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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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IST1NTD and CHMP1B tube assembly. (a) Assembly of wild type (WT) IST1NTD or three different IST1NTD mutants with the designated amino acid substitutions at the tip of the α1/α2 hairpin (mutated residues are underlined in Fig. 4c). IST1NTD proteins were diluted from concentrated protein stocks in high salt buffers to final concentrations of 62 µM. Salt concentrations in the assembly buffers are provided in the inset key and protein assembly was followed by light scattering at 330 nm. (b) Transmission electron microscopic images of a subset of the wild type and mutant IST1NTD assembly reactions from a. Note that IST1NTD only assembled under low salt conditions (compare upper and middle left panels) and that IST1NTD proteins with different point mutations at the tip of the α1/α2 hairpin did not assemble in either high salt (not shown) or low salt conditions (right panels). An expanded view of a single IST1NTD tube is shown in the lower left panel. Scale bars are 500 nm. (c) Transmission electron microscopic images of CHMP1B tubes assembled under low salt conditions. A field of tubes is shown in the left panel and an expanded view of a single CHMP1B tube is shown in the right panel. Scale bars are 500 nm.
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Figure 6: IST1NTD and CHMP1B tube assembly. (a) Assembly of wild type (WT) IST1NTD or three different IST1NTD mutants with the designated amino acid substitutions at the tip of the α1/α2 hairpin (mutated residues are underlined in Fig. 4c). IST1NTD proteins were diluted from concentrated protein stocks in high salt buffers to final concentrations of 62 µM. Salt concentrations in the assembly buffers are provided in the inset key and protein assembly was followed by light scattering at 330 nm. (b) Transmission electron microscopic images of a subset of the wild type and mutant IST1NTD assembly reactions from a. Note that IST1NTD only assembled under low salt conditions (compare upper and middle left panels) and that IST1NTD proteins with different point mutations at the tip of the α1/α2 hairpin did not assemble in either high salt (not shown) or low salt conditions (right panels). An expanded view of a single IST1NTD tube is shown in the lower left panel. Scale bars are 500 nm. (c) Transmission electron microscopic images of CHMP1B tubes assembled under low salt conditions. A field of tubes is shown in the left panel and an expanded view of a single CHMP1B tube is shown in the right panel. Scale bars are 500 nm.

Mentions: CHMP3 can co-polymerize with truncated CHMP2A proteins to form 50 nm diameter helical tubes in vitro6. As noted above, both IST1 and IST1NTD became insoluble under low ionic strength conditions, and we therefore tested whether these proteins assembled into regular structures. Assembly conditions were initially surveyed using light scattering to follow complex formation. As shown in Fig. 6a, monomeric IST1NTD spontaneously polymerized into high molecular weight complexes that strongly scattered light at 330 nm when the protein was diluted from a high salt buffer (350 mM NaCl) into a low salt buffer (100 mM NaCl). In contrast, IST1NTD did not scatter light when diluted into an otherwise equivalent high salt buffer (350 mM NaCl).


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)

IST1NTD and CHMP1B tube assembly. (a) Assembly of wild type (WT) IST1NTD or three different IST1NTD mutants with the designated amino acid substitutions at the tip of the α1/α2 hairpin (mutated residues are underlined in Fig. 4c). IST1NTD proteins were diluted from concentrated protein stocks in high salt buffers to final concentrations of 62 µM. Salt concentrations in the assembly buffers are provided in the inset key and protein assembly was followed by light scattering at 330 nm. (b) Transmission electron microscopic images of a subset of the wild type and mutant IST1NTD assembly reactions from a. Note that IST1NTD only assembled under low salt conditions (compare upper and middle left panels) and that IST1NTD proteins with different point mutations at the tip of the α1/α2 hairpin did not assemble in either high salt (not shown) or low salt conditions (right panels). An expanded view of a single IST1NTD tube is shown in the lower left panel. Scale bars are 500 nm. (c) Transmission electron microscopic images of CHMP1B tubes assembled under low salt conditions. A field of tubes is shown in the left panel and an expanded view of a single CHMP1B tube is shown in the right panel. Scale bars are 500 nm.
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Figure 6: IST1NTD and CHMP1B tube assembly. (a) Assembly of wild type (WT) IST1NTD or three different IST1NTD mutants with the designated amino acid substitutions at the tip of the α1/α2 hairpin (mutated residues are underlined in Fig. 4c). IST1NTD proteins were diluted from concentrated protein stocks in high salt buffers to final concentrations of 62 µM. Salt concentrations in the assembly buffers are provided in the inset key and protein assembly was followed by light scattering at 330 nm. (b) Transmission electron microscopic images of a subset of the wild type and mutant IST1NTD assembly reactions from a. Note that IST1NTD only assembled under low salt conditions (compare upper and middle left panels) and that IST1NTD proteins with different point mutations at the tip of the α1/α2 hairpin did not assemble in either high salt (not shown) or low salt conditions (right panels). An expanded view of a single IST1NTD tube is shown in the lower left panel. Scale bars are 500 nm. (c) Transmission electron microscopic images of CHMP1B tubes assembled under low salt conditions. A field of tubes is shown in the left panel and an expanded view of a single CHMP1B tube is shown in the right panel. Scale bars are 500 nm.
Mentions: CHMP3 can co-polymerize with truncated CHMP2A proteins to form 50 nm diameter helical tubes in vitro6. As noted above, both IST1 and IST1NTD became insoluble under low ionic strength conditions, and we therefore tested whether these proteins assembled into regular structures. Assembly conditions were initially surveyed using light scattering to follow complex formation. As shown in Fig. 6a, monomeric IST1NTD spontaneously polymerized into high molecular weight complexes that strongly scattered light at 330 nm when the protein was diluted from a high salt buffer (350 mM NaCl) into a low salt buffer (100 mM NaCl). In contrast, IST1NTD did not scatter light when diluted into an otherwise equivalent high salt buffer (350 mM NaCl).

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