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Structural basis for ESCRT-III protein autoinhibition.

Bajorek M, Schubert HL, McCullough J, Langelier C, Eckert DM, Stubblefield WM, Uter NT, Myszka DG, Hill CP, Sundquist WI - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member.The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains.Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah, Salt Lake City, Utah, USA.

ABSTRACT
Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission. The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains. Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

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CHMP3 activation in vitro. (a) Ribbon diagram showing the locations of mutated CHMP3 residues at the tip of the α1/α2 hairpin (purple) and activating mutations on either side of the interface between the core α2 helix (cyan) and the autoinhibitory α5 helix (magenta). The red arrow suggests how the closed CHMP3 conformation might convert into an open conformation by dissociation of the autoinhibitory α5 helix from the core. (b) GST pull-down analyses of the binary CHMP3/CHMP2A interaction. Pure recombinant CHMP2A (lower panel, anti-CHMP2A) was tested for binding to a glutathione sepharose matrix (lane 2, negative control) or to immobilized wild type (lane 3) or mutant GST-CHMP3 proteins (lanes 4–8). Both proteins were detected by Western blotting, and input CHMP2A (0.3%) is shown in lane 1 for reference. (c) EM analyses of helical CHMP3-CHMP2A assembly. Different panels show assemblies formed by 1:1 mixtures CHMP2A with: full length, wild type CHMP3 (panel 1, negative control, no assembly), CHMP31–150 core domain (panel 2, positive control), activated CHMP3V48D,A64D (panel 3), activated CHMP3I168D,L169D (panel 4), and activated and tip mutant CHMP3I168D,L169D,V59D,V62D (panel 5, no assemblies). Arrows highlight rings (1), tubes (2), and cones/tapered tubes (3). Scale bars are 100 nm.
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Figure 7: CHMP3 activation in vitro. (a) Ribbon diagram showing the locations of mutated CHMP3 residues at the tip of the α1/α2 hairpin (purple) and activating mutations on either side of the interface between the core α2 helix (cyan) and the autoinhibitory α5 helix (magenta). The red arrow suggests how the closed CHMP3 conformation might convert into an open conformation by dissociation of the autoinhibitory α5 helix from the core. (b) GST pull-down analyses of the binary CHMP3/CHMP2A interaction. Pure recombinant CHMP2A (lower panel, anti-CHMP2A) was tested for binding to a glutathione sepharose matrix (lane 2, negative control) or to immobilized wild type (lane 3) or mutant GST-CHMP3 proteins (lanes 4–8). Both proteins were detected by Western blotting, and input CHMP2A (0.3%) is shown in lane 1 for reference. (c) EM analyses of helical CHMP3-CHMP2A assembly. Different panels show assemblies formed by 1:1 mixtures CHMP2A with: full length, wild type CHMP3 (panel 1, negative control, no assembly), CHMP31–150 core domain (panel 2, positive control), activated CHMP3V48D,A64D (panel 3), activated CHMP3I168D,L169D (panel 4), and activated and tip mutant CHMP3I168D,L169D,V59D,V62D (panel 5, no assemblies). Arrows highlight rings (1), tubes (2), and cones/tapered tubes (3). Scale bars are 100 nm.

Mentions: Like the IST1-CHMP1 pair, CHMP2A and CHMP3 bind one another preferentially. To investigate this interaction further, we used GST pulldown assays to test for binary interactions between CHMP2A and GST-CHMP3 (Figs. 7a,b). These experiments were performed at low protein concentrations where higher order assemblies were not detectable (35µM CHMP2A, ~7 µM GST-CHMP3). CHMP2A protein bound GST-CHMP3 (Fig. 7b, lane 3) but did not bind a matrix control (lane 2). CHMP2A also bound the truncated GST-CHMP31–150 protein (lane 4), indicating that CHMP2A interacted with the CHMP3 core and that the binary interaction was not dramatically affected by removal of the terminal CHMP3 autoinhibitory sequences. In contrast, a double point mutation (V59D, V62D) that disrupted the exposed hydrophobic surface on one side of the tip of the CHMP3 α1/ α2 hairpin eliminated CHMP2A binding (lane 5). These data suggest that CHMP2A and CHMP3 interact through a tip-to-tip type interaction, and demonstrate that the binary CHMP2A-CHMP3 and IST1-CHMP1B pairs interact via different binding surfaces and therefore form structurally distinct complexes.


Structural basis for ESCRT-III protein autoinhibition.

Bajorek M, Schubert HL, McCullough J, Langelier C, Eckert DM, Stubblefield WM, Uter NT, Myszka DG, Hill CP, Sundquist WI - Nat. Struct. Mol. Biol. (2009)

CHMP3 activation in vitro. (a) Ribbon diagram showing the locations of mutated CHMP3 residues at the tip of the α1/α2 hairpin (purple) and activating mutations on either side of the interface between the core α2 helix (cyan) and the autoinhibitory α5 helix (magenta). The red arrow suggests how the closed CHMP3 conformation might convert into an open conformation by dissociation of the autoinhibitory α5 helix from the core. (b) GST pull-down analyses of the binary CHMP3/CHMP2A interaction. Pure recombinant CHMP2A (lower panel, anti-CHMP2A) was tested for binding to a glutathione sepharose matrix (lane 2, negative control) or to immobilized wild type (lane 3) or mutant GST-CHMP3 proteins (lanes 4–8). Both proteins were detected by Western blotting, and input CHMP2A (0.3%) is shown in lane 1 for reference. (c) EM analyses of helical CHMP3-CHMP2A assembly. Different panels show assemblies formed by 1:1 mixtures CHMP2A with: full length, wild type CHMP3 (panel 1, negative control, no assembly), CHMP31–150 core domain (panel 2, positive control), activated CHMP3V48D,A64D (panel 3), activated CHMP3I168D,L169D (panel 4), and activated and tip mutant CHMP3I168D,L169D,V59D,V62D (panel 5, no assemblies). Arrows highlight rings (1), tubes (2), and cones/tapered tubes (3). Scale bars are 100 nm.
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Figure 7: CHMP3 activation in vitro. (a) Ribbon diagram showing the locations of mutated CHMP3 residues at the tip of the α1/α2 hairpin (purple) and activating mutations on either side of the interface between the core α2 helix (cyan) and the autoinhibitory α5 helix (magenta). The red arrow suggests how the closed CHMP3 conformation might convert into an open conformation by dissociation of the autoinhibitory α5 helix from the core. (b) GST pull-down analyses of the binary CHMP3/CHMP2A interaction. Pure recombinant CHMP2A (lower panel, anti-CHMP2A) was tested for binding to a glutathione sepharose matrix (lane 2, negative control) or to immobilized wild type (lane 3) or mutant GST-CHMP3 proteins (lanes 4–8). Both proteins were detected by Western blotting, and input CHMP2A (0.3%) is shown in lane 1 for reference. (c) EM analyses of helical CHMP3-CHMP2A assembly. Different panels show assemblies formed by 1:1 mixtures CHMP2A with: full length, wild type CHMP3 (panel 1, negative control, no assembly), CHMP31–150 core domain (panel 2, positive control), activated CHMP3V48D,A64D (panel 3), activated CHMP3I168D,L169D (panel 4), and activated and tip mutant CHMP3I168D,L169D,V59D,V62D (panel 5, no assemblies). Arrows highlight rings (1), tubes (2), and cones/tapered tubes (3). Scale bars are 100 nm.
Mentions: Like the IST1-CHMP1 pair, CHMP2A and CHMP3 bind one another preferentially. To investigate this interaction further, we used GST pulldown assays to test for binary interactions between CHMP2A and GST-CHMP3 (Figs. 7a,b). These experiments were performed at low protein concentrations where higher order assemblies were not detectable (35µM CHMP2A, ~7 µM GST-CHMP3). CHMP2A protein bound GST-CHMP3 (Fig. 7b, lane 3) but did not bind a matrix control (lane 2). CHMP2A also bound the truncated GST-CHMP31–150 protein (lane 4), indicating that CHMP2A interacted with the CHMP3 core and that the binary interaction was not dramatically affected by removal of the terminal CHMP3 autoinhibitory sequences. In contrast, a double point mutation (V59D, V62D) that disrupted the exposed hydrophobic surface on one side of the tip of the CHMP3 α1/ α2 hairpin eliminated CHMP2A binding (lane 5). These data suggest that CHMP2A and CHMP3 interact through a tip-to-tip type interaction, and demonstrate that the binary CHMP2A-CHMP3 and IST1-CHMP1B pairs interact via different binding surfaces and therefore form structurally distinct complexes.

Bottom Line: Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member.The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains.Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Utah, Salt Lake City, Utah, USA.

ABSTRACT
Endosomal sorting complexes required for transport-III (ESCRT-III) subunits cycle between two states: soluble monomers and higher-order assemblies that bind and remodel membranes during endosomal vesicle formation, midbody abscission and enveloped virus budding. Here we show that the N-terminal core domains of increased sodium tolerance-1 (IST1) and charged multivesicular body protein-3 (CHMP3) form equivalent four-helix bundles, revealing that IST1 is a previously unrecognized ESCRT-III family member. IST1 and its ESCRT-III binding partner, CHMP1B, both form higher-order helical structures in vitro, and IST1-CHMP1 interactions are required for abscission. The IST1 and CHMP3 structures also reveal that equivalent downstream alpha5 helices can fold back against the core domains. Mutations within the CHMP3 core-alpha5 interface stimulate the protein's in vitro assembly and HIV-inhibition activities, indicating that dissociation of the autoinhibitory alpha5 helix from the core activates ESCRT-III proteins for assembly at membranes.

Show MeSH
Related in: MedlinePlus