scholarly journals Structures of the fungal dynamin-related protein Vps1 reveal a unique, open helical architecture

2018 ◽  
Vol 217 (10) ◽  
pp. 3608-3624 ◽  
Author(s):  
Natalia V. Varlakhanova ◽  
Frances J.D. Alvarez ◽  
Tyler M. Brady ◽  
Bryan A. Tornabene ◽  
Christopher J. Hosford ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Self Assembly ◽  
Related Protein ◽  
Membrane Remodeling ◽  
Helical Structures ◽  
Gtp Hydrolysis ◽  
Cellular Targets ◽  
Related Proteins

Dynamin-related proteins (DRPs) are large multidomain GTPases required for diverse membrane-remodeling events. DRPs self-assemble into helical structures, but how these structures are tailored to their cellular targets remains unclear. We demonstrate that the fungal DRP Vps1 primarily localizes to and functions at the endosomal compartment. We present crystal structures of a Vps1 GTPase–bundle signaling element (BSE) fusion in different nucleotide states to capture GTP hydrolysis intermediates and concomitant conformational changes. Using cryoEM, we determined the structure of full-length GMPPCP-bound Vps1. The Vps1 helix is more open and flexible than that of dynamin. This is due to further opening of the BSEs away from the GTPase domains. A novel interface between adjacent GTPase domains forms in Vps1 instead of the contacts between the BSE and adjacent stalks and GTPase domains as seen in dynamin. Disruption of this interface abolishes Vps1 function in vivo. Hence, Vps1 exhibits a unique helical architecture, highlighting structural flexibilities of DRP self-assembly.

scholarly journals Dnm1 forms spirals that are structurally tailored to fit mitochondria

2005 ◽  
Vol 170 (7) ◽  
pp. 1021-1027 ◽  
Author(s):  
Elena Ingerman ◽  
Edward M. Perkins ◽  
Michael Marino ◽  
Jason A. Mears ◽  
J. Michael McCaffery ◽  
...  
Keyword(s):  
Self Assembly ◽  
Membrane Dynamics ◽  
Nucleotide Hydrolysis ◽  
Mitochondrial Division ◽  
Cellular Processes ◽  
Self Assembling ◽  
Related Proteins ◽  
Rate Limiting ◽  
Common Function

Dynamin-related proteins (DRPs) are large self-assembling GTPases whose common function is to regulate membrane dynamics in a variety of cellular processes. Dnm1, which is a yeast DRP (Drp1/Dlp1 in humans), is required for mitochondrial division, but its mechanism is unknown. We provide evidence that Dnm1 likely functions through self-assembly to drive the membrane constriction event that is associated with mitochondrial division. Two regulatory features of Dnm1 self-assembly were also identified. Dnm1 self-assembly proceeded through a rate-limiting nucleation step, and nucleotide hydrolysis by assembled Dnm1 structures was highly cooperative with respect to GTP. Dnm1 formed extended spirals, which possessed diameters greater than those of dynamin-1 spirals but whose sizes, remarkably, were equal to those of mitochondrial constriction sites in vivo. These data suggest that Dnm1 has evolved to form structures that fit the dimensions of mitochondria.

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

2014 ◽  
Vol 204 (5) ◽  
pp. 793-806 ◽  
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.

scholarly journals Structural Insights into Bak Activation and Oligomerisation

2014 ◽  
Vol 70 (a1) ◽  
pp. C1166-C1166
Author(s):  
Jason Brouwer ◽  
Adeline Robin ◽  
Geoff Thompson ◽  
Ahmad Wardak ◽  
Ruth Kluck ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Death ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Conformational Changes ◽  
Mitochondrial Outer Membrane ◽  
Related Protein ◽  
Mitochondrial Outer Membrane Permeabilisation ◽  
Structural Insights ◽  
Analogous Mechanism

Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. This activation can occur when certain BH3-only proteins directly interact with Bak and Bax. A recent crystal structure by Czabotar et al. (2013) revealed a novel conformational change for Bax upon activation by BH3-only peptides. Distinguishing characteristics of BH3-only proteins capable of directly activating Bax were also elucidated. Here we describe complementary studies on the related protein Bak. We identify specific BH3-only peptides capable of inducing Bak dimerisation and describe crystal structures that provide key insights into Bak activation and oligomerisation. These structures demonstrate that Bak undergoes similar conformational changes upon activation to those observed with Bax. Altogether our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to BH3-only peptides.

Crystallographic studies of elongation factor G

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1209-1216 ◽  
Author(s):  
Anders Liljas ◽  
Arnthor Ævarsson ◽  
Salam Al-Karadaghi ◽  
Maria Garber ◽  
Julia Zheltonosova ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Change ◽  
Conformational Changes ◽  
Elongation Factor ◽  
Elongation Factors ◽  
Elongation Factor G ◽  
Conformational States ◽  
Gtp Hydrolysis ◽  
Large Conformational Change ◽  
Factor G

The elongation factors G (EF-G) and Tu (EF-Tu) go through a number of conformation states in their functional cycles. Since they both are GTPases, have similar G domains and domains II, and have similar interactions with the nucleotides, then GTP hydrolysis must occur in similar ways. The crystal structures of two conformational states are known for EF-G and three are known for EF-Tu. The conformations of EF-G∙GDP and EF-Tu∙GTP are closely related. EF-Tu goes through a large conformational change upon GTP cleavage. This conformational change is to a large extent due to an altered interaction between the G domain and domains II and III. A number of kirromycin-resistant mutations are situated at the interface between domains I and III. The interface between the G domain and domain V in EF-G corresponds with this dynamic interface in EF-Tu. The contact area in EF-G is small and dominated by interactions between charged amino acids, which are part of a system that is observed to undergo conformational changes. Furthermore, a number of fusidic acid resistant mutants have been identified in this area. All of this evidence makes it likely that EF-G undergoes a large conformational change in its functional cycle. If the structures and conformational states of the elongation factors are related to a scheme in which the ribosome oscillates between two conformations, the pretranslocational and posttranslocational states, a model is arrived at in which EF-Tu drives the reaction in one direction and EF-G in the opposite. This may lead to the consequence that the GTP state of one factor is similar to the GDP state of the other. At the GTP hydrolysis state, the structures of the factors will be close to superimposable.Key words: elongation factor G, elongation factor Tu, crystal structures, conformational changes, ribosomal conformation.

scholarly journals Structures of collagen IV globular domains: insight into associated pathologies, folding and network assembly

IUCrJ ◽  
2018 ◽  
Vol 5 (6) ◽  
pp. 765-779 ◽  
Author(s):  
Patricia Casino ◽  
Roberto Gozalbo-Rovira ◽  
Jesús Rodríguez-Díaz ◽  
Sreedatta Banerjee ◽  
Ariel Boutaud ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Self Assembly ◽  
Molecular Mechanisms ◽  
Collagen Iv ◽  
Basement Membranes ◽  
Missense Mutations ◽  
The Core ◽  
Extracellular Structures ◽  
Insight Into

Basement membranes are extracellular structures of epithelia and endothelia that have collagen IV scaffolds of triple α-chain helical protomers that associate end-to-end, forming networks. The molecular mechanisms by which the noncollagenous C-terminal domains of α-chains direct the selection and assembly of the α1α2α1 and α3α4α5 hetero-oligomers found in vivo remain obscure. Autoantibodies against the noncollagenous domains of the α3α4α5 hexamer or mutations therein cause Goodpasture's or Alport's syndromes, respectively. To gain further insight into oligomer-assembly mechanisms as well as into Goodpasture's and Alport's syndromes, crystal structures of noncollagenous domains produced by recombinant methods were determined. The spontaneous formation of canonical homohexamers (dimers of trimers) of these domains of the α1, α3 and α5 chains was shown and the components of the Goodpasture's disease epitopes were viewed. Crystal structures of the α2 and α4 noncollagenous domains generated by recombinant methods were also determined. These domains spontaneously form homo-oligomers that deviate from the canonical architectures since they have a higher number of subunits (dimers of tetramers and of hexamers, respectively). Six flexible structural motifs largely explain the architectural variations. These findings provide insight into noncollagenous domain folding, while supporting the in vivo operation of extrinsic mechanisms for restricting the self-assembly of noncollagenous domains. Intriguingly, Alport's syndrome missense mutations concentrate within the core that nucleates the folding of the noncollagenous domain, suggesting that this syndrome, when owing to missense changes, is a folding disorder that is potentially amenable to pharmacochaperone therapy.

scholarly journals Structural heterogeneity in biliverdin modulates spectral properties of Sandercyanin fluorescent protein

2021 ◽  
Author(s):  
Swagatha Ghosh ◽  
Sayan Mondal ◽  
Keerti Yadav ◽  
Shantanu Aggarwal ◽  
Wayne F. Schaefer ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Spectral Properties ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Binding Pocket ◽  
Structural Heterogeneity ◽  
Wild Type ◽  
Absorbing States

Sandercyanin, a blue homo-tetrameric lipocalin protein purified from Canadian walleye (Stizostedion vitreus), is the first far-red fluorescent protein reported in vertebrates. Sandercyanin binds non-covalently to biliverdin IXα (BLA) and fluoresces at 675nm on excitation at 375nm and 635nm. Sandercyanin fluorescence can be harnessed for many in vivo applications when engineered into a stable monomeric form. Here, we report the spectral properties and crystal structures of engineered monomeric Sandercyanin-BLA complexes. Compared to wild-type protein, monomeric Sandercyanin (~18kDa) binds BLA with similar affinities and show a broad red- shifted absorbance spectra but possess reduced quantum efficiency. Crystal structures reveal D-ring pyrrole of BLA rotated around the C14-C15 bond, which is stabilized by neighboring aromatic residues and increased water-mediated polar contacts in the BLA-binding pocket. A tetrameric Sandercyanin variant (Tyr-142-Ala) co-displaying red- and far-red absorbing states, and reduced fluorescence shows similar conformational changes in BLA binding pocket. Our results suggest that D-ring flexibility of BLA and its rearrangement reduces the fluorescence quantum-yield of monomeric Sandercyanin. Structures of monomeric Sandercyanin could be utilized as prototypes to generate bright BLA-inducible fluorescent proteins. Further, our study postulates a mechanism for modulating photo-states in BLA- bound lipocalins, known only in phytochromes till date.

Cooperative self-assembly of cyanines on carboxymethylamylose and other anionic scaffolds as tools for fluorescence-based biochemical sensing

2006 ◽  
Vol 78 (12) ◽  
pp. 2313-2323 ◽  
Author(s):  
David G. Whitten ◽  
Komandoor E. Achyuthan ◽  
Gabriel P. Lopez ◽  
Oh-Kil Kim
Keyword(s):  
Conformational Changes ◽  
Self Assembly ◽  
Random Coil ◽  
Helical Structures ◽  
Force Microscopy ◽  
Helix Formation ◽  
Biochemical Sensing ◽  
Sensing Applications ◽  
Atomic Force ◽  
J Aggregates

We recently found that certain cyanines form tight complexes with carboxymethylamylose (CMA) in aqueous solutions and that in these complexes the cyanine exists as a strongly fluorescent and stable J-aggregate. Cyanine dyes are characterized by their ability to form J-aggregates showing very narrow absorption and fluorescence spectra relative to the monomer. Although they have found uses in sensing applications, the practicability has been limited in many cases due to the low quantum efficiencies for J-aggregate fluorescence. The CMA-cyanine complex is formed by a cooperative self-assembly in which both components undergo conformational changes during the association. The CMA exists as a random coil in solution prior to complex formation; helix formation is prevented due to repulsion of the charges on the carboxymethylated glucose units. The cyanine exists as a nonfluorescent monomer in the same solutions. A helical atomic force microscopy image and large induced circular dichroism (CD) spectra of the cyanine J-aggregate indicate that the self-assembly is a superhelix scaffold of CMA decorated with J-aggregates of the cyanine. Similar behavior was also observed with carboxymethylated cellulose (CMC). Enzymatic disruption of the helical structures (e.g., by the use of amylase to disrupt the structure of CMA helix) leads to the disappearance of the J-aggregate-associated fluorescence. The photophysical behavior and applications of this complex for sensing are discussed.

scholarly journals Two Caenorhabditis elegans calponin-related proteins have overlapping functions to maintain cytoskeletal integrity and are essential for reproduction

2020 ◽  
Author(s):  
Shoichiro Ono ◽  
Kanako Ono
Keyword(s):  
Caenorhabditis Elegans ◽  
Actin Filaments ◽  
Body Wall ◽  
The Body ◽  
Body Wall Muscle ◽  
Related Protein ◽  
Somatic Gonad ◽  
Related Proteins

AbstractMulticellular organisms have multiple genes encoding calponins and calponin-related proteins, and some of these are known to regulate actin cytoskeletal dynamics and contractility. However, functional similarities and differences among these proteins are largely unknown. In the nematode Caenorhabditis elegans, UNC-87 is a calponin-related protein with seven calponin-like (CLIK) motifs and is required for maintenance of contractile apparatuses in muscle cells. Here, we report that CLIK-1, another calponin-related protein that also contains seven CLIK motifs, has an overlapping function with UNC-87 to maintain actin cytoskeletal integrity in vivo and has both common and different actin-regulatory activities in vitro. CLIK-1 is predominantly expressed in the body wall muscle and somatic gonad, where UNC-87 is also expressed. unc-87 mutation causes cytoskeletal defects in the body wall muscle and somatic gonad, whereas clik-1 depletion alone causes no detectable phenotypes. However, simultaneous depletion of clik-1 and unc-87 caused sterility due to ovulation failure by severely affecting the contractile actin networks in the myoepithelial sheath of the somatic gonad. In vitro, UNC-87 bundles actin filaments. However, CLIK-1 binds to actin filaments without bundling them and is antagonistic to UNC-87 in filament bundling. UNC-87 and CLIK-1 share common functions to inhibit cofilin binding and allow tropomyosin binding to actin filaments, suggesting that both proteins stabilize actin filaments. Thus, partially redundant functions of UNC-87 and CLIK-1 in ovulation is likely mediated by their common actin-regulatory activities, but their distinct activities in actin bundling suggest that they also have different biological functions.

scholarly journals Bacterial actin MreB forms antiparallel double filaments

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Fusinita van den Ent ◽  
Thierry Izoré ◽  
Tanmay AM Bharat ◽  
Christopher M Johnson ◽  
Jan Löwe
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Cell Shape ◽  
Antimicrobial Agents ◽  
Nucleotide Hydrolysis ◽  
Site Specific ◽  
E Coli ◽  
Parallel Fashion

Filaments of all actin-like proteins known to date are assembled from pairs of protofilaments that are arranged in a parallel fashion, generating polarity. In this study, we show that the prokaryotic actin homologue MreB forms pairs of protofilaments that adopt an antiparallel arrangement in vitro and in vivo. We provide an atomic view of antiparallel protofilaments of Caulobacter MreB as apparent from crystal structures. We show that a protofilament doublet is essential for MreB's function in cell shape maintenance and demonstrate by in vivo site-specific cross-linking the antiparallel orientation of MreB protofilaments in E. coli. 3D cryo-EM shows that pairs of protofilaments of Caulobacter MreB tightly bind to membranes. Crystal structures of different nucleotide and polymerisation states of Caulobacter MreB reveal conserved conformational changes accompanying antiparallel filament formation. Finally, the antimicrobial agents A22/MP265 are shown to bind close to the bound nucleotide of MreB, presumably preventing nucleotide hydrolysis and destabilising double protofilaments.

scholarly journals Electrostatic lateral interactions drive ESCRT-III heteropolymer assembly

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sudeep Banjade ◽  
Shaogeng Tang ◽  
Yousuf H Shah ◽  
Scott D Emr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Self Assembly ◽  
Electrostatic Interactions ◽  
Membrane Remodeling ◽  
Lateral Interactions ◽  
Vesicle Biogenesis ◽  
Flexible Architectures ◽  
Functional Defects

Self-assembly of ESCRT-III complex is a critical step in all ESCRT-dependent events. ESCRT-III hetero-polymers adopt variable architectures, but the mechanisms of inter-subunit recognition in these hetero-polymers to create flexible architectures remain unclear. We demonstrate in vivo and in vitro that the Saccharomyces cerevisiae ESCRT-III subunit Snf7 uses a conserved acidic helix to recruit its partner Vps24. Charge-inversion mutations in this helix inhibit Snf7-Vps24 lateral interactions in the polymer, while rebalancing the charges rescues the functional defects. These data suggest that Snf7-Vps24 assembly occurs through electrostatic interactions on one surface, rather than through residue-to-residue specificity. We propose a model in which these cooperative electrostatic interactions in the polymer propagate to allow for specific inter-subunit recognition, while sliding of laterally interacting polymers enable changes in architecture at distinct stages of vesicle biogenesis. Our data suggest a mechanism by which interaction specificity and polymer flexibility can be coupled in membrane-remodeling heteropolymeric assemblies.

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