scholarly journals Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck

2016 ◽  
Vol 113 (38) ◽  
pp. E5552-E5561 ◽  
Author(s):  
Tatiana B. Stanishneva-Konovalova ◽  
Charlotte F. Kelley ◽  
Tania L. Eskin ◽  
Emily M. Messelaar ◽  
Steven A. Wasserman ◽  
...  
Keyword(s):  
Bound States ◽  
Membrane Binding ◽  
Structural Basis ◽  
Synaptic Growth ◽  
Sh3 Domains ◽  
Actin Organization ◽  
Bar Domain ◽  
Src Homology

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott–Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR–membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.

scholarly journals Eps15 membrane-binding and -bending activity acts redundantly with Fcho1 during clathrin-mediated endocytosis

2016 ◽  
Vol 27 (17) ◽  
pp. 2675-2687 ◽  
Author(s):  
Lei Wang ◽  
Adam Johnson ◽  
Michael Hanna ◽  
Anjon Audhya
Keyword(s):  
Genetic Interaction ◽  
Critical Role ◽  
Membrane Binding ◽  
Accessory Proteins ◽  
Membrane Remodeling ◽  
Bar Domain ◽  
C Elegans ◽  
Accessory Factor

Clathrin coat assembly on membranes requires cytosolic adaptors and accessory proteins, which bridge triskeleons with the lipid bilayer and stabilize lattice architecture throughout the process of vesicle formation. In Caenorhabditis elegans, the prototypical AP-2 adaptor complex, which is activated by the accessory factor Fcho1 at the plasma membrane, is dispensable during embryogenesis, enabling us to define alternative mechanisms that facilitate clathrin-mediated endocytosis. Here we uncover a synthetic genetic interaction between C. elegans Fcho1 (FCHO-1) and Eps15 (EHS-1), suggesting that they function in a parallel and potentially redundant manner. Consistent with this idea, we find that the FCHO-1 EFC/F-BAR domain and the EHS-1 EH domains exhibit highly similar membrane-binding and -bending characteristics in vitro. Furthermore, we demonstrate a critical role for EHS-1 when FCHO-1 membrane-binding and -bending activity is specifically eliminated in vivo. Taken together, our data highlight Eps15 as an important membrane-remodeling factor, which acts in a partially redundant manner with Fcho proteins during the earliest stages of clathrin-mediated endocytosis.

scholarly journals The human GRB2 and Drosophila Drk genes can functionally replace the Caenorhabditis elegans cell signaling gene sem-5.

1993 ◽  
Vol 4 (11) ◽  
pp. 1175-1188 ◽  
Author(s):  
M J Stern ◽  
L E Marengere ◽  
R J Daly ◽  
E J Lowenstein ◽  
M Kokel ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Signaling ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Sh3 Domains ◽  
Signaling Systems ◽  
Src Homology ◽  
And Function

Mutations in the Caenorhabditis elegans gene sem-5 affect cell signaling processes involved in guiding a class of cell migrations and inducing vulval cell fates. The sem-5 sequence encodes a protein comprised almost exclusively of SH2 and SH3 domains (SH, src homology region) that are found together in many signaling proteins and nonreceptor tyrosine kinases. A human protein, GRB2, was identified by its ability to associate with the activated human epidermal growth factor receptor (hEGFR). The GRB2 and Sem-5 proteins share an identical architecture of their SH2 and SH3 domains and 58% amino acid sequence identity. Here we demonstrate that GRB2 and a Drosophila sem-5-like gene Drk can specifically rescue sem-5 mutants. We also show that Sem-5, like GRB2, can bind to the activated hEGFR in vitro. We further correlate the abilities of several mutant variants of GRB2 and Sem-5 to bind to the hEGFR in vitro with their abilities to functionally replace sem-5 in vivo. These data indicate that GRB2 and Drk are functional homologues of Sem-5 and demonstrate the high degree of conservation of both structure and function between signaling systems throughout evolution.

scholarly journals Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Christopher McDonald ◽  
Goran Jovanovic ◽  
Oscar Ces ◽  
Martin Buck
Keyword(s):  
Elastic Stress ◽  
Membrane Binding ◽  
Membrane Integrity ◽  
Regulatory Function ◽  
Membrane Stress ◽  
Inner Membrane ◽  
Thylakoid Biogenesis ◽  
Stress Dependent

ABSTRACTPhage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled,in vitromethodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing rolesin vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. Thisin vitrorecapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mountedin vivowhen a cell's inner membrane experiences increased SCE stress.IMPORTANCEAll cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response.

scholarly journals Kinetic analysis of GTP hydrolysis catalysed by the Arf1-GTP–ASAP1 complex

2007 ◽  
Vol 402 (3) ◽  
pp. 439-447 ◽  
Author(s):  
Ruibai Luo ◽  
Bijan Ahvazi ◽  
Diana Amariei ◽  
Deborah Shroder ◽  
Beatriz Burrola ◽  
...  
Keyword(s):  
Binary Complex ◽  
Gtp Hydrolysis ◽  
Gtpase Activating Proteins ◽  
Steady State Kinetics ◽  
Catalytically Active ◽  
Ras Family ◽  
Src Homology ◽  
Significant Conformational Change

Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a kcat of 57±5 s−1 and a Km of 2.2±0.5 μM determined by steady-state kinetics and a kcat of 56±7 s−1 determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4−), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than Km. Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the kcat. Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP–ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.

scholarly journals Crystal structure of human carbonic anhydrase II at 1.95 Å resolution in complex with 667-coumate, a novel anti-cancer agent

2005 ◽  
Vol 385 (3) ◽  
pp. 715-720 ◽  
Author(s):  
Matthew D. LLOYD ◽  
Richard L. PEDERICK ◽  
Ramanathan NATESH ◽  
L. W. Lawrence WOO ◽  
Atul PUROHIT ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Phase 1 ◽  
Structural Basis ◽  
Pharmacokinetic Studies ◽  
Reversible Binding ◽  
X Ray Crystallography ◽  
Hydrophobic Binding ◽  
Cancer Agent

CA (carbonic anhydrase) catalyses the reversible hydration of carbon dioxide into bicarbonate, and at least 14 isoforms have been identified in vertebrates. The role of CA type II in maintaining the fluid and pH balance has made it an attractive drug target for the treatment of glaucoma and cancer. 667-Coumate is a potent inhibitor of the novel oncology target steroid sulphatase and is currently in Phase 1 clinical trials for hormone-dependent breast cancer. It also inhibits CA II in vitro. In the present study, CA II was crystallized with 667-coumate and the structure was determined by X-ray crystallography at 1.95 Å (1 Å=0.1 nm) resolution. The structure reported here is the first for an inhibitor based on a coumarin ring and shows ligation of the sulphamate group to the active-site zinc at 2.15 Å through a nitrogen anion. The first two rings of the coumarin moiety are bound within the hydrophobic binding site of CA II. Important residues contributing to binding include Val-121, Phe-131, Val-135, Leu-141, Leu-198 and Pro-202. The third seven-membered ring is more mobile and is located in the channel leading to the surface of the enzyme. Pharmacokinetic studies show enhanced stability of 667-coumate in vivo and this has been ascribed to binding of CA II in erythrocytes. This result provides a structural basis for the stabilization and long half-life of 667-coumate in blood compared with its rapid disappearance in plasma, and suggests that reversible binding of inhibitors to CA may be a general method of delivering this type of labile drug.

Methods to study the structure of misfolded protein states in systemic amyloidosis

2021 ◽  
Vol 49 (2) ◽  
pp. 977-985
Author(s):  
Marcus Fändrich ◽  
Matthias Schmidt
Keyword(s):  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Systemic Amyloidosis ◽  
Structural Basis ◽  
Amyloid A ◽  
Serum Amyloid A Protein ◽  
Proteolytic Stability

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

The ADP/ATP carrier is the 32-kilodalton receptor for an NH2-terminally myristylated src peptide but not for pp60src polypeptide

1993 ◽  
Vol 13 (5) ◽  
pp. 3084-3092
Author(s):  
C T Sigal ◽  
M D Resh
Keyword(s):  
Binding Capacity ◽  
Membrane Binding ◽  
Mitochondrial Fraction ◽  
Fatty Acyl ◽  
Affinity Tag ◽  
Peptide Probe ◽  
The Cross ◽  
Docking Protein

Membrane binding of pp60src is initiated via its myristylated NH2 terminus. To identify a candidate pp60src docking protein or receptor in the membrane, a radiolabelled peptide corresponding to the pp60src NH2-terminal membrane binding domain was cross-linked to fibroblast membranes and found to specifically label a 32-kDa protein. This protein was purified by appending an affinity tag to the peptide probe so that the cross-linked complex could be isolated via affinity chromatography. Microsequencing indicated that the 32-kDa protein was the mitochondrial ADP/ATP carrier (AAC). This result was further confirmed by the ability of an antibody to the AAC to immunoprecipitate the cross-linked complex, by the ability of certain inhibitors of the AAC to block cross-linking, and by membrane fractionation to show that complex formation occurred essentially exclusively in the mitochondrial fraction. While the AAC bound the myristyl-src peptide in a specific manner both in vitro and in vivo, its localization to the inner membrane of the mitochondrion precludes its being a pp60src binding protein. An analysis of pp60v-src binding in vitro was consistent with this expectation. Thus, use of a myristyl-src peptide revealed an unexpected and previously unidentified binding capacity of the AAC, most likely related to the ability of long-chain fatty acyl coenzyme As to serve as AAC inhibitors. The amphipathic nature of the pp60src NH2 terminus suggests alternative strategies for uncovering pp60src membrane binding species.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Mechanosensation and Mechanotransduction in Natural Killer Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Matteo Santoni ◽  
Federica Maggi ◽  
Maria Beatrice Morelli ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Tyrosine Phosphatase ◽  
Cell Interaction ◽  
Structural Basis ◽  
Adhesive Interactions

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

Structural characterization of amorphous calcium carbonate-binding protein: an insight into the mechanism of amorphous calcium carbonate formation

2013 ◽  
Vol 453 (2) ◽  
pp. 179-186 ◽  
Author(s):  
Jingtan Su ◽  
Xiao Liang ◽  
Qiang Zhou ◽  
Guiyou Zhang ◽  
Hongzhong Wang ◽  
...  
Keyword(s):  
Calcium Carbonate ◽  
Binding Sites ◽  
Binding Protein ◽  
Homology Modelling ◽  
Size Exclusion ◽  
Structural Basis ◽  
Amorphous Calcium Carbonate ◽  
Carbonate Formation

ACC (amorphous calcium carbonate) plays an important role in biomineralization process for its function as a precursor for calcium carbonate biominerals. However, it is unclear how biomacromolecules regulate the formation of ACC precursor in vivo. In the present study, we used biochemical experiments coupled with bioinformatics approaches to explore the mechanisms of ACC formation controlled by ACCBP (ACC-binding protein). Size-exclusion chromatography, chemical cross-linking experiments and negative staining electron microscopy reveal that ACCBP is a decamer composed of two adjacent pentamers. Sequence analyses and fluorescence quenching results indicate that ACCBP contains two Ca2+-binding sites. The results of in vitro crystallization experiments suggest that one Ca2+-binding site is critical for ACC formation and the other site affects the ACC induction efficiency. Homology modelling demonstrates that the Ca2+-binding sites of pentameric ACCBP are arranged in a 5-fold symmetry, which is the structural basis for ACC formation. To the best of our knowledge, this is the first report on the structural basis for protein-induced ACC formation and it will significantly improve our understanding of the amorphous precursor pathway.

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