This readme.txt file was generated on 20220510 by Cristina Olivieri Recommended citation for the data: Olivieri, Cristina; Veglia, Gianluigi. (2022). Changes in protein kinase A conformational entropy by ATP-competitive inhibitors. Retrieved from the Data Repository for the University of Minnesota, https://doi.org/10.13020/kswg-6d07 ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset Changes in protein kinase A conformational entropy by ATP-competitive inhibitors 2. Author Information Principal Investigator Contact Information Name: Gianlugi Veglia Institution: Biochemistry, Molecular Biology, and Biophysics (BMBB) Department, University of Minnesota Twin-Cities Address: 321 Church Street SE, Minneapolis, MN 55455 Email: vegli001@umn.edu ORCID: orcid.org/0000-0002-2795-6964 Associate or Co-investigator Contact Information Name: Cristina Olivieri Institution: BMBB Department, University of Minnesota Twin-Cities Address: 312 Church Street SE, NHH 5-256, Minneapolis, MN 55455 Email: colivier2umn.edu ORCID: orcid.org/0000-0002-2957-5452 3. Date published or finalized for release: 2022 4. Date of data collection (single date, range, approximate date) 2015-2021 5. Geographic location of data collection (where was data collected?): BMBB Department, Structural Biology Division, University of Minnestota Twin-Cities, Minneapolis, Minnesota, MN 55455 6. Information about funding sources that supported the collection of the data: Sponsorship: NIH GM100310 and S10 OD021536 to Gianluigi Veglia 7. Overview of the data (abstract): NMR characterization of the structural and dynamic changes of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) in complex with balanol and H89, two ATP-competitive inhibitors. This study aims to unveil how nucleotide or inibitor binding can be use to modulate the activity, the structure and the dynamics of PKA-C. These data are part of a paper that has been sent to a peer-review journal. -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: Attribution-NonCommercial-NoDerivs 3.0 United States 2. Links to publications that cite or use the data: The paper is under review at Science Advances (AAAS). The title of the pubblication is: "ATP-Competitive Inhibitors Modulate the Substrate Binding Cooperativity of a Kinase by Altering its Conformational Entropy" 3. Was data derived from another source? If yes, list source(s): Yes. Some of the data used for this pubblication/repository were previously pubblished in: Kim J, Li G, Walters MA, Taylor SS, Veglia G. Uncoupling Catalytic and Binding Functions in the Cyclic AMP-Dependent Protein Kinase A. Structure. 2016 Mar 1;24(3):353-63. doi: 10.1016/j.str.2015.11.016. Epub 2016 Jan 28. PMID: 26833386; PMCID: PMC4775281. 4. Terms of Use: Data Repository for the U of Minnesota (DRUM) By using these files, users agree to the Terms of Use. https://conservancy.umn.edu/pages/drum/policies/#terms-of-use --------------------- DATA & FILE OVERVIEW --------------------- 1. File List A. Filename: NMR_List_file.xlsx Short description: Proton and Nitrogen chemical shift files from NMR TROSY-HSQC spectra of PKA-C bound to balanol and H89, with or w/t pseudo-substrate peptide (PKI) B. Filename: Conformational_entropy.xlsx Short description: Calculation of the conformational energy derived from the fast-dynamics of the methyl group side chains of Isoleucine, Leucine, and Valin (ILV) residues of PKA-C bound to nucleotide/inhibitors C. Filename: Correlation_score.xlsx Short description: Correlation scores vs. residue derived from CHESCA analysis for the balanol/ and H89/PKA-C complexes 2. Relationship between files: The 1H (proton) - 15N (nitrogen) chemical shift reported in NMR_List_file.xlsx has been used to calculate the correlation score (Correlation_score.xlsx) and the CHESCA matrix. The matlab script used for these analysis has been previously deposited in DRUM (https://doi.org/10.13020/7DB9-VD97 - file: CHESCA.m) -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. Description of methods used for collection/generation of data: NMR experiments. NMR measurements were performed on a Bruker Avance spectrometer operating at a 1H Larmor frequency of 900 MHz equipped with a cryogenic probe or on a Bruker Avance III 850 MHz spectrometer equipped with a TCI cryoprobe. All data were acquired at 300 K. The typical NMR sample was prepared in the following buffer 20 mM KH2PO4 (pH 6.5), 90 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM NaN3, and 10% D2O. The inhibitor-bound complexes were prepared by saturating the enzyme with 1.35 mM balanol and 2 mM H89. PKA-C concentration was usually 200 uM. For the spectra of the ternary complex (PKA-C/inhibitor/psuedosubstrate), the enzyme-pesudosubstrate molar ratio was 1:1.2. The 2D backbone amide spectrum was acquired using the CLEAN-TROSY version of the original TROSY-HSQC pulse sequence on the 850 MHz spectrometer. The data were acquired using 120 (t1) x 2048 (t2) complex points using 16 scans and a recycle delay of 2.0 seconds. The order parameters were recorded using triple quantum-based 1H relaxation violated choerence transfer cross-correlation experiment. The methyl-labeled PKA-C (ILV) samples concentration ranged from 200 to 250 uM. The correlation time used for the calculation of the order parameters was determined using dynamic light scattering (DLS). Single quantum (SQ) and triple quantum (TQ) filtered spectra were acquired with relaxation delays (T) of 3, 6, 9, 12, 15, 20, 25, 30, 35 ms using 2048 x 160 complex data points and with a recycle delay of 2 seconds. 2. Methods for processing the data: The NMR spectra were processed usign NMRPipe and analyzed using Sparky. 3. Instrument- or software-specific information needed to interpret the data: Topship (Bruker) used to acquire the NMR spectra, given by the company upon request. NMRPipe can be downloaded here: https://spin.niddk.nih.gov/NMRPipe/install/legacy.html NMRFAM-SParky can be downloaded here: https://nmrfam.wisc.edu/nmrfam-sparky-distribution/ The chesca analysis has been implemented in NMRFAM-Sparky, but for our analysis we have used the deposited MatLab script 4. Standards and calibration information, if appropriate: Read the methods section 5. Environmental/experimental conditions: Read the methods section 6. Describe any quality-assurance procedures performed on the data: All the spectra were acquired at least 2 times and before each SQ/TQ acquisition the pulse sequence and spectrometer functionality were tested unsing a standard ubiquitin sample by the operator. 7. People involved with sample collection, processing, analysis and/or submission: Dr. Cristina Olivieri, Dr. Jonggul Kim, Dr. Geoffrey Li ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: [FILENAME] ----------------------------------------- NMR_List_file.xlsx 1. Number of variables: 6 (each sheet) 2. Number of cases/rows: 260 (each sheet) 3. Missing data codes: Not applicable. 4. Variable List A. Name: Assignment Description: name of the amide residue B. Name: w1 (ppm) Description: nitrogen chemical shift C. Name: w2 (ppm) Description: proton chemical shift D. Name: S/N Description: signal to noise ratio directly calculated by NMRFAM-Sparky E. Name: lw1 (hz) Description: nitrogen lineweight in hertz F. Name: lw2 (hz) Description: proton lineweight in hertz 5. Tabs in spreadsheet A. PKA-C_Balanol Description: Backbone chemical shift list file for PKA-C bound to Balanol B. PKA-C_Balanol_PKI Description: Backbone chemical shift list file for PKA-C bound to Balanol/PKI C. PKA-C_H89 Description: Backbone chemical shift list file for PKA-C bound to H89 D. PKA-C_H89_PKI Description: Backbone chemical shift list file for PKA-C bound to H89/PKI Correlation_score.xlsx (CorrelationScore_per_residue tab) 1. Number of variables: 6 2. Number of cases/rows: 351 3. Missing data codes: A. Code: 0 Description: Not possible to calculate the value. 4. Variable List A. Name: #residue Description: residue number B. Name: ATPgN Description: correlation score calculated for ATPgN C. Name:Adenosine Description: correlation score calculated for Adenosine D. Name: Balanol Description: correlation score calculated for Balanol E. Name: AMP Description: correlation score calculated for AMP F. Name: H89 Description: correlation score calculated for H89 5. Description of CorrelationScore_per_residue: Correlation score per residue calculated for each nucleotide/inibitor PKA-C complex Correlation_score.xlsx (lnSigma_vs_CorrScoreVs tab) 1. Number of variables:5 2. Number of cases/rows: 5 3. Missing data codes: Not applicable. 4. Variable List A. Name: (column A) Description: list of the nucleotide/inhibitor analyzed B. Name: lnσ (column B) Description: natural log of the log of the cooperativity coofficient (σ) C. Name:lnσ (column C) Description: error associated to the natural log of the cooperativity coofficient (σ) D. Name: (column D) Description: Average of the correlation score calculated for each nucleotide/inhibitor (the raw data are reported in the same excel file, different sheet) E. Name: Normalized (column E) Description: normalized value of the correlation score in respect to the ATPgN 5. Description of InSigma_vs_CorrScoreVs tab: Linear correlation between the average of the correlation score and the natural log of the binding coefficient (σ) calculated for each nucleotide/inhibitor PKA-C complex Conformational_entropy.xlsx (MethylOrdPar_O2 tab) 1. Number of variables:15 2. Number of cases/rows: 53 3. Missing data codes: Code/symbol: 0 Definition: Not possible to calculate the value. Code/symbol: [blank] Definition: Not possible to calculate the value. 4. Variable List A. Name: #Residue (column A) Description: number of the residue B. Name: APO (column B) Description: order parameter (O2) calculated for the apo for of PKA-C C. Name: (column C) Description: error associated to the O2 reported in column B D. Name: H89 (column D) Description: order parameter (O2) calculated for PKA-C/H89 complex E. Name: (column E) Description: error associated to the O2 reported in column D F. Name: AMP (column F) Description: order parameter (O2) calculated for PKA-C/AMP complex G. Name: (column G) Description: error associated to the O2 reported in column F H. Name: Balanol (column H) Description: order parameter (O2) calculated for PKA-C/balanol complex I. Name: (column I) Description: error associated to the O2 reported in column H J. Name: Adenosine (column J) Description: order parameter (O2) calculated for PKA-C/Adenosine complex K. Name: (column K) Description: error associated to the O2 reported in column J L. Name: ADP (column L) Description: order parameter (O2) calculated for PKA-C/ADP complex M. Name: (column M) Description: error associated to the O2 reported in column L N. Name: ATPgN (column N) Description: order parameter (O2) calculated for PKA-C/ATPgN complex O. Name: (column O) Description: error associated to the O2 reported in column N 5. Description of MethylOrdPar_O2 tab: Methyl order parameter calculated for the ILV residues of PKA-C in the apo state and bound to nucleotide/inhibitor. The description of how these values are calculated is reported in the Methods section of the paper. Conformational_entropy.xlsx (MethylOrdPar_deltaO2) 1. Number of variables:13 2. Number of cases/rows: 53 3. Missing data codes: Not applicable 4. Variable List A. Name: #residue (column A) Description: number of the residue B. Name: H89 (column B) Description: subtraction of the O2 value calculated for apo from the one calculated for H89 Basically column D - Column B from MethylOrdPar_O2 sheet C. Name: (column C) Description: error associated to the ΔO2 reported in column B D. Name: AMP (column D) Description: subtraction of the O2 value calculated for apo from the one calculated for AMP Basically column F - Column B from MethylOrdPar_O2 sheet E. Name: (column E) Description: error associated to the ΔO2 reported in column D F. Name: Balanol (column F) Description: subtraction of the O2 value calculated for apo from the one calculated for Balanol Basically column H- Column B from MethylOrdPar_O2 sheet G. Name: (column G) Description: error associated to the ΔO2 reported in column F H. Name: Adenosine (column H) Description: subtraction of the O2 value calculated for apo from the one calculated for adenosine Basically column J - Column B from MethylOrdPar_O2 sheet I. Name: (column I) Description: error associated to the ΔO2 reported in column H J. Name: ADP (column J) Description: subtraction of the O2 value calculated for apo from the one calculated for ADP Basically column L - Column B from MethylOrdPar_O2 sheet K. Name: (column K) Description: error associated to the ΔO2 reported in column J L. Name: ATPgN (column L) Description: subtraction of the O2 value calculated for apo from the one calculate for ATPgN Basically column N - Column B from MethylOrdPar_O2 sheet M. Name: (column M) Description: error associated to the ΔO2 reported in column L 5. Description of MethylOrdPar_deltaO2 tab: Difference between the O2 values of each residue for PKA-C bound to nucleotide/inhibitor from the apo form. Conformational_entropy.xlsx (Conf_Energy_Model Free Analysis tab) Description: Calculation of the conformational entropy (ΔSconf) accordingly to Stetz MA, Caro JA, Kotaru S, Yao X, Marques BS, Valentine KG, Wand AJ. Characterization of Internal Protein Dynamics and Conformational Entropy by NMR Relaxation. Methods Enzymol. 2019;615:237-284. doi: 10.1016/bs.mie.2018.09.010. Epub 2018 Dec 8. PMID: 30638531; PMCID: PMC6364297. Column A to H row 2 to 8 : Calculation of ΔSconf in kilo calorie (kcal) 1. Number of variables: 8 2. Number of cases/rows: 7 3. Missing data codes: Not applicable 4. Variable List A. Name: Nucleotide/inhibitor (column A) Description: list of the nucleotide/inhibitor analyzed B. Name: Nchi (column B) Description: total number of the torsional angler for methyl-bearing residue tested C. Name: sd (column C) Description: scaling factor expressed in cal/mol*K D. Name: <ΔO2> (column D) Description: average of the differenze of methyl order parameter from the nucleotide/inhibitor-bound form from the apo form of PKA-C. These values are the averaged from the data reported in the sheet : MethylOrdPar_deltaO2 E. Name: error (column E) Description: error associated to the values reported in colum D F. Name: ΔSconf (column F) Description: conformational entropy value calculated, expressed in kcal G. Name: error (column G) Description: error associated to the values reported in colum G H. Name: -TΔS (column H) Description: Conformational entropy * temperature Column A to I row 14 to 20 : Calculation of ΔSconf in kilo Joule (kJ) 1. Number of variables:9 2. Number of cases/rows: 7 3. Missing data codes: Not applicable 4. Variable List A. Name: Nucleotide/inhibitor (column A) Description: list of the nucleotide/inhibitor analyzed B. Name: Nchi (column B) Description: total number of the torsional angler for methyl-bearing residue tested C. Name: sd (column C) Description: scaling factor expressed in J/mol*K D. Name: <ΔO2> (column D) Description: average of the differenze of methyl order parameter from the nucleotide/inhibitor-bound form from the apo form of PKA-C. These values are the averaged from the data reported in the sheet : MethylOrdPar_deltaO2 E. Name: error (column E) Description: error associated to the values reported in colum D F. Name: ΔSconf (column F) Description: conformational entropy value calculated, expressed in kJ G. Name: error (column G) Description: error associated to the values reported in colum G I. Name: -TΔS (column I) Description: Conformational entropy * temperature H. Name: error (column H) Description: error associated to the values reported in colum I Column A to C row 23 to 29 : Calculation of ΔSconf in kilo Joule (kJ) 1. Number of variables: 3 2. Number of cases/rows: 7 3. Missing data codes: Not applicable 4. Variable List A. Name: Nucleotide/inhibitor (column A) Description: list of the nucleotide/inhibitor analyzed B. Name: -TΔS (kJ/mol) (column B) Description: values conformational entopy calculated in colum I, expressed in kJ/mol C. Name: σ (column C) Description: cooperativity coofficient of each nucleotide/inhibitor Conformational_entropy.xlsx (from CoopCoeff_to_Free Energy tab) 1. Number of variables: 11 2. Number of cases/rows: 7 3. Missing data codes: Not applicable 4. Variable List A. Name: Nucleotide/inhibitor (column A) Description: list of the nucleotide/inhibitor analyzed B. Name: σ (column B) Description: cooperativity coofficient of each nucleotide/inhibitor C. Name: error (column C) Description: error associated to the values reported in colum B D. Name: RT in cal/molK and kJ/mol (column D) Description: product of the molar gas constant (R) and the temperature (T) E. Name: ddG (column E) Description: difference in the gibbs free energy in cal/mol F. Name: errror(column F) Description: error associated to colum E G. Name: ddG (column G) Description: difference in the gibbs free energy in kcal/mol H. Name: errror(column H) Description: error associated to colum G J. Name: ddG (column J) Description: difference in the gibbs free energy in kJ/mol K. Name: errror (column K) Description: error associated to colum J