Structural insights into membrane remodeling by SNX1

The sorting nexin (SNX) family of proteins deform the membrane to generate transport carriers in endosomal pathways. Here, we elucidate how a prototypic member, SNX1, acts in this process. Performing cryoelectron microscopy, we find that SNX1 assembles into a protein lattice that consists of helical rows of SNX1 dimers wrapped around tubular membranes in a crosslinked fashion. We also visualize the details of this structure, which provides a molecular understanding of how various parts of SNX1 contribute to its ability to deform the membrane. Moreover, we have compared the SNX1 structure with a previously elucidated structure of an endosomal coat complex formed by retromer coupled to a SNX, which reveals how the molecular organization of the SNX in this coat complex is affected by retromer. The comparison also suggests insight into intermediary stages of assembly that results in the formation of the retromer-SNX coat complex on the membrane.

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Selective packaging of mitochondrial proteins into extracellular vesicles prevents the release of mitochondrial DAMPs

Nature Communications ◽

10.1038/s41467-021-21984-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kiran Todkar ◽

Lilia Chikhi ◽

Véronique Desjardins ◽

Firas El-Mortada ◽

Geneviève Pépin ◽

...

Keyword(s):

Extracellular Vesicles ◽

Mitochondrial Proteins ◽

Control Mechanisms ◽

Mitochondrial Content ◽

Sorting Nexin ◽

Inflammatory Conditions ◽

Selective Targeting ◽

Molecular Patterns ◽

Damage Associated Molecular Patterns ◽

Insight Into

AbstractMost cells constitutively secrete mitochondrial DNA and proteins in extracellular vesicles (EVs). While EVs are small vesicles that transfer material between cells, Mitochondria-Derived Vesicles (MDVs) carry material specifically between mitochondria and other organelles. Mitochondrial content can enhance inflammation under pro-inflammatory conditions, though its role in the absence of inflammation remains elusive. Here, we demonstrate that cells actively prevent the packaging of pro-inflammatory, oxidized mitochondrial proteins that would act as damage-associated molecular patterns (DAMPs) into EVs. Importantly, we find that the distinction between material to be included into EVs and damaged mitochondrial content to be excluded is dependent on selective targeting to one of two distinct MDV pathways. We show that Optic Atrophy 1 (OPA1) and sorting nexin 9 (Snx9)-dependent MDVs are required to target mitochondrial proteins to EVs, while the Parkinson’s disease-related protein Parkin blocks this process by directing damaged mitochondrial content to lysosomes. Our results provide insight into the interplay between mitochondrial quality control mechanisms and mitochondria-driven immune responses.

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Cargo induces retromer-mediated membrane remodeling on membranes

Molecular Biology of the Cell ◽

10.1091/mbc.e18-06-0339 ◽

2018 ◽

Vol 29 (22) ◽

pp. 2709-2719 ◽

Cited By ~ 6

Author(s):

Latha Kallur Purushothaman ◽

Christian Ungermann

Keyword(s):

Plasma Membrane ◽

Simple Model ◽

Membrane Proteins ◽

Golgi Complex ◽

Exact Order ◽

Membrane Remodeling ◽

Sorting Nexin ◽

Low Concentrations ◽

Sorting Station ◽

Specific Trigger

Endosomes serve as a central sorting station of lipids and proteins that arrive via vesicular carrier from the plasma membrane and the Golgi complex. At the endosome, retromer complexes sort selected receptors and membrane proteins into tubules or vesicles that bud off the endosome. The mature endosome finally fuses with the lysosome. Retromer complexes consist of a cargo selection complex (CSC) and a membrane remodeling part (sorting nexin [SNX]-Bin/amphiphysin/Rvs [BAR], or Snx3 in yeast) and different assemblies of retromer mediate recycling of different cargoes. Due to this complexity, the exact order of events that results in carrier formation is not yet understood. Here, we reconstituted this process on giant unilamellar vesicles together with purified retromer complexes from yeast and selected cargoes. Our data reveal that the membrane remodeling activity of both Snx3 and the SNX-BAR complex is strongly reduced at low concentrations, which can be reactivated by CSC. At even lower concentrations, these complexes still associate with membranes, but only remodel membranes in the presence of their specific cargoes. Our data thus favor a simple model, where cargo functions as a specific trigger of retromer-mediated sorting on endosomes.

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Hsp70 molecular chaperones: multifunctional allosteric holding and unfolding machines

Biochemical Journal ◽

10.1042/bcj20170380 ◽

2019 ◽

Vol 476 (11) ◽

pp. 1653-1677 ◽

Cited By ~ 19

Author(s):

Eugenia M. Clerico ◽

Wenli Meng ◽

Alexandra Pozhidaeva ◽

Karishma Bhasne ◽

Constantine Petridis ◽

...

Keyword(s):

Molecular Chaperones ◽

Protein Homeostasis ◽

Nucleotide Exchange ◽

Cellular Functions ◽

The Past ◽

Hsp70 Family ◽

Nucleotide Exchange Factors ◽

Client Proteins ◽

Structural Insights ◽

Insight Into

AbstractThe Hsp70 family of chaperones works with its co-chaperones, the nucleotide exchange factors and J-domain proteins, to facilitate a multitude of cellular functions. Central players in protein homeostasis, these jacks-of-many-trades are utilized in a variety of ways because of their ability to bind with selective promiscuity to regions of their client proteins that are exposed when the client is unfolded, either fully or partially, or visits a conformational state that exposes the binding region in a regulated manner. The key to Hsp70 functions is that their substrate binding is transient and allosterically cycles in a nucleotide-dependent fashion between high- and low-affinity states. In the past few years, structural insights into the molecular mechanism of this allosterically regulated binding have emerged and provided deep insight into the deceptively simple Hsp70 molecular machine that is so widely harnessed by nature for diverse cellular functions. In this review, these structural insights are discussed to give a picture of the current understanding of how Hsp70 chaperones work.

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Genetic and Molecular Organization of the Alkylbenzene Catabolism Operon in the Psychrotrophic Strain Pseudomonas putida 01G3

Applied and Environmental Microbiology ◽

10.1128/aem.67.1.453-458.2001 ◽

2001 ◽

Vol 67 (1) ◽

pp. 453-458 ◽

Cited By ~ 15

Author(s):

P. A. Chablain ◽

A. L. Zgoda ◽

C.-O. Sarde ◽

N. Truffaut

Keyword(s):

Growth Rate ◽

Pseudomonas Putida ◽

Low Temperatures ◽

Multiple Alignment ◽

Molecular Organization ◽

Substrate Specificities ◽

Psychrotrophic Strain ◽

Insight Into

ABSTRACT The 11-kb sequence encompassing the alkylbenzene upper pathway inPseudomonas putida 01G3, a psychrotrophic strain able to degrade alkylbenzenes at low temperatures, was characterized. Together with a potential regulator (EbdR), six putative enzymes (EbdAaAbAcAdBC) were identified, and they exhibited highly significant similarities with enzymes implicated in the equivalent pathway in P. putida RE204. ebd genes appeared to be preferentially induced by ethylbenzene. Multiple-alignment data and growth rate measurements led us to classify 01G3 and closely related strains in two groups with distinct substrate specificities. Close to identified genes, remnants of IS5-like elements provided insight into the evolution of catabolic sequences through rearrangements from a less complex ancestral cluster.

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Fission of SNX-BAR–coated endosomal retrograde transport carriers is promoted by the dynamin-related protein Vps1

The Journal of Cell Biology ◽

10.1083/jcb.201309084 ◽

2014 ◽

Vol 204 (5) ◽

pp. 793-806 ◽

Cited By ~ 45

Author(s):

Richard J. Chi ◽

Jingxuan Liu ◽

Matthew West ◽

Jing Wang ◽

Greg Odorizzi ◽

...

Keyword(s):

Retrograde Transport ◽

Related Protein ◽

Membrane Remodeling ◽

Functional Link ◽

Bar Domain ◽

Endosomal Sorting ◽

Sorting Nexin ◽

Bar Domain Proteins

Retromer is an endosomal sorting device that orchestrates capture and packaging of cargo into transport carriers coated with sorting nexin BAR domain proteins (SNX-BARs). We report that fission of retromer SNX-BAR–coated tubules from yeast endosomes is promoted by Vps1, a dynamin-related protein that localizes to endosomes decorated by retromer SNX-BARs and Mvp1, a SNX-BAR that is homologous to human SNX8. Mvp1 exhibits potent membrane remodeling activity in vitro, and it promotes association of Vps1 with the endosome in vivo. Retrograde transport carriers bud from the endosome coated by retromer and Mvp1, and cargo export is deficient in mvp1- and vps1-null cells, but with distinct endpoints; cargo export is delayed in mvp1-null cells, but cargo export completely fails in vps1-null cells. The results indicate that Mvp1 promotes Vps1-mediated fission of retromer- and Mvp1-coated tubules that bud from the endosome, revealing a functional link between the endosomal sorting and fission machineries to produce retrograde transport carriers.

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An in silico structural insights into Plasmodium LytB protein and its inhibition

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314082096 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1791-C1791

Author(s):

Rajabrata Bhunya ◽

Suman Nandy ◽

Alpana Seal

Keyword(s):

In Silico ◽

3D Structure ◽

Three Dimensional ◽

Comparative Modeling ◽

Binding Energies ◽

E Coli ◽

Dimethylallyl Diphosphate ◽

In Silico Study ◽

Structural Insights ◽

Insight Into

In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new antimalarial compounds or inhibitors. Here a systematic in-silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three dimensional (3D) structure of Plasmodium LytB taking E. Coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand dataset containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, 5 were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any "drug like" molecule and the knowledge of Plasmodium LytB inhibitory mechanism which can provide valuable support for the antimalarial inhibitor design in future.

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Structural and functional analysis of LIM domain-dependent recruitment of paxillin to focal adhesions

10.21203/rs.3.rs-42943/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Marta Ripamonti ◽

Nicolas Liaudet ◽

Bernhard Wehrle-Haller

Keyword(s):

Plasma Membrane ◽

Focal Adhesions ◽

Bimolecular Fluorescence Complementation ◽

Molecular Organization ◽

Structural Function ◽

Lim Domain ◽

Mechanical Coupling ◽

Lim Domains ◽

Β3 Integrin ◽

Structural Insights

Abstract The LIM domain-dependent localization of the adapter protein paxillin to focal adhesions (FAs) is not mechanistically understood. Here, by combining molecular biology with photoactivation and FA-isolation experiments, we demonstrate a specific contribution of each LIM domain and reveal the existence of multiple paxillin docking sites in the FA-complex. Mutation of β3 integrin at a putative paxillin binding site leads to rapid inward sliding of FAs, correlating with enhanced paxillin dissociation rates. Mechanical coupling of paxillin to integrins or the plasma membrane arrests the FAs sliding, thereby disclosing an essential structural function of the LIM-array for the maturation of integrin/talin clusters. Moreover, via bimolecular fluorescence complementation, we determine a precise spatial orientation of the paxillin LIM domains, juxtaposing the positively charged LIM4 to the plasma membrane and the extremity of the β3 integrin tail, providing structural insights into the molecular organization of FAs.

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Structural insights on TRPV5 gating by endogenous modulators

10.1101/338798 ◽

2018 ◽

Cited By ~ 1

Author(s):

Taylor E.T. Hughes ◽

Ruth A. Pumroy ◽

Aysenur Torun Yazici ◽

Marina A. Kasimova ◽

Edwin C. Fluck ◽

...

Keyword(s):

Transient Receptor Potential ◽

Receptor Potential ◽

Transient Receptor Potential Channel ◽

Cryo Electron Microscopy ◽

Calcium Reabsorption ◽

Conformational Rearrangements ◽

Transient Receptor ◽

Endogenous Modulators ◽

Structural Insights ◽

Insight Into

ABSTRACTTRPV5 is a transient receptor potential channel involved in calcium reabsorption. Here we investigate the interaction of two endogenous modulators with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and calmodulin (CaM) have been shown to directly bind to TRPV5 and activate or inactivate the channel, respectively. Using cryo-electron microscopy (cryo-EM), we determined TRPV5 structures in the presence of dioctanoyl PI(4,5)P2 and CaM. The PI(4,5)P2 structure revealed a novel binding site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These interactions with PI(4,5)P2 induce conformational rearrangements in the lower gate, opening the channel. The CaM structure revealed two TRPV5 C-terminal peptides anchoring a single CaM molecule and that calcium inhibition is mediated through a cation-π interaction between Lys116 on the C-lobe of calcium-activated CaM and Trp583 at the intracellular gate of TRPV5. Overall, this investigation provides insight into the endogenous modulation of TRPV5, which has the potential to guide drug discovery.

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Structural insights into the architecture and assembly of eukaryotic flagella

Microbial Cell ◽

10.15698/mic2020.11.734 ◽

2020 ◽

Vol 7 (11) ◽

pp. 289-299

Author(s):

Narcis-Adrian Petriman ◽

Esben Lorentzen

Keyword(s):

Structure And Function ◽

Eukaryotic Cells ◽

Repetitive Nature ◽

Unicellular Organisms ◽

Cilia And Flagella ◽

And Function ◽

Signaling Components ◽

Structural Insights ◽

Insight Into

Cilia and flagella are slender projections found on most eukaryotic cells including unicellular organisms such as Chlamydomonas, Trypanosoma and Tetrahymena, where they serve motility and signaling functions. The cilium is a large molecular machine consisting of hundreds of different proteins that are trafficked into the organelle to organize a repetitive microtubule-based axoneme. Several recent studies took advantage of improved cryo-EM methodology to unravel the high-resolution structures of ciliary complexes. These include the recently reported purification and structure determination of axonemal doublet microtubules from the green algae Chlamydomonas reinhardtii, which allows for the modeling of more than 30 associated protein factors to provide deep molecular insight into the architecture and repetitive nature of doublet microtubules. In addition, we will review several recent contributions that dissect the structure and function of ciliary trafficking complexes that ferry structural and signaling components between the cell body and the cilium organelle.

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Mechanistic insight into substrate processing and allosteric inhibition of human p97

10.1101/2021.02.08.430295 ◽

2021 ◽

Author(s):

Man Pan ◽

Yuanyuan Yu ◽

Huasong Ai ◽

Qingyun Zheng ◽

Yuan Xie ◽

...

Keyword(s):

Conformational Changes ◽

Cellular Protein ◽

Power Stroke ◽

Cryo Electron Microscopy ◽

D2 Domain ◽

The Central Nervous System ◽

Degenerative Disorder ◽

Substrate Processing ◽

Structural Insights ◽

Insight Into

ABSTRACTp97, also known as valosin-containing protein (VCP), processes ubiquitinated substrates and plays a central role in cellular protein homeostasis. Mutations in human p97 are associated with multisystem proteinopathy (MSP), a dominantly inherited degenerative disorder that can affect muscle, bone and the central nervous system. It is also a drug target for cancer therapy with various inhibitors developed over the past decade. Despite significant structural insights into the fungal homologue of p97, Cdc48, little is known about how human p97 processes its substrates and how the activity is allosterically affected by inhibitors. Here, we report a series of cryo-electron microscopy (cryo-EM) structures of substrate-engaged human p97 complex with resolutions ranging from 2.9 to 3.8 Å that captured “power stroke”-like motions of both the D1 and D2 ATPase rings of p97. The structures elucidated how the unfolded substrate is engaged in the pore at atomic level. Critical conformational changes of the inter-subunit signaling (ISS) motifs were revealed, providing molecular insights into substrate translocation. Furthermore, we also determined cryo-EM structures of human p97 in complex with NMS-873, the most potent p97 inhibitor, at a resolution of 2.4 Å. The structures showed that NMS-873 binds at a cryptic groove in the D2 domain and interacts with the ISS motif, preventing its conformational change, thus blocking substrate translocation allosterically. Finally, using NMS-873 at a substoichiometric concentration, we captured a series of intermediate states, suggesting how the cofactor Npl4 coordinates with the D1 ring of p97 to initiate the translocation.

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