Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 May 22;5(5):eaav8421.
doi: 10.1126/sciadv.aav8421. eCollection 2019 May.

HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C

Miranda P Collier  1 , T Reid Alderson  1 , Carin P de Villiers  2 , Daisy Nicholls  1 , Heidi Y Gastall  1 , Timothy M Allison  1   3 , Matteo T Degiacomi  1   4 , He Jiang  2 , Georg Mlynek  5 , Dieter O Fürst  6 , Peter F M van der Ven  6 , Kristina Djinovic-Carugo  5   7 , Andrew J Baldwin  1 , Hugh Watkins  2 , Katja Gehmlich  2   8 , Justin L P Benesch  1
Affiliations

HspB1 phosphorylation regulates its intramolecular dynamics and mechanosensitive molecular chaperone interaction with filamin C

Miranda P Collier et al. Sci Adv. .

Abstract

Mechanical force-induced conformational changes in proteins underpin a variety of physiological functions, typified in muscle contractile machinery. Mutations in the actin-binding protein filamin C (FLNC) are linked to musculoskeletal pathologies characterized by altered biomechanical properties and sometimes aggregates. HspB1, an abundant molecular chaperone, is prevalent in striated muscle where it is phosphorylated in response to cues including mechanical stress. We report the interaction and up-regulation of both proteins in three mouse models of biomechanical stress, with HspB1 being phosphorylated and FLNC being localized to load-bearing sites. We show how phosphorylation leads to increased exposure of the residues surrounding the HspB1 phosphosite, facilitating their binding to a compact multidomain region of FLNC proposed to have mechanosensing functions. Steered unfolding of FLNC reveals that its extension trajectory is modulated by the phosphorylated region of HspB1. This may represent a posttranslationally regulated chaperone-client protection mechanism targeting over-extension during mechanical stress.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. FLNC and HspB1 are up-regulated in MLP KO mouse heart and interact via domains 18 to 21 in a phosphorylation-dependent manner.
(A) Schematic of FLNC composition and localization in striated muscle at the Z-disc and intercalated disc. Gray lines, multiple Z-disc proteins, including α-actinin, desmin, etc. Adaptors: talin, vinculin, catenins, etc. PM, plasma membrane. ABD, actin-binding domain of FLNC. Not pictured: possible localization at costameres. (B) Frozen sections of ventricular tissue from WT and MLP KO mice. Sections were stained for FLNC and counterstained to visualize sarcomeres (myomesin) and intercalated discs (N-cadherin). Scale bar, 10 μm. FLNC is primarily localized at intercalated discs and sarcomeric Z-discs (blue and red striped arrows, respectively) and is more abundant in the DCM tissue. (C) Immunoprecipitation (IP) from MLP KO ventricular tissue lysate using an FLNC-specific antibody or isotype control and lysate control. Molecular masses from positions of markers. Asterisk indicates signal from antibody chains. HspB1 coprecipitates with FLNC, evidencing an interaction between the endogenous proteins. (D) Western blots from WT and MLP KO mouse heart homogenates for FLNC, HspB1, PHspB1, and GAPDH (glyceraldehyde phosphate dehydrogenase) as a loading control. Both FLNC and HspB1 were up-regulated in MLP KO hearts, and HspB1 was phosphorylated at Ser86 (equivalent to human Ser82). (E) Recombinant EEF-tagged FLNCd18–21 mixed with T7-tagged HspB1 and immunoprecipitated. T7 antibody (but not an isotype control) pulled down both HspB1 and FLNCd18–21, indicating binding between the recombinant proteins. Neither protein is pulled down when T7-HspB1 is omitted from the sample. (F) Pelleted fraction of mixtures of recombinant FLNCd18–21 and WT or 3PHspB1 over the course of 90-min incubation at 25°C. Measurements by densitometry of SDS–polyacrylamide gel electrophoresis (PAGE).
Fig. 2
Fig. 2. HspB177–171undergoes dynamical changes leading to partial dissociation upon phosphomimicry.
(A) Overlaid 1H-15N heteronuclear single-quantum coherence (HSQC) spectra of HspB184–171 (gray), HspB177–171 (pink), and 2PHspB177–171 (maroon). More signals are present in the region 7.8 to 8.6 ppm upon phosphomimicry. (B) N-H order parameters (S2) derived using assigned backbone chemical shifts from 2PHspB177–171 (maroon) and from MD simulations of HspB184–171 (gray) confirm that the PPR and β2 strand are highly dynamic (top). A plot of Rex, a qualitative metric of microsecond-millisecond motions probed by 15N CPMG relaxation dispersion, for HspB184–171 (gray) and 2PHspB177–171 (maroon) reveals a significant increase in dynamics in residues 95 to 110 (bottom). Secondary structure elements from PDB 4MJH are indicated. Rex data are means ± SD. (C) Snapshots from a 1-μs MD simulation of HspB184–171 showing the complete dissociation of a β2 strand. Residues are colored according to Rex [from (B)], showing the region of increased dynamics to be in the β3-β4 loop on the side of the ACD. (D and E) Native mass spectra and stoichiometric quantification of HspB177–171 WT (pink), 1P (PSer82, yellow), and 2P (PSer78-PSer82, maroon). The construct populates a monomer-dimer equilibrium and phosphomimicry shifts the oligomeric state toward the monomer. Data are means ± SD.
Fig. 3
Fig. 3. Crystal structure of the HspB1 ACD in complex with a partial peptide mimic of the PPR.
(A) HspB1 ACD dimer with peptide PPR mimic bound. Dotted line indicates the antiparallel interdimer interface. (B) Areas indicated by black arrows in (A) showing two locations of the β2 strand: bound to β3 (left) or dissociated from the ACD and reaching across the β4-β8 groove of a neighboring monomer (right). (C) PPR binding [red arrow in (A)], shown as an overlay of the four peptide-bound monomers in the asymmetric unit. Whereas the ACDs overlay closely, the peptides appear heterogeneous and flexible. Rotated view shows β2 strand divergences, sometimes arcing toward the peptide binding site and in one instance partially modeled as continuation of the peptide.
Fig. 4
Fig. 4. (P)HspB180–88 and FLNCd18–21 form a complex that extends in a phosphorylation-dependent manner.
(A) The location of HspB1 region 80 to 88, the FLNCd18–21 binding epitope used as a peptide in subsequent IM-MS experiments, is indicated on each subunit of the HspB184–170 dimer structure. (B) Contour plots of native mass spectra of FLNCd18–21 obtained upon titrating the unphosphorylated (top) and phosphorylated (middle) peptides. Association of multiple peptides is observed, based on the emergence of new peaks in the mass spectra, appearing as additional horizontal streaks in the plot. Pink lines denote peptide-bound FLNC; line thickness designates charge series and hence the same stoichiometry. Binding curves, fit using standard methods and assuming up to three equivalent binding sites, reveal similar affinities of each peptide to FLNCd18–21 (bottom). Data are means ± SEM. (C) Dissociation of the peptide was affected by activation in the gas phase, with the phosphorylated peptide remaining bound to higher potentials than the unphosphorylated equivalent. Breakdown curves (derived from tandem MS data, see fig. S5) reveal clear differences between the peptides—shown are continuous single experiments, with error bars representing one SD as calculated from repeats. Mass spectra underlying dashed lines in (B) are projected to the right. At 40 V, binding differences are negligible, but at 140 V, all of the unphosphorylated peptide has been ejected but the phosphopeptide has not. (D) Coulombically steered unfolding of the 13+ charge stage of FLNCd18–21 unbound, bound to one HspB180–88, and bound to one PHspB180–88. Bottom, overlaid arrival time distributions at 30 and 90 V, highlighting the persistence of the intermediate state with bound phosphopeptide (*). Lines on unfolding plots designate the activation required to transition half of the intermediate to the final state. (E) Schematic of force-induced changes to FLNC underlying the unfolding plots and relation to HspB1 peptide binding. The right panel contextualizes mechanical stress and the full-length proteins.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Luo T., Mohan K., Iglesias P. A., Robinson D. N., Molecular mechanisms of cellular mechanosensing. Nat. Mater. 12, 1064–1071 (2013). - PMC - PubMed
    1. Tarone G., Brancaccio M., Keep your heart in shape: Molecular chaperone networks for treating heart disease. Cardiovasc. Res. 102, 346–361 (2014). - PubMed
    1. Willis M. S., Patterson C., Proteotoxicity and cardiac dysfunction—Alzheimer’s disease of the heart? N. Engl. J. Med. 368, 455–464 (2013). - PubMed
    1. Ferrer I., Olivé M., Molecular pathology of myofibrillar myopathies. Expert Rev. Mol. Med. 10, e25 (2008). - PubMed
    1. Haslbeck M., Weinkauf S., Buchner J., Small heat shock proteins: Simplicity meets complexity. J. Biol. Chem. 294, 2121–2132 (2018). - PMC - PubMed

Publication types

MeSH terms