Myasnikov, A. | Search bioRxiv

bioRxiv2025

Molecular mechanism of substrate transport by human peroxisomal ABCD3

Peroxisomes are eukaryotic oxidative organelles involved in numerous metabolic functions that include fatty acid oxidation, bile acid synthesis, and detoxification of reactive oxygen species. ATP-binding cassette transporters of the D subfamily (ABCD1-3) mediate the import of CoA thioesters of fatty acids into the peroxisome. ABCD3, the most abundant of these transporters in the peroxisomal membrane, facilitates the transport of a broad spectrum of substrates including branched-chain fatty acids, very long-chain fatty acids, bile salt intermediates, and dicarboxylic acids. Mutations in ABCD3 are associated with defects in congenital bile acid synthesis and variants of Zellweger syndrome. The structural and functional details of the human ABCD3 transporter remain unclear, despite its significance. In this study, we report the cryogenic sample electron microscopy (cryo-EM) structures of full-length human ABCD3 in its apo state and bound to one of the physiological substrates (phytanoyl-CoA) at resolutions of 3.33 [A] and 3.13 [A], respectively. Our biochemical assays reveal that substrate binding induces ATPase activity in ABCD3, suggesting a substrate-dependent conformational change. Structural comparison of the apo and substrate bound states demonstrate that the substrate interaction brings nucleotide-binding domains closer, providing a mechanistic basis of substrate induced ATPase activity. These findings offer critical insights into the transport mechanism of ABCD3 and lay a structural foundation for understanding its role in peroxisomal metabolite import and related diseases. Significance StatementPeroxisomes are involved in essential cellular metabolic processes that include fatty acid oxidation, bile acid synthesis, and detoxification. The ABCD subfamily of membrane transporter proteins transport fatty acyl-CoA molecules from the cytosol into peroxisomes. Among the ABCD subfamily members, ABCD3 transports branched chain fatty acids and a range of other important metabolites. Dysfunction in ABCD3 has been associated with several inherited and acquired diseases. Using cryo-EM, we determined high-resolution structures of full-length human ABCD3 in both unbound and phytanoyl-CoA-bound forms. We show that substrate binding stimulates ATP hydrolysis activity by apposition of the two nucleotide binding domains of ABCD3, adding structural insight into the mechanism of action of ABCD3 and its role in peroxisomal function and disease.

biochemistry↗

bioRxiv2024

Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution

Cryo-transmission electron microscopy (cryo-EM) of frozen hydrated specimens is an efficient method for the structural analysis of purified biological molecules. However, cryo-EM and cryo-electron tomography are limited by the low signal-to-noise ratio (SNR) of recorded images, making detection of smaller particles challenging. For dose-resilient samples often studied in the physical sciences, electron ptychography - a coherent diffractive imaging technique using 4D scanning transmission electron microscopy (4D-STEM) - has recently demonstrated excellent SNR and resolution down to tens of picometers for thin specimens imaged at room temperature. Here we applied 4D-STEM and ptychographic data analysis to frozen hydrated proteins, reaching sub-nanometer resolution 3D reconstructions. We employed low-dose cryo-EM with an aberration-corrected, convergent electron beam to collect 4D-STEM data for our reconstructions. The high frame rate of the electron detector allowed us to record large datasets of electron diffraction patterns with substantial overlaps between the interaction volumes of adjacent scan positions, from which the scattering potentials of the samples were iteratively reconstructed. The reconstructed micrographs show strong SNR enabling the reconstruction of the structure of apoferritin protein at up to 5.8 [A] resolution. We also show structural analysis of the Phi92 capsid and sheath, tobacco mosaic virus, and bacteriorhodopsin at slightly lower resolutions.

biophysics↗

bioRxiv2022

DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage.

Structural maintenance of chromosomes (SMC) complexes fold DNA by loop extrusion to support chromosome segregation, genome maintenance, and gene expression. Wadjet systems (JetABCD/MksBEFG/EptABCD) are derivative SMC complexes with proposed roles in bacterial immunity against selfish DNA elements. Here, we show that JetABCD systems restrict extrachromosomal circular DNA with an upper size limit of about 100 kb, while a linear plasmid evades restriction. Recombinant preparations of a JetABCD complex cleave circular DNA regardless of its helical topology but not linear DNA; cleavage occurs at random positions and depends on ATP as well as the SMC ATPase. We solve a structure of the JetABCD core by cryo-EM revealing an alternative dimer-of-dimers configuration. The two SMC DNA motor units face in opposite orientations--rather than the same as observed with MukBEF--possibly representing a restriction-specific state of JetABCD. We hypothesize that JetABCD is a DNA-shape-specific endonuclease and present a model for the activation of DNA cleavage exclusively when extrusion of an entire plasmid has been completed by a single JetABCD complex. The self-stalling of two opposing DNA motor units may serve as signal to trigger DNA cleavage. Complete extrusion by a single complex cannot be achieved on the much larger chromosome, explaining how self-DNA may evade cleavage.

molecular biology↗

bioRxiv2022

Cryo-EM structures and binding of mouse ACE2 to SARS-CoV-2 variants of concern

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations.[1] There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.[2] Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors.[3,4] Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor. AUTHOR SUMMARYThe SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.

immunology↗

bioRxiv2021

Structural analysis of the Spike of the Omicron SARS-COV-2 variant by Cryo-EM and implications for immune evasion

Investigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations.[1] There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected.[2] Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors.[3,4] Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor. AUTHOR SUMMARYThe SARS-CoV-2 virus can infect different types of animals beyond humans. The virus uses its Spike protein on its surface to bind to cells. These cells have a protein called ACE2 that the Spike protein recognizes. Animals have slightly different ACE2 receptors compared to humans. Mice are widely used as a research animal and live in the same environments as humans so scientists are particularly interested. Understanding how Spike proteins binds to the mouse ACE2 receptor allows us to understand the impact of immune evading mutations found in new variants. We use a high resolution imaging technique called cryo-electron microscopy to look at how different Spike variants bind to the ACE2 receptor from mouse at a resolution where we can see the amino acids. We can see directly the individual amino acids and mutations on the Spike protein that interact with the mouse ACE2 receptor. Many of the mutations found in variants of concern also increase the strength of binding to the mouse ACE2 receptor. This result suggests that mutations in the Spike protein of future variants may have an additional effect in influencing how it binds to not only human ACE2 receptors but to mice and also different animals.

immunology↗

bioRxiv2020

GR chaperone cycle mechanism revealed by cryo-EM: inactivation of GR by GR:Hsp90:Hsp70:Hop client-loading complex

Maintaining a healthy proteome is fundamental for organism survival1,2. Integral to this are Hsp90 and Hsp70 molecular chaperones that together facilitate the folding, remodeling and maturation of Hsp90s many "client" proteins3-7. The glucocorticoid receptor (GR) is a model client strictly dependent upon Hsp90/Hsp70 for activity8-13. Chaperoning GR involves a cycle of inactivation by Hsp70, formation of an inactive GR:Hsp90:Hsp70:Hop "loading" complex, conversion to an active GR:Hsp90:p23 "maturation" complex, and subsequent GR release14. Unfortunately, a molecular understanding of this intricate chaperone cycle is lacking for any client. Here, we report the cryo-EM structure of the GR loading complex, in which Hsp70 loads GR onto Hsp90, revealing the molecular basis of direct Hsp90/Hsp70 coordination. The structure reveals two Hsp70s--one delivering GR and the other scaffolding Hop. Unexpectedly, the Hop cochaperone interacts with all components of the complex including GR, poising Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized via an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR maturation complex (Noddings et al., 2020), we present the first complete molecular mechanism of chaperone-dependent client remodeling, establishing general principles of client recognition, inhibition, transfer and activation.

biochemistry↗

bioRxiv2020

Cryo-EM reveals the dynamic interplay between mitochondrial Hsp90 and SdhB folding intermediates

TRAP1 is a mitochondrion specific Hsp90, a ubiquitous chaperone family that mediates the folding and maturation of hundreds of "client" proteins. Through the interaction with client proteins, TRAP1 regulates mitochondrial protein homeostasis, oxidative phosphorylation/glycolysis balance, and plays a critical role in mitochondrial dynamics and disease. However, the molecular mechanism of client protein recognition and remodeling by TRAP1 remains elusive. Here we established the succinate dehydrogenase B subunit (SdhB) from mitochondrial complex II as a client protein for TRAP1 amenable to detailed biochemical and structural investigation. SdhB accelerates the rate of TRAP1 dimer closure and ATP hydrolysis by 5-fold. Cryo-EM structures of the TRAP1:SdhB complex show TRAP1 stabilizes SdhB folding intermediates by trapping an SdhB segment in the TRAP1 lumen. Unexpectedly, client protein binding induces an asymmetric to symmetric transition in the TRAP1 closed state. Our results highlight a client binding mechanism conserved throughout Hsp90s that transcends the need for cochaperones and provide molecular insights into how TRAP1 modulates protein folding within mitochondria. Our structures also suggest a potential role for TRAP1 in Fe-S cluster biogenesis and mitochondrial protein import and will guide small molecule development for therapeutic intervention in specific TRAP1 client interactions.

biophysics↗

bioRxiv2019

High-resolution cryo-electron microscopy structure of the Escherichia coli 50S subunit and validation of nucleotide modifications

Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides positions, a task that has previously been accomplished by X-ray crystallography. Here we present a cryo-electron microscopy (cryo-EM) structure of Escherichia coli (E. coli) 50S subunit at an average resolution of 2.2[A] as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for identification of post-transcriptional rRNA modifications using a cryo-EM map. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.

biochemistry↗