Browsing by Subject "Protein Structure, Quaternary"

Now showing 1 - 5 of 5
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    Curved FtsZ protofilaments generate bending forces on liposome membranes.
    (The EMBO journal, 2009-11) Osawa, Masaki; Anderson, David E; Erickson, Harold P
    We have created FtsZ-YFP-mts where an amphipathic helix on the C-terminus tethers FtsZ to the membrane. When incorporated inside multi-lamellar tubular liposomes, FtsZ-YFP-mts can assemble Z rings that generate a constriction force. When added to the outside of liposomes, FtsZ-YFP-mts bound and produced concave depressions, bending the membrane in the same direction as the Z ring inside liposomes. Prominent membrane tubules were then extruded at the intersections of concave depressions. We tested the effect of moving the membrane-targeting sequence (mts) from the C-terminus to the N-terminus, which is approximately 180 degrees from the C-terminal tether. When mts-FtsZ-YFP was applied to the outside of liposomes, it generated convex bulges, bending the membrane in the direction opposite to the concave depressions. We conclude that FtsZ protofilaments have a fixed direction of curvature, and the direction of membrane bending depends on which side of the bent protofilament the mts is attached to. This supports models in which the FtsZ constriction force is generated by protofilament bending.
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    FtsZ filament capping by MciZ, a developmental regulator of bacterial division.
    (Proceedings of the National Academy of Sciences of the United States of America, 2015-04-06) Bisson-Filho, Alexandre W; Discola, Karen F; Castellen, Patrícia; Blasios, Valdir; Martins, Alexandre; Sforça, Maurício L; Garcia, Wanius; Zeri, Ana Carolina M; Erickson, Harold P; Dessen, Andréa; Gueiros-Filho, Frederico J
    Cytoskeletal structures are dynamically remodeled with the aid of regulatory proteins. FtsZ (filamentation temperature-sensitive Z) is the bacterial homolog of tubulin that polymerizes into rings localized to cell-division sites, and the constriction of these rings drives cytokinesis. Here we investigate the mechanism by which the Bacillus subtilis cell-division inhibitor, MciZ (mother cell inhibitor of FtsZ), blocks assembly of FtsZ. The X-ray crystal structure reveals that MciZ binds to the C-terminal polymerization interface of FtsZ, the equivalent of the minus end of tubulin. Using in vivo and in vitro assays and microscopy, we show that MciZ, at substoichiometric levels to FtsZ, causes shortening of protofilaments and blocks the assembly of higher-order FtsZ structures. The findings demonstrate an unanticipated capping-based regulatory mechanism for FtsZ.
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    Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9.
    (Nature, 2011-11-23) McLellan, JS; Pancera, M; Carrico, C; Gorman, J; Julien, JP; Khayat, R; Louder, R; Pejchal, R; Sastry, M; Dai, K; O'Dell, S; Patel, N; Shahzad ul Hussan, S; Yang, Y; Zhang, B; Zhou, T; Zhu, J; Boyington, JC; Chuang, GY; Diwanji, D; Georgiev, I; Kwon, YD; Lee, D; Louder, MK; Moquin, S; Schmidt, SD; Yang, ZY; Bonsignori, M; Crump, JA; Kapiga, SH; Sam, NE; Haynes, BF; Burton, DR; Koff, WC; Walker, LM; Phogat, S; Wyatt, R; Orwenyo, J; Wang, LX; Arthos, J; Bewley, CA; Mascola, JR; Nabel, GJ; Schief, WR; Ward, AB; Wilson, IA; Kwong, PD
    Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded β-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which-with PG9-involves a site of vulnerability comprising just two glycans and a strand.
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    The structure of irisin reveals a novel intersubunit β-sheet fibronectin type III (FNIII) dimer: implications for receptor activation.
    (The Journal of biological chemistry, 2013-11) Schumacher, Maria A; Chinnam, Nagababu; Ohashi, Tomoo; Shah, Riddhi Sanjay; Erickson, Harold P
    Irisin was recently identified as a putative myokine that is induced by exercise. Studies suggest that it is produced by cleavage of the FNDC5 (fibronectin domain-containing protein 5) receptor; irisin corresponds to the extracellular receptor ectodomain. Data suggesting that irisin stimulates white-to-brown fat conversion have led to the hypothesis that it does so by binding an unknown receptor, thus functioning as a myokine. As brown fat promotes energy dissipation, myokines that elicit the transformation of white to brown fat have potentially profound benefits in the treatment of obesity and metabolic disorders. Understanding the molecular basis for such exercise-induced phenomena is thus of considerable interest. Moreover, FNDC5-like receptors are highly conserved and have been shown to be critical for neuronal development. However, the structural and molecular mechanisms utilized by these proteins are currently unknown. Here, we describe the crystal structure and biochemical characterization of the FNDC5 ectodomain, corresponding to the irisin myokine. The 2.28 Å structure shows that irisin consists of an N-terminal fibronectin III (FNIII)-like domain attached to a flexible C-terminal tail. Strikingly, the FNIII-like domain forms a continuous intersubunit β-sheet dimer, previously unobserved for any FNIII protein. Biochemical data confirm that irisin is a dimer and that dimerization is unaffected by glycosylation. This finding suggests a possible mechanism for receptor activation by the irisin domain as a preformed myokine dimer ligand or as a paracrine or autocrine dimerization module on FNDC5-like receptors.
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    Visualization of arrestin recruitment by a G-protein-coupled receptor.
    (Nature, 2014-08-14) Shukla, Arun K; Westfield, Gerwin H; Xiao, Kunhong; Reis, Rosana I; Huang, Li-Yin; Tripathi-Shukla, Prachi; Qian, Jiang; Li, Sheng; Blanc, Adi; Oleskie, Austin N; Dosey, Anne M; Su, Min; Liang, Cui-Rong; Gu, Ling-Ling; Shan, Jin-Ming; Chen, Xin; Hanna, Rachel; Choi, Minjung; Yao, Xiao Jie; Klink, Bjoern U; Kahsai, Alem W; Sidhu, Sachdev S; Koide, Shohei; Penczek, Pawel A; Kossiakoff, Anthony A; Woods, Virgil L; Kobilka, Brian K; Skiniotis, Georgios; Lefkowitz, Robert J
    G-protein-coupled receptors (GPCRs) are critically regulated by β-arrestins, which not only desensitize G-protein signalling but also initiate a G-protein-independent wave of signalling. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor (β2AR)-G-protein complex, has provided novel insights into the structural basis of receptor activation. However, complementary information has been lacking on the recruitment of β-arrestins to activated GPCRs, primarily owing to challenges in obtaining stable receptor-β-arrestin complexes for structural studies. Here we devised a strategy for forming and purifying a functional human β2AR-β-arrestin-1 complex that allowed us to visualize its architecture by single-particle negative-stain electron microscopy and to characterize the interactions between β2AR and β-arrestin 1 using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and chemical crosslinking. Electron microscopy two-dimensional averages and three-dimensional reconstructions reveal bimodal binding of β-arrestin 1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of crosslinked residues suggest engagement of the finger loop of β-arrestin 1 with the seven-transmembrane core of the receptor. In contrast, focal areas of raised HDX levels indicate regions of increased dynamics in both the N and C domains of β-arrestin 1 when coupled to the β2AR. A molecular model of the β2AR-β-arrestin signalling complex was made by docking activated β-arrestin 1 and β2AR crystal structures into the electron microscopy map densities with constraints provided by HDX-MS and crosslinking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented here provides a framework for better understanding the basis of GPCR regulation by arrestins.