Glaza, Przemyslaw

Ranaweera, Chathurange B.

Zolkiewski, Michal

K-State Research Exchange

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/326010764Interaction of substrate-mimicking peptides with the AAA+ ATPase ClpB from Escherichia coliPoster · January 2018DOI: 10.13140/RG.2.2.19936.79363CITATIONS0READS323 authors, including:Some of the authors of this publication are also working on these related projects:Mechanism of Substrate Binding to the Molecular Chaperone ClpB View projectBiochemical and physiological characterization of human protease HtrA3, UMO-2013/09/B/NZ1/01068 View projectChathuranaga RanaweeraKansas State University14 PUBLICATIONS   32 CITATIONS   SEE PROFILEPrzemysław GlazaCaptor Therapeutics Inc.19 PUBLICATIONS   206 CITATIONS   SEE PROFILEAll content following this page was uploaded by Chathuranaga Ranaweera on 27 June 2018.The user has requested enhancement of the downloaded file.Interaction of substrate-mimicking peptides with the AAA+ ATPase ClpB from Escherichia coliChathurange B. Ranaweera, Przemyslaw Glaza, and Michal ZolkiewskiDepartment of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, KS4. The N-terminal domain of ClpB is not required for peptide binding2. Prediction of aggregation-prone segments in ClpB-binding peptides6. Aggregation-prone segments are not necessary to support the peptide binding to ClpB3. ATP, but not ADP, stimulates peptide binding to ClpB7. Summary of the ClpB-peptide interaction affinities 5. Aggregation-prone segments are not sufficient to support the peptide binding to ClpBTruncated peptides that contain only theaggregation-prone segments in B1 and B2interact with ClpB with a lower affinity than thefull-length B1 and B2.This experiment was performed using ClpB-trapin the presence of ATP.B1 and B2 bind to wild type ClpB in the presenceof ATPγS. The N-terminally truncated ΔNClpBbinds the peptides with a higher affinity than thefull-length ClpB.Binding of the peptides to the ClpB-trap variant (3)was associated with an increase in the FITCfluorescence anisotropy. The binding required ATP,which is consistent with the substrate-bindingmechanism of the AAA+ ATPases. The peptidebinding to ClpB showed positive cooperativity,consistent with the linkage between substratebinding and ClpB self-association into hexamers.We investigated the peptides B1 and B2, which havebeen shown to mimic ClpB substrates (1). TheTANGO algorithm (2) predicts that B1 and B2contain discrete aggregation-prone sequencesegments. To test the role of the aggregation-pronesegments in supporting binding to ClpB, we alsoproduced truncated and scrambled derivatives of B1and B2. The peptides have been labeled with FITCat their N termini.All experiments were performed using ClpB-trap inthe presence of ATP. The non-linear least-squaresfitting was performed using GraphPad Prismsoftware.The affinity of the scrambled peptides towards ClpBdoes not correlate with the presence of aggregation-prone regions. This experiment was performedusing ClpB-trap in the presence of ATP.1. ClpB is an energy-dependent protein disaggregase8. ConclusionsPeptide Kd [nM] Hill	CoefficientB1 47 2.4B1T 329 1.7B2 13.2 2.5B2T1 912 1.1B2T2 >1000 0.5N1 7.3 1.4N2 17.6 1.7N3 10.2 0.9N4 11.5 1.3ClpB reactivates aggregated proteins in cooperation with DnaK. The ClpB monomer contains two AAA+ ATP-binding domains (D1, D2), the coiled-coil domain, and the N-terminal domain. The ClpB-mediated protein disaggregation is linked to translocation of substrates through the central channel in the hexameric ClpB.• We tested the hypothesis that the predictedaggregation-prone segments in peptides mediatethe substrate recognition by ClpB. We found thatthe aggregation-prone segments are neithersufficient nor necessary for the peptideinteractions with ClpB.• Our results suggest that the substrate recognitionmechanism of ClpB may rely on global surfaceproperties of aggregated proteins rather than onlocal sequence motifs.References:1. Schlieker et al., Nat. Str. Mol. Biol., 11, 607, 2004.2. Fernandez-Escamilla et al., Nat. Biotech., 22,1302. 2004.3. Barnett et al., J. Biol. Chem., 280, 34940, 2005.Funding:National Institutes of Health, R56 AI121366View publication stats

Interaction of substrate-mimicking peptides with the AAA+ ATPase ClpBfrom Escherichia coli

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Interaction of substrate-mimicking peptides with the AAA+ ATPase ClpBfrom Escherichia coli

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