Spin.niddk.nih.gov



Supplementary Information for:Conditional disorder in small heat-shock proteinsT. Reid Alderson1,2, Jinfa Ying2, Ad Bax2*, Justin L. P. Benesch1*, Andrew J. Baldwin1*1Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, South Parks Road, Oxford, OX1 3QZ, UK2Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA* Correspondence to: andrew.baldwin@chem.ox.ac.uk, bax@, justin.benesch@chem.ox.ac.ukKeywords: NMR, molecular chaperone, small heat-shock protein, conditional disorder, 15N relaxation, residual dipolar couplingsSupplementary Figure 1. Histidine residues in the ACD and a pH titration followed by NMR. (A) Crystal structure of the HSP27 ACD dimer (PDB: 4mjh) with the dotted line denoting separate monomers. Each ACD monomer contains four histidine residues that are located in the β2 strand (H90), the region between β3 and β4 (H103), the β5 strand (H124), and the loop between the β5 strand and the β6+7 strand (H131). Previous NMR data indicate that protonation of H124 at pH values below 7 is responsible for shifting the dimer-monomer equilibrium toward the monomer ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/s41467-019-08557-8","PMID":"30842409","abstract":"The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mam-malian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.","author":[{"dropping-particle":"","family":"Alderson","given":"T Reid","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Roche","given":"Julien","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gastall","given":"Heidi Y","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dias","given":"David M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Priti?anac","given":"Iva","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ying","given":"Jinfa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bax","given":"Ad","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Benesch","given":"Justin L P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Baldwin","given":"Andrew J","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature Communications","id":"ITEM-1","issued":{"date-parts":[["2019"]]},"page":"1-16","title":"Local unfolding of the HSP27 monomer regulates chaperone activity","type":"article-journal","volume":"10"},"uris":[""]},{"id":"ITEM-2","itemData":{"DOI":"10.1007/s12192-017-0783-z","ISSN":"1355-8145","author":[{"dropping-particle":"","family":"Clouser","given":"Amanda F.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Klevit","given":"Rachel E.","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Cell Stress and Chaperones","id":"ITEM-2","issued":{"date-parts":[["2017","3","22"]]},"page":"1-7","publisher":"Springer Netherlands","title":"pH-dependent structural modulation is conserved in the human small heat shock protein HSBP1","type":"article-journal"},"uris":[""]},{"id":"ITEM-3","itemData":{"DOI":"10.7554/eLife.07304","ISSN":"2050-084X","PMID":"25962097","abstract":"Small heat shock proteins (sHSPs) are essential 'holdase' chaperones that form large assemblies and respond dynamically to pH and temperature stresses to protect client proteins from aggregation. While the alpha-crystallin domain (ACD) dimer of sHSPs is the universal building block, how the ACD transmits structural changes in response to stress to promote holdase activity is unknown. We found that the dimer interface of HSPB5 is destabilized over physiological pHs and a conserved histidine (His-104) controls interface stability and oligomer structure in response to acidosis. Destabilization by pH or His-104 mutation shifts the ACD from dimer to monomer but also results in a large expansion of HSPB5 oligomer states. Remarkably, His-104 mutant-destabilized oligomers are efficient holdases that reorganize into structurally distinct client-bound complexes. Our data support a model for sHSP function wherein cell stress triggers small perturbations that alter the ACD building blocks to unleash a cryptic mode of chaperone action.","author":[{"dropping-particle":"","family":"Rajagopal","given":"Ponni","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Tse","given":"Eric","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Borst","given":"Andrew J","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Delbecq","given":"Scott P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Shi","given":"Lei","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Southworth","given":"Daniel R","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Klevit","given":"Rachel E","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"eLife","id":"ITEM-3","issued":{"date-parts":[["2015","1"]]},"title":"A conserved histidine modulates HSPB5 structure to trigger chaperone activity in response to stress-related acidosis.","type":"article-journal","volume":"4"},"uris":[""]}],"mendeley":{"formattedCitation":"[1–3]","plainTextFormattedCitation":"[1–3]","previouslyFormattedCitation":"[1–3]"},"properties":{"noteIndex":0},"schema":""}[1–3]. (B) 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 (residues 84-171) at a total protein concentration of 250 ?M in 30 mM sodium phosphate, 2 mM EDTA buffer as a function of pH. The C137S mutation was included to prevent disulfide formation. The pH values are indicated in the lower-right corner of each panel. The resonance from G116 near 105/8.8 ppm is boxed and the M and D refer to monomer and dimer, respectively, as determined previously ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1038/s41467-019-08557-8","PMID":"30842409","abstract":"The small heat-shock protein HSP27 is a redox-sensitive molecular chaperone that is expressed throughout the human body. Here, we describe redox-induced changes to the structure, dynamics, and function of HSP27 and its conserved α-crystallin domain (ACD). While HSP27 assembles into oligomers, we show that the monomers formed upon reduction are highly active chaperones in vitro, but are susceptible to self-aggregation. By using relaxation dispersion and high-pressure nuclear magnetic resonance (NMR) spectroscopy, we observe that the pair of β-strands that mediate dimerisation partially unfold in the monomer. We note that numerous HSP27 mutations associated with inherited neuropathies cluster to this dynamic region. High levels of sequence conservation in ACDs from mam-malian sHSPs suggest that the exposed, disordered interface present in free monomers or oligomeric subunits may be a general, functional feature of sHSPs.","author":[{"dropping-particle":"","family":"Alderson","given":"T Reid","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Roche","given":"Julien","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Gastall","given":"Heidi Y","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Dias","given":"David M","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Priti?anac","given":"Iva","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ying","given":"Jinfa","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bax","given":"Ad","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Benesch","given":"Justin L P","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Baldwin","given":"Andrew J","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Nature Communications","id":"ITEM-1","issued":{"date-parts":[["2019"]]},"page":"1-16","title":"Local unfolding of the HSP27 monomer regulates chaperone activity","type":"article-journal","volume":"10"},"uris":[""]}],"mendeley":{"formattedCitation":"[1]","plainTextFormattedCitation":"[1]","previouslyFormattedCitation":"[1]"},"properties":{"noteIndex":0},"schema":""}[1]. Resonances from interfacial residues are circled and annotated in the pH 7 spectrum. The circles are present in the remaining spectra to highlight the location of the resonance frequencies at pH 7. Spectra were recorded on a 14.1 T spectrometer with the temperature unit set to 25 °C. Notably, only ca. 60 resonances are observed in the pH 5 spectrum compared to 75 resonances at pH 7. Supplementary Figure 2. Inter-molecular association of the cHSP27 monomer at elevated concentrations. 2D 1H-15N HSQC spectra of 15N-labeled cHSP27 monomer in 30 mM sodium phosphate, 2 mM EDTA buffer at pH 4.2 and 298 K. The protein concentration was 0.15 mM (A) or 0.45 mM (B). The spectra were acquired on a spectrometer operating at 14.1 T with identical parameters and processed in the same manner.Supplementary Figure 3. Resonance assignments and secondary chemical shifts of the acid-unfolded form of cHSP27. (A) Assigned spectrum of acid-unfolded cHSP27 at pH 3 at a static magnetic field of 14.1 T and 25 °C. (B-D) Secondary chemical shifts, Δδ, for 1HN, 13CO, and 13Cα. Neighbor-corrected random coil chemical shifts ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1007/s10858-018-0166-5","ISSN":"1573-5001","PMID":"29399725","abstract":"Chemical shifts contain important site-specific information on the structure and dynamics of proteins. Deviations from statistical average values, known as random coil chemical shifts (RCCSs), are extensively used to infer these relationships. Unfortunately, the use of imprecise reference RCCSs leads to biased inference and obstructs the detection of subtle structural features. Here we present a new method, POTENCI, for the prediction of RCCSs that outperforms the currently most authoritative methods. POTENCI is parametrized using a large curated database of chemical shifts for protein segments with validated disorder; It takes pH and temperature explicitly into account, and includes sequence-dependent nearest and next-nearest neighbor corrections as well as second-order corrections. RCCS predictions with POTENCI show root-mean-square values that are lower by 25-78%, with the largest improvements observed for 1Hα and 13C'. It is demonstrated how POTENCI can be applied to analyze subtle deviations from RCCSs to detect small populations of residual structure in intrinsically disorder proteins that were not discernible before. POTENCI source code is available for download, or can be deployed from the URL .","author":[{"dropping-particle":"","family":"Nielsen","given":"Jakob Toudahl","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Mulder","given":"Frans A A","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of Biomolecular NMR","id":"ITEM-1","issue":"3","issued":{"date-parts":[["2018"]]},"page":"141-165","title":"POTENCI: prediction of temperature, neighbor and pH-corrected chemical shifts for intrinsically disordered proteins.","type":"article-journal","volume":"70"},"uris":[""]}],"mendeley":{"formattedCitation":"[4]","plainTextFormattedCitation":"[4]","previouslyFormattedCitation":"[4]"},"properties":{"noteIndex":0},"schema":""}[4] were subtracted from the experimentally measured values. The uniformly small values indicate that the protein is disordered, but the small deviations from zero are suggestive of transient secondary structure. Slightly negative ΔδCα values are suggestive of low populations of extended conformations. Supplementary Figure 4. Parameters obtained from the modelfree analysis of the cHSP27 monomer. The 15N relaxation data (Figure 3) from the cHSP27 monomer were fit to Lipari-Szabo models that included a local τc value. (A) The locally fitted τc values are shown as a function of residue number. Excluding the disordered N-terminal region and the locally unfolded interfacial region, the average value of τc, <τc>, is 5.89 ± 0.8 ns, which closely agrees with the value of the global τc determined by 15N R2/R1 ratios (6.0 ± 0.8 ns). (B) τe values describe rapid internal motion of N-H bond vectors on the picosecond timescale. The residues that required τe values are shown here. (C) Exchange contributions to R2 mainly cluster to the interfacial region, suggesting evidence of conformational exchange on the millisecond timescale that was not suppressed by the 1 kHz spin lock applied during the 15N R1ρ experiments.Supplementary Figure 5. Analysis of 1DNH couplings for the cHSP27 dimer and monomer (A) Correlation plot showing the measured values of 1DNH for the cHSP27 dimer (x-axis) or monomer (y-axis) in solutions of 4.2% PEG-hexanol (see Methods). The solid line indicates y = x. The graph on the left shows residues only in secondary structural elements (n = 36), and the graph on the right depicts all residues (n = 75). The values of Pearson’s correlation coefficient (R2) are indicated above each graph. (B) Comparison of experimentally measured (y-axis) and predicted (x-axis) values of 1DNH for the cHSP27 dimer or monomer (C). The solid lines depict y = x. The graphs on the left show residues located in regions of secondary structure and the graphs on the right depict all residues. The Q factors ADDIN CSL_CITATION {"citationItems":[{"id":"ITEM-1","itemData":{"DOI":"10.1021/ja9812610","ISSN":"0002-7863","author":[{"dropping-particle":"","family":"Cornilescu","given":"Gabriel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Marquardt","given":"John L.","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Ottiger","given":"Marcel","non-dropping-particle":"","parse-names":false,"suffix":""},{"dropping-particle":"","family":"Bax","given":"Ad","non-dropping-particle":"","parse-names":false,"suffix":""}],"container-title":"Journal of the American Chemical Society","id":"ITEM-1","issue":"27","issued":{"date-parts":[["1998","7"]]},"page":"6836-6837","title":"Validation of Protein Structure from Anisotropic Carbonyl Chemical Shifts in a Dilute Liquid Crystalline Phase","type":"article-journal","volume":"120"},"uris":[""]}],"mendeley":{"formattedCitation":"[5]","plainTextFormattedCitation":"[5]"},"properties":{"noteIndex":0},"schema":""}[5] are indicated above each graph. Table S1. 1DNH values for the cHSP27 monomer and dimer recorded at 900 MHz in samples that were aligned in 4.3% PEG-hexanol. Columns that contain a superscript refer to the monomer and those with b refer to the dimer. The values under the column “Predicted (Hz)” refer to those back-calculated from the crystal structure using the fitted alignment tensor. See Methods for additional details.ResRDC (Hz)aError (Hz)aPredicted (Hz)aRDC (Hz)bError (Hz)bPredicted (Hz)b851.90.01N/AN/AN/AN/A863.20.0120.80.10.218.8873.60.0217.57.40.123.7883.20.0119.25.30.113.7895.20.025.710.90.122.3905.00.0214.5N/AN/A8.9914.20.0211.49.51.820.0926.10.039.615.40.520.2934.90.1019.90.30.523.79413.70.2021.014.00.420.79515.50.5610.712.00.8-7.79615.30.4017.0N/AN/A28.99721.00.5218.225.50.624.39817.80.3019.529.70.728.49920.20.3220.219.20.524.110019.80.3520.229.50.729.31014.80.97-2.6N/AN/A0.41021.20.919.624.23.026.01033.20.396.2-3.11.817.010418.60.41-22.5-4.81.0-9.010512.01.88-6.9-2.92.310.51070.33.21-1.2-8.61.6-11.11080.40.535.123.72.921.61098.50.4117.625.61.223.611018.50.4520.712.00.316.011121.40.5221.425.10.524.311219.10.3017.28.20.32.711319.50.3219.720.40.223.311416.50.2817.811.00.615.711517.10.4217.60.61.64.011617.80.4211.79.71.61.71178.50.5719.222.90.914.311813.20.6218.424.71.224.511919.70.7017.74.40.05.512020.90.5021.320.51.725.812118.31.0314.03.54.03.312220.20.5018.320.70.721.312315.10.9011.614.44.012.712417.80.9017.413.15.312.312510.90.3516.517.94.012.11264.30.085.723.61.022.012710.00.2017.122.01.227.91287.60.2518.816.73.527.61292.70.30-16.9-7.84.0-36.01300.50.46-31.0-29.12.7-31.91313.40.28-26.5-37.33.2-44.51328.10.25-9.8-11.93.2-19.61336.30.32-10.42.83.6-25.51348.60.6717.825.41.622.91358.50.6020.312.51.617.11366.80.3620.6N/AN/A23.313711.60.3619.714.13.913.813815.30.2219.920.82.712.213918.20.4218.823.61.211.414021.91.1021.126.63.426.014118.80.4912.920.32.5-1.314218.60.6620.623.41.323.514318.30.3417.78.90.311.81445.00.280.423.20.419.71470.20.20-6.113.80.214.514811.60.2518.8-1.70.311.41493.30.28-4.414.00.211.915117.90.36-15.3-27.41.5-19.015213.50.3012.123.82.423.21538.10.219.322.41.120.81546.40.316.424.50.424.215518.30.2220.925.80.324.515619.50.2618.126.70.414.315719.90.2821.422.80.323.915820.50.2618.023.20.424.4160-26.80.44-31.6-37.31.8-41.816112.00.2420.6-6.10.719.916212.60.2113.718.70.617.216315.80.506.16.11.0-5.316422.00.4119.232.50.629.416517.80.4418.124.40.525.516612.60.4015.028.30.728.716711.40.549.225.90.723.91696.50.010.021.50.318.91716.60.01N/A13.10.1N/ASupplementary ReferencesADDIN Mendeley Bibliography CSL_BIBLIOGRAPHY [1]T.R. Alderson, J. Roche, H.Y. Gastall, D.M. Dias, I. Priti?anac, J. Ying, A. Bax, J.L.P. Benesch, A.J. Baldwin, Local unfolding of the HSP27 monomer regulates chaperone activity, Nat. Commun. 10 (2019) 1–16. doi:10.1038/s41467-019-08557-8.[2]A.F. Clouser, R.E. Klevit, pH-dependent structural modulation is conserved in the human small heat shock protein HSBP1, Cell Stress Chaperones. (2017) 1–7. doi:10.1007/s12192-017-0783-z.[3]P. Rajagopal, E. Tse, A.J. Borst, S.P. Delbecq, L. Shi, D.R. Southworth, R.E. Klevit, A conserved histidine modulates HSPB5 structure to trigger chaperone activity in response to stress-related acidosis., Elife. 4 (2015). doi:10.7554/eLife.07304.[4]J.T. Nielsen, F.A.A. Mulder, POTENCI: prediction of temperature, neighbor and pH-corrected chemical shifts for intrinsically disordered proteins., J. Biomol. NMR. 70 (2018) 141–165. doi:10.1007/s10858-018-0166-5.[5]G. Cornilescu, J.L. Marquardt, M. Ottiger, A. Bax, Validation of Protein Structure from Anisotropic Carbonyl Chemical Shifts in a Dilute Liquid Crystalline Phase, J. Am. Chem. Soc. 120 (1998) 6836–6837. doi:10.1021/ja9812610. ................
................

In order to avoid copyright disputes, this page is only a partial summary.

Google Online Preview   Download