Redox regulation of brain selective kinases BRSK1/2: Implications for dynamic control of the eukaryotic AMPK family through Cys-based mechanisms

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.

Methods
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).