Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity

Gudrun Rutsdottir, Johan Härmark, Yoran Weide, Hans Hebert, Morten I. Rasmussen, Sven Wernersson, Michal Respondek, Mikael Akke, Peter Højrup, Philip J.B. Koeck, Christopher A.G. Söderberg*, Cecilia Emanuelsson

*Corresponding author for this work

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Abstract

Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

Original languageEnglish
JournalJournal of Biological Chemistry
Volume292
Issue number19
Pages (from-to)8103-8121
ISSN0021-9258
DOIs
Publication statusPublished - 2017

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Small Heat-Shock Proteins
Heat-Shock Proteins
Protein C
Plant Structures
Oligomerization
Workflow
Model structures
X ray scattering
Oligomers
Methionine
Dimers
Mass spectrometry
Proteins
Agglomeration
Nuclear magnetic resonance
X-Rays

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Rutsdottir, Gudrun ; Härmark, Johan ; Weide, Yoran ; Hebert, Hans ; Rasmussen, Morten I. ; Wernersson, Sven ; Respondek, Michal ; Akke, Mikael ; Højrup, Peter ; Koeck, Philip J.B. ; Söderberg, Christopher A.G. ; Emanuelsson, Cecilia. / Structural model of dodecameric heat-shock protein Hsp21 : Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 19. pp. 8103-8121.
@article{161034c975c04541a2dc96479b84e571,
title = "Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity",
abstract = "Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.",
author = "Gudrun Rutsdottir and Johan H{\"a}rmark and Yoran Weide and Hans Hebert and Rasmussen, {Morten I.} and Sven Wernersson and Michal Respondek and Mikael Akke and Peter H{\o}jrup and Koeck, {Philip J.B.} and S{\"o}derberg, {Christopher A.G.} and Cecilia Emanuelsson",
year = "2017",
doi = "10.1074/jbc.M116.766816",
language = "English",
volume = "292",
pages = "8103--8121",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "19",

}

Rutsdottir, G, Härmark, J, Weide, Y, Hebert, H, Rasmussen, MI, Wernersson, S, Respondek, M, Akke, M, Højrup, P, Koeck, PJB, Söderberg, CAG & Emanuelsson, C 2017, 'Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity', Journal of Biological Chemistry, vol. 292, no. 19, pp. 8103-8121. https://doi.org/10.1074/jbc.M116.766816

Structural model of dodecameric heat-shock protein Hsp21 : Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity. / Rutsdottir, Gudrun; Härmark, Johan; Weide, Yoran; Hebert, Hans; Rasmussen, Morten I.; Wernersson, Sven; Respondek, Michal; Akke, Mikael; Højrup, Peter; Koeck, Philip J.B.; Söderberg, Christopher A.G.; Emanuelsson, Cecilia.

In: Journal of Biological Chemistry, Vol. 292, No. 19, 2017, p. 8103-8121.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Structural model of dodecameric heat-shock protein Hsp21

T2 - Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity

AU - Rutsdottir, Gudrun

AU - Härmark, Johan

AU - Weide, Yoran

AU - Hebert, Hans

AU - Rasmussen, Morten I.

AU - Wernersson, Sven

AU - Respondek, Michal

AU - Akke, Mikael

AU - Højrup, Peter

AU - Koeck, Philip J.B.

AU - Söderberg, Christopher A.G.

AU - Emanuelsson, Cecilia

PY - 2017

Y1 - 2017

N2 - Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

AB - Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

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U2 - 10.1074/jbc.M116.766816

DO - 10.1074/jbc.M116.766816

M3 - Journal article

C2 - 28325834

AN - SCOPUS:85019453209

VL - 292

SP - 8103

EP - 8121

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 19

ER -