DOI: 10.5061/dryad.gb5mkkx1j
title: Redox regulation of brain selective kinases BRSK1/2: Implications for dynamic control of the eukaryotic AMPK family through Cys-based mechanisms

description: In eukaryotes, protein kinase signaling is regulated by a diverse array of
 post-translational modifications (PTMs), including phosphorylation of
 Ser/Thr residues and oxidation of cysteine (Cys) residues. While
 regulation by activation segment phosphorylation of Ser/Thr residues is
 well understood, relatively little is known about how oxidation of
 cysteine residues modulate catalysis. In this study, we investigate redox
 regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and
 detail how broad catalytic activity is directly regulated through
 reversible oxidation and reduction of evolutionarily conserved Cys
 residues within the catalytic domain. We show that redox-dependent control
 of BRSKs is a dynamic and multilayered process involving oxidative
 modifications of several Cys residues, including the formation of
 intra-molecular disulfide bonds involving a pair of Cys residues near the
 catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific
 Cys within an unusual CPE motif at the end of the activation segment.
 Consistently, mutation of the CPE-Cys increases catalytic activity in
 vitro and drives phosphorylation of the BRSK substrate Tau in cells.
 Molecular modeling and molecular dynamics simulations indicate that
 oxidation of the CPE-Cys destabilizes a conserved salt bridge network
 critical for allosteric activation. The occurrence of spatially proximal
 Cys amino acids in diverse Ser/Thr protein kinase families suggests that
 disulfide mediated control of catalytic activity may be a prevalent
 mechanism for regulation within the broader AMPK family.
description: The starting model for molecular dynamics (MD) simulations was
 selected to provide an accurate representation of the protein kinase in
 its active-like conformation. To achieve this, we utilized an AlphaFold
 model of the BRSK2 kinase domain, corresponding to residues 14-267, in an
 active-like conformation. The average pLDDT score for the portion of the
 AlphaFold model employed in MD simulations was calculated to be 89.18%,
 indicating high confidence and accuracy (Jumper et al. 2021). Starting
 structures were prepared using the CHARMM-GUI interface which allowed for
 incorporation and parameterization of T-Loop phosphorylation, cysteine to
 alanine mutation, and oxidative cysteine modification (Brooks et al. 2009;
 Lee et al. 2016; Jo et al. 2014). Cysteine 176 (T+2) and 183 (CPE motif)
 were each mutated to alanine, sulfenic acid, or sulfonic acid forms. The
 protein was solvated in a cubic box of TIP3P water molecules, and
 counterions were added to maintain neutrality. The final systems contained
 ~ 54,000 atoms. Prior to production runs, the system
 was subjected to minimization and equilibration protocols, using
 previously described parameters (Yeung et al. 2021; Venkat et al. 2023).
 Initially, a steepest descent energy minimization was performed to relax
 the system, followed by equilibration at constant volume and temperature
 (NVT) and constant pressure and temperature (NPT). Each equilibration
 stage was carried out for 125 ps with 1 fs time steps. Following
 equilibration, long-range electrostatics were calculated via particle mesh
 Ewald (PME) algorithms using the GROMACS MD engine (Van Der Spoel et al.
 2005). Three 100 ns production molecular dynamics (MD) replicates were
 conducted at a 2fs time-step using the CHARMM36 forcefield for each
 starting model (Brooks et al. 2009). The resultant MDs were visualized
 with PyMOL (Schrodinger 2015) and analyzed in the Python environment
 (Michaud-Agrawal et al. 2011).
description: # Redox regulation of brain selective kinases BRSK1/2: Implications for
 dynamic control of the eukaryotic AMPK family through Cys-based mechanisms
 This dataset contains all of the molecular dynamics input files and
 associated GROMACs trajectories used in the generation of figures for the
 manuscript. ## Description of the data and file structure for folder
 "BRSK2\_MD\_FROM\_PAPER.zip" The dataset is arranged in folders
 representing each modification of the BRSK2 AlphaFold (brsk2_af)
 structure. Molecular dynamics simulations were performed in triplicate
 with each replicate enumerated in the folder name. The suffix following
 brsk2 denotes the residue and modification type. e.g. brsk2_af_a_183
 represents a modified brsk2 AlphaFold structure with an alanine at
 position 183. The following key can be used to interpret the titles of the
 files: brsk2_af - the original BRSK2 AlphaFold structure which all
 modifications are based on. brsk2_af_a183 - Cysteine 183 mutated to
 Alanine brsk2_af_cso183 - Cysteine 183 modified to S-HydroxyCysteine
 brsk2_af_csx183 - Cysteine 183 modified to sulfenic acid brsk2_af_ocs183 -
 Cysteine 183 modified to sulfonic acid brsk2_af_pT174 - Threonine 174
 modified to phosphorylated Theonine brsk2_af_pT174_a176 - Theonine 174
 modified to phosphorylated Threonine and Cysteine 176 mutated to Alanine
 brsk2_af_cso183 - Threonine 174 modified to phosphorylated Theonine and
 Cysteine 183 modified to S-HydroxyCysteine brsk2_af_csx183 - Threonine 174
 modified to phosphorylated Theonine and Cysteine 183 modified to sulfenic
 acid brsk2_af_ocs183 - Threonine 174 modified to phosphorylated Theonine
 and Cysteine 183 modified to sulfonic acid Each folder contains a README
 generated by CHARMM-GUI outlining the steps neccessary for the repetition
 of the GROMACs simulation, associated parameter files in the folder
 "toppar" and the folder "MD" which contains the
 processed trajectory and compressed output from the associated GROMACs
 simulation. Folder Organization: brsk2_af_* ├── MD -folder containing
 processed trajectory │ ├── A.pdb - frame zero from the trajectory │ ├──
 cluster_whole.xtc - trajectory centerecd on protein │ ├──
 ft_cluster_whole.xtc - trajectory centered on protein and cleaned │ ├──
 trj.pdb - trajectory converted to pdb format │ └── whole.xtc - entire
 trajectory compressed to 1 ns time steps ├── toppar - folder containing
 topology for GROMACS run │ ├── CLA.itp - Topology file for chloride (Cl⁻)
 ions │ ├── forcefield.itp – Main force field parameter file │ ├── POT.itp
 – Topology file for potassium (K⁺) ions │ ├── PROB.itp – Topology file for
 protein │ └── TIP3.itp – Topology file for TIP3P water model ├── index.ndx
 - GROMACS index ├── README - CHARMM-GUI generated instructions for running
 GROMACS simulation ├── step3_input PDB/GROMACS format input for
 simulations ├── step3_input.psf - input protein structure file ├──
 step4.0_minimization.mdf - GROMACS settings for minimization ├──
 step4.1_equilibration.mdf - GROMACS settings for equilibration ├──
 step5_production.mdf - GROMACS settings for production └── topol.top -
 GROMACS topology file ## Sharing/Access information Data was derived from
 the following sources: GROMACs inputs were generated using CHARMM-GUI -
 [https://www.charmm-gui.org/](https://www.charmm-gui.org/) ##
 Code/Software Included in this data set the the jupyternotebook
 brsk_md_from_paper.IPYNB which was used to derive the contact maps for
 figures as well as the image BRSK2_pT174_average representing the output
 from the notebook.