Structural basis for activation of plasma-membrane Ca2+-ATPase by calmodulin

AbstractHedgehog protein signals mediate tissue patterning and maintenance via binding to and inactivation of their common receptor Patched, a twelve-transmembrane protein that otherwise would suppress activity of the seven-transmembrane protein, Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. The extracellular domain mediates association of Patched monomers in an unusual dimeric architecture that implies curvature in the associated membrane. A central conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Patched expression indeed reduces cholesterol activity in the inner leaflet of the plasma membrane, in a manner antagonized by Hedgehog stimulation and with implications for regulation of Smoothened.

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Fine-Structural Localization of Adenosine Triphosphatase in the Rectum of Calliphora

Journal of Cell Science ◽

10.1242/jcs.3.1.17 ◽

1968 ◽

Vol 3 (1) ◽

pp. 17-32

Author(s):

M. J. BERRIDGE ◽

B. L. GUPTA

Keyword(s):

Plasma Membrane ◽

Adenosine Triphosphatase ◽

Plasma Membranes ◽

Fluid Transport ◽

Microsomal Fraction ◽

Adenosine Diphosphate ◽

Structural Basis ◽

Osmotic Gradient ◽

Ph Optimum ◽

Structural Localization

Adenosine triphosphatase (ATPase) activity in the rectal papillae of Calliphora has been studied by biochemical and histochemical techniques. The microsomal fraction contained a Mg2+-activated ATPase with a pH optimum of 8.0. The enzyme was not stimulated by the addition of Na+ plus K+ and was insensitive to ouabain. Histochemical studies using modifications of the Wachstein-Meisel method showed that at pH 7.2 this Mg2+-activated ATPase was specifically localized on the intracellular surface of the lateral plasma membranes. A similar though less intense reaction was obtained with adenosine diphosphate and inosine triphosphate, but not with guanosine triphosphate, uridine triphosphate or β-glycerophosphate as substrates. At an acid pH (6.6-6.8), very little reaction occurred on the lateral plasma membrane but some reaction product was present in mitochondria and nuclei. Very little enzyme activity was found in the flattened rectal epithelium. These results are discussed in relation to the available data on transport ATPases and on the structural basis of fluid transport by rectal papillae. It is proposed that the ATPase localized on the stacks of lateral plasma membrane may be involved with ion secretion into the intercellular spaces to create the osmotic gradient necessary to extract water from the lumen.

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Fine-Structural Aspects of Fertilization in Chlamydomonas Reinhardi

Journal of Cell Science ◽

10.1242/jcs.3.1.115 ◽

1968 ◽

Vol 3 (1) ◽

pp. 115-128

Author(s):

I. FRIEDMANN ◽

A. L. COLWIN ◽

LAURA H. COLWIN

Keyword(s):

Plasma Membrane ◽

Mating Type ◽

Starch Content ◽

The Body ◽

Structural Basis ◽

Basal Bodies ◽

Cytoplasmic Organelle ◽

Animal Phyla ◽

Small Structure ◽

Specialized Structure

Gametes of C. reinhardi lack the cell wall which vegetative cells possess. Just below the cell apex gametes form a fertilization tubule which is up to 2 µ long and 0.2 µ in diameter; its plasma membrane and that of the apex have slender tubular projections. At the base of the fertilization tubule regularly lies the choanoid body, a collar-shaped cytoplasmic organelle; the plasma membrane overlying the body appears as an electron-dense ring. Gametes possess two ‘free’ basal bodies in addition to the basal bodies of the two flagella. In the initial stage of union the conjugating cells are connected by the fertilization tubule whose plasma membrane is continuous with that of both copulants. At one end of the tubule lies a conspicuous choanoid body, but at the other end is a small structure which possibly is a homologue of the choanoid body. Subsequently, the fertilization tubule shortens and widens until finally no tubule exists and the apical ends of the two protoplasts adjoin. The merging cells then bend like a jack-knife and lateral alignment of the protoplasts occurs. This four-flagellated zygote becomes motile at about the time when the flagellar bases of the former gametes seem to approach each other and when fibrillar elements of the flagellar roots come into contact. In the motile zygote the nuclei do not fuse but remain ensheathed in the cup-shaped plastids of the two gametes. A mating of plus (+) and minus (-) strains cultured, respectively, for high and low starch content suggested that gametes of only the plus (+) mating type contain the choanoid body. Since it appears that the gamete containing the choanoid body also produces the fertilization tubule, it is inferred that gametes of only the plus (+) mating type produce the fertilization tubule. Should further investigation support this inference, it would be established that there is a structural basis for designating the plus (+) mating type as male and the minus (-) type as female. Fertilization involves fusion of the gamete membranes through the mediation of a specialized structure (the fertilization tubule) and in this respect there are similarities to certain aspects of fertilization in animal phyla. The relation of the fertilization tubule to the protoplasmic bridge of other species of Chlamydomonas is discussed.

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Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0602818103 ◽

2006 ◽

Vol 103 (30) ◽

pp. 11364-11369 ◽

Cited By ~ 397

Author(s):

J. S. Saad ◽

J. Miller ◽

J. Tai ◽

A. Kim ◽

R. H. Ghanam ◽

...

Keyword(s):

Plasma Membrane ◽

Virus Assembly ◽

Structural Basis ◽

Gag Proteins ◽

Hiv 1

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Structural basis for plant plasma membrane protein dynamics and organization into functional nanodomains

eLife ◽

10.7554/elife.26404 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 46

Author(s):

Julien Gronnier ◽

Jean-Marc Crowet ◽

Birgit Habenstein ◽

Mehmet Nail Nasir ◽

Vincent Bayle ◽

...

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Super Resolution ◽

Lipid Binding ◽

Single Particle Tracking ◽

Structural Basis ◽

Binding Motif ◽

Supramolecular Organization ◽

Hormonal Crosstalk ◽

Definition Of

Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.

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Model for the architecture of caveolae based on a flexible, net-like assembly of Cavin1 and Caveolin discs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1616838113 ◽

2016 ◽

Vol 113 (50) ◽

pp. E8069-E8078 ◽

Cited By ~ 48

Author(s):

Miriam Stoeber ◽

Pascale Schellenberger ◽

C. Alistair Siebert ◽

Cedric Leyrat ◽

Ari Helenius ◽

...

Keyword(s):

Plasma Membrane ◽

Complex Formation ◽

Membrane Binding ◽

Coiled Coil ◽

Structural Basis ◽

Membrane Domains ◽

Coiled Coil Domain ◽

Regular Dodecahedron ◽

Plasma Membrane Domains ◽

Electron Cryotomography

Caveolae are invaginated plasma membrane domains involved in mechanosensing, signaling, endocytosis, and membrane homeostasis. Oligomers of membrane-embedded caveolins and peripherally attached cavins form the caveolar coat whose structure has remained elusive. Here, purified Cavin1 60S complexes were analyzed structurally in solution and after liposome reconstitution by electron cryotomography. Cavin1 adopted a flexible, net-like protein mesh able to form polyhedral lattices on phosphatidylserine-containing vesicles. Mutating the two coiled-coil domains in Cavin1 revealed that they mediate distinct assembly steps during 60S complex formation. The organization of the cavin coat corresponded to a polyhedral nano-net held together by coiled-coil segments. Positive residues around the C-terminal coiled-coil domain were required for membrane binding. Purified caveolin 8S oligomers assumed disc-shaped arrangements of sizes that are consistent with the discs occupying the faces in the caveolar polyhedra. Polygonal caveolar membrane profiles were revealed in tomograms of native caveolae inside cells. We propose a model with a regular dodecahedron as structural basis for the caveolae architecture.

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Conformational dynamics of auto-inhibition in the ER calcium sensor STIM1

eLife ◽

10.7554/elife.66194 ◽

2021 ◽

Vol 10 ◽

Author(s):

Stijn van Dorp ◽

Ruoyi Qiu ◽

Ucheor B Choi ◽

Minnie M Wu ◽

Michelle Yen ◽

...

Keyword(s):

Plasma Membrane ◽

Single Molecule ◽

Conformational Dynamics ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Bound State ◽

Structural Basis ◽

Resting Cells ◽

Dual Roles ◽

Er Calcium

The dimeric ER Ca2+ sensor STIM1 controls store-operated Ca2+ entry (SOCE) through the regulated binding of its CRAC activation domain (CAD) to Orai channels in the plasma membrane. In resting cells, the STIM1 CC1 domain interacts with CAD to suppress SOCE, but the structural basis of this interaction is unclear. Using single-molecule Förster resonance energy transfer (smFRET) and protein crosslinking approaches, we show that CC1 interacts dynamically with CAD in a domain-swapped configuration with an orientation predicted to sequester its Orai-binding region adjacent to the ER membrane. Following ER Ca2+ depletion and release from CAD, cysteine crosslinking indicates that the two CC1 domains become closely paired along their entire length in the active Orai-bound state. These findings provide a structural basis for the dual roles of CC1: sequestering CAD to suppress SOCE in resting cells and propelling it towards the plasma membrane to activate Orai and SOCE after store depletion.

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Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

10.1101/638890 ◽

2019 ◽

Author(s):

Cosmo Z. Buffalo ◽

Christina M. Stürzel ◽

Elena Heusinger ◽

Dorota Kmiec ◽

Frank Kirchhoff ◽

...

Keyword(s):

Plasma Membrane ◽

Binding Site ◽

Host Defense ◽

Structural Basis ◽

Viral Infectivity ◽

Mhc I ◽

Human Tetherin ◽

Clathrin Adaptor ◽

Α Helix ◽

Tetherin Antagonism

AbstractTetherin is a host defense that physically prevents escape of virions from the plasma membrane. Human tetherin lacks the motif DIWK antagonized by SIV, the antecedent of HIV. Here, we reconstituted the AP-2 clathrin adaptor complex with a simian tetherin and SIV Nef and determined its structure by cryo-EM. Nef refolds the first α-helix of the β2 subunit of AP-2 to a β hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of MHC-I, CD3, and CD4, but overlaps the site for SERINC5 restricting viral infectivity. The structure explains the dependence of SIVs on the host tetherin DIWK sequence and the consequent barrier to human transmission.

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The structural basis of the oncogenic mutant K-Ras4B homodimers

10.1101/2020.09.07.285783 ◽

2020 ◽

Author(s):

Kayra Kosoglu ◽

Meltem Eda Omur ◽

Hyunbum Jang ◽

Ruth Nussinov ◽

Ozlem Keskin ◽

...

Keyword(s):

Plasma Membrane ◽

Structural Basis ◽

Ras Proteins ◽

Oncogenic Ras ◽

Extracellular Stimuli ◽

Prediction Server ◽

Physical Interactions ◽

Multiple Conformations ◽

Dynamics Simulations ◽

Membrane Signaling

AbstractRas proteins activate their effectors through physical interactions in response to the various extracellular stimuli at the plasma membrane. Oncogenic Ras forms dimer and nanoclusters at the plasma membrane, boosting the downstream MAPK signal. It was reported that K-Ras4B can dimerize through two major interfaces: (i) the effector lobe interface, mapped to Switch I and effector binding regions; (ii) the allosteric lobe interface involving α3 and α4 helices. Recent experiments showed that constitutively active, oncogenic mutant K-Ras4BG12D dimers are enriched in the plasma membrane. Here, we perform molecular dynamics simulations of K-Ras4BG12D homodimers aiming to quantify the two major interfaces in atomic level. To examine the effect of mutations on dimerization, two double mutations, K101D/R102E on the allosteric lobe and R41E/K42D on the effector lobe interfaces were added to the K-Ras4BG12D dimer simulations. We observed that the effector lobe K-Ras4BG12D dimer is stable, while the allosteric lobe dimer alters its helical interface during the simulations, presenting multiple conformations. The K101D/R102E mutations slightly weakens the allosteric lobe interface. However, the R41E/K42D mutations disrupt the effector lobe interface. Using the homo-oligomers prediction server, we obtained trimeric, tetrameric, and pentameric complexes with the allosteric lobe K-Ras4BG12D dimers. However, the allosteric lobe dimer with the K101D/R102E mutations is not capable of generating multiple higher order structures. Our detailed interface analysis may help to develop inhibitor design targeting functional Ras dimerization and high order oligomerization at the membrane signaling platform.

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Structural basis of diverse membrane target recognitions by ankyrins

eLife ◽

10.7554/elife.04353 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 40

Author(s):

Chao Wang ◽

Zhiyi Wei ◽

Keyu Chen ◽

Fei Ye ◽

Cong Yu ◽

...

Keyword(s):

Nervous System ◽

Plasma Membrane ◽

Binding Sites ◽

Cell Adhesion Molecules ◽

Structural Basis ◽

Membrane Domains ◽

Regulatory Domain ◽

Multiple Target ◽

Membrane Target ◽

Target Binding

Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target proteins with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins' membrane targets, with implications for other proteins containing extended ANK repeat domains.

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