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane packaging defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function. One-Sentence SummaryUsing cryo-EM, we solved the structure of the CORVET tethering complex and explained how it promotes endosome fusion.

biochemistry↗

bioRxiv2023

Conformational variability of cyanobacterial ChlI, the AAA+ motor of magnesium chelatase involved in chlorophyll biosynthesis

Magnesium chelatase is a conserved enzyme complex responsible for the first committed step of chlorophyll biosynthesis in photosynthetic organisms, which is the addition of magnesium to the chlorophyll precursor, protoporphyrin IX. The complex is composed of the catalytic subunit ChlH, the bridging subunit ChlD, and the subunit ChlI, which serves as the motor that drives the entire complex. Although the enzyme is well-characterized functionally, high-resolution structures are available only for individual subunits. Hence, the full assembly and the molecular mechanism of the enzyme complex remains unknown. Here, we used cryo-EM, supported by biochemical analysis and mass photometry, to determine structures of the ChlI motor subunit of magnesium chelatase under turnover conditions in the presence of ATP. Our data reveal the molecular details of ChlI oligomerization and conformational dynamics upon ATP binding and hydrolysis. These findings provide new insights into the mechanistic function of ChlI and its implications for the entire magnesium chelatase complex machinery.

biophysics↗

bioRxiv2022

Structure of the lysosomal membrane fusion machinery

Lysosomes are of central importance in cellular recycling, nutrient signaling 1,2 and endocytosis, and are tightly connected to autophagy 3 and the invasion of pathogenic bacteria and viruses 1,4. Lysosomal fusion events are fundamental to cell survival and require HOPS, a conserved heterohexameric tethering complex 5,6. HOPS recognizes and binds small membrane-associated GTPases on lysosomes and organelles, and assembles membrane bound SNAREs for fusion 7,8. Through tethering, HOPS brings membranes in close proximity to each other and significantly increases fusion efficacy by catalysing SNARE assembly. Consequently, different HOPS mutations are causative for severe diseases 6. Despite its fundamental cellular duties, it remained speculative how HOPS fulfils its function as high-resolution structural data were unavailable. Here, we used cryo-electron microscopy to reveal the structure of HOPS. In the complex, two central subunits form the backbone and an assembly hub for the functional domains. Two GTPase binding units extend to opposing ends, while the SNARE binding module points to the side, resulting in a triangular shape of the complex. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities. We show that HOPS complex variants with mutations proximal to the backbone can still tether membranes but fail to efficiently promote fusion indicating, that the observed integrity of HOPS is essential to its function. In our model, the core of HOPS acts as a counter bearing between the flexible GTPase binding domains. This positions the SNARE binding module exactly between the GTPase anchored membranes to promote fusion. Our structural and functional analysis reveals the link between the spectacular architecture of HOPS and its mechanism that couples membrane tethering and SNARE assembly, to catalyse lysosomal fusion.

biophysics↗

bioRxiv2018

Glycolipid composition of the heterocyst envelope of Anabaena sp. PCC 7120 is crucial for diazotrophic growth and relies on the UDP-galactose 4-epimerase HgdA

The nitrogenase complex in the heterocysts of the filamentous freshwater cyanobacterium Anabaena sp. PCC 7120 fixes atmospheric nitrogen to allow diazotrophic growth. The heterocyst cell envelope protects the nitrogenase from oxygen and consists of a polysaccharide and a glycolipid layer that are formed by a complex process involving the recruitment of different proteins. Here we studied the function of the putative nucleoside-diphosphate-sugar epimerase HgdA, which along with HgdB and HgdC is essential for deposition of the glycolipid layer and growth without a combined nitrogen source. Using site-directed mutagenesis and single homologous recombination approach, we performed a thoroughly functional characterization of HgdA and confirmed that the glycolipid layer of the hgdA mutant heterocyst is aberrant as shown by transmission electron microscopy and chemical analysis. The hgdA gene was expressed during late stages of the heterocyst differentiation. GFP-tagged HgdA protein localized inside the heterocysts. The purified HgdA protein had UDP-galactose 4-epimerase activity in vitro. This enzyme could be responsible for synthesis of heterocyst-specific glycolipid precursors, which could be transported over the cell wall by the ABC transporter components HgdB/HgdC.\n\nImportanceThe phototrophic multicellular bacterium Anabaena sp. PCC 7120 is an important model organism for investigations of critical processes such as photosynthesis, nitrogen fixation, and cell differentiation in prokaryotes. The cyanobacterium utilizes specific cells, called heterocysts, to fix atmospheric nitrogen. Heterocyst function requires a specific cell envelope. We focused on unstudied aspects of heterocyst envelope formation and revealed the role of the UDP-galactose 4-epimerase HgdA in this process.

microbiology↗