Limits...
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.

Show MeSH

Related in: MedlinePlus

Mutational analyses of IST1-CHMP1B interactions. (a) Sensorgrams showing different concentrations of purified CHMP1B binding to immobilized GST-IST1NTD. Triplicate measurements in response units (RU) are shown for each CHMP1B concentration. (b) Representative biosensor binding isotherms showing CHMP1B binding to wild type (WT) and mutant GST-IST1NTD proteins with strong (S), intermediate (I) and weak (W) binding affinities. IST1NTD mutations and estimated dissociation constants are given in the inset. Errors represent either standard deviations from multiple independent measurements (n≥3), or standard deviations derived from single isotherms measured in triplicate (values in parentheses report the standard deviations in the final digit of the measurement).(c) Ribbon diagram showing the location of all IST1NTD mutations tested for CHMP1 binding. Mutated residues are shown explicitly, and the binding affinities of the mutant proteins are color-coded as follows: blue, strong (S) binding (binding affinities within 1.5-fold of wild type IST1NTD); green, intermediate (I) binding (binding affinity reduced 1.5–8 fold); magenta, weak (W) binding (binding affinity reduced ≥8-fold).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2712734&req=5

Figure 4: Mutational analyses of IST1-CHMP1B interactions. (a) Sensorgrams showing different concentrations of purified CHMP1B binding to immobilized GST-IST1NTD. Triplicate measurements in response units (RU) are shown for each CHMP1B concentration. (b) Representative biosensor binding isotherms showing CHMP1B binding to wild type (WT) and mutant GST-IST1NTD proteins with strong (S), intermediate (I) and weak (W) binding affinities. IST1NTD mutations and estimated dissociation constants are given in the inset. Errors represent either standard deviations from multiple independent measurements (n≥3), or standard deviations derived from single isotherms measured in triplicate (values in parentheses report the standard deviations in the final digit of the measurement).(c) Ribbon diagram showing the location of all IST1NTD mutations tested for CHMP1 binding. Mutated residues are shown explicitly, and the binding affinities of the mutant proteins are color-coded as follows: blue, strong (S) binding (binding affinities within 1.5-fold of wild type IST1NTD); green, intermediate (I) binding (binding affinity reduced 1.5–8 fold); magenta, weak (W) binding (binding affinity reduced ≥8-fold).

Mentions: Many ESCRT-III proteins bind one another, and previous studies have shown that the N-terminal domain of IST1 binds preferentially to the C-terminal region of the CHMP1 subset of ESCRT-III proteins11–14. To map the IST1 binding surface more precisely, we created an ensemble of IST1 proteins with point mutations at conserved, surface-exposed residues and tested them for CHMP1A and CHMP1B interactions using: 1) immunoprecipitation (IP) reactions in 293T cells (not shown), 2) GST-pulldown experiments with recombinant proteins (not shown), and 3) biosensor binding experiments in which pure recombinant CHMP1B bound immobilized GST-IST1NTD (Fig. 4).


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)

Mutational analyses of IST1-CHMP1B interactions. (a) Sensorgrams showing different concentrations of purified CHMP1B binding to immobilized GST-IST1NTD. Triplicate measurements in response units (RU) are shown for each CHMP1B concentration. (b) Representative biosensor binding isotherms showing CHMP1B binding to wild type (WT) and mutant GST-IST1NTD proteins with strong (S), intermediate (I) and weak (W) binding affinities. IST1NTD mutations and estimated dissociation constants are given in the inset. Errors represent either standard deviations from multiple independent measurements (n≥3), or standard deviations derived from single isotherms measured in triplicate (values in parentheses report the standard deviations in the final digit of the measurement).(c) Ribbon diagram showing the location of all IST1NTD mutations tested for CHMP1 binding. Mutated residues are shown explicitly, and the binding affinities of the mutant proteins are color-coded as follows: blue, strong (S) binding (binding affinities within 1.5-fold of wild type IST1NTD); green, intermediate (I) binding (binding affinity reduced 1.5–8 fold); magenta, weak (W) binding (binding affinity reduced ≥8-fold).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2712734&req=5

Figure 4: Mutational analyses of IST1-CHMP1B interactions. (a) Sensorgrams showing different concentrations of purified CHMP1B binding to immobilized GST-IST1NTD. Triplicate measurements in response units (RU) are shown for each CHMP1B concentration. (b) Representative biosensor binding isotherms showing CHMP1B binding to wild type (WT) and mutant GST-IST1NTD proteins with strong (S), intermediate (I) and weak (W) binding affinities. IST1NTD mutations and estimated dissociation constants are given in the inset. Errors represent either standard deviations from multiple independent measurements (n≥3), or standard deviations derived from single isotherms measured in triplicate (values in parentheses report the standard deviations in the final digit of the measurement).(c) Ribbon diagram showing the location of all IST1NTD mutations tested for CHMP1 binding. Mutated residues are shown explicitly, and the binding affinities of the mutant proteins are color-coded as follows: blue, strong (S) binding (binding affinities within 1.5-fold of wild type IST1NTD); green, intermediate (I) binding (binding affinity reduced 1.5–8 fold); magenta, weak (W) binding (binding affinity reduced ≥8-fold).
Mentions: Many ESCRT-III proteins bind one another, and previous studies have shown that the N-terminal domain of IST1 binds preferentially to the C-terminal region of the CHMP1 subset of ESCRT-III proteins11–14. To map the IST1 binding surface more precisely, we created an ensemble of IST1 proteins with point mutations at conserved, surface-exposed residues and tested them for CHMP1A and CHMP1B interactions using: 1) immunoprecipitation (IP) reactions in 293T cells (not shown), 2) GST-pulldown experiments with recombinant proteins (not shown), and 3) biosensor binding experiments in which pure recombinant CHMP1B bound immobilized GST-IST1NTD (Fig. 4).

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