DOI: 10.5061/dryad.8931zcrxb
title: Molecular Dynamics Simulations and associated data for: Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases

description: Catalytic signaling outputs of protein kinases are dynamically regulated
 by an array of structural mechanisms, including allosteric interactions
 mediated by intrinsically disordered segments flanking the conserved
 catalytic domain. The Doublecortin Like Kinases (DCLKs) are a family of
 microtubule-associated proteins characterized by a flexible C-terminal
 autoregulatory 'tail’ segment that varies in length across the
 various human DCLK isoforms. However, the mechanism whereby these
 isoform-specific variations contribute to unique modes of autoregulation
 is not well understood. Here, we employ a combination of statistical
 sequence analysis, molecular dynamics simulations and in vitro mutational
 analysis to define hallmarks of DCLK family evolutionary divergence,
 including analysis of splice variants within the DCLK1 sub-family, which
 arise through alternative codon usage and serve to ‘supercharge’ the
 inhibitory potential of the DCLK1 C-tail. We identify co-conserved motifs
 that readily distinguish DCLKs from all other Calcium Calmodulin Kinases
 (CAMKs), and a ‘Swiss-army’ assembly of distinct motifs that tether the
 C-terminal tail to conserved ATP and substrate-binding regions of the
 catalytic domain to generate a scaffold for auto-regulation through C-tail
 dynamics. Consistently, deletions and mutations that alter C-terminal tail
 length or interfere with co-conserved interactions within the catalytic
 domain alter intrinsic protein stability, nucleotide/inhibitor-binding,
 and catalytic activity, suggesting isoform-specific regulation of activity
 through alternative splicing. Our studies provide a detailed framework for
 investigating kinome–wide regulation of catalytic output through
 cis-regulatory events mediated by intrinsically disordered segments,
 opening new avenues for the design of mechanistically-divergent DCLK1
 modulators, stabilizers or degraders.
description: PDB constructs were generated by retrieving structural models
 RCSB and the ΑlphaFold2 database. Post-translational modifications were
 performed in PyMOL using the PyTMs plugin. All structures were solvated
 using the TIP3P water model. Energy minimization was run for a maximum of
 10,000 steps, performed using the steepest-descent algorithm, followed by
 the conjugate-gradient algorithm. The system was heated from 0K to a
 temperature of 300K. After two equilibration steps that each lasted 20
 picoseconds, 1 microsecond long simulations were run at a two femtosecond
 timestep. Long-range electrostatics were calculated via particle mesh
 Ewald (PME) algorithms using the GROMACS MD engine. We utilized the
 CHARMM36 force field. The resulting output was visualized using VMD 1.9.3.
 All molecular dynamics analysis was conducted using scripts coded in
 Python using the MDAnalysis module.
description: # Molecular Dynamics Simulations and associated data for Mechanistic and
 evolutionary insights into isoform-specific 'supercharging' in
 DCLK family kinases
 [https://doi.org/10.5061/dryad.8931zcrxb](https://doi.org/10.5061/dryad.8931zcrxb) MD Data (topology, coordinates, etc) for visualizing and analyzing MD Simulations discussed in the article: Mechanistic and evolutionary insights into isoform-specific 'supercharging' in DCLK family kinases, accepted in eLife. ## Description of the data and file structure The top zip folder contains three folders of different MDs run for DCLK1 (DCLK1.1, DCLK1.2, DCLK1.2\_pT688). Each folder contains 3 replicates (rep1, rep2, rep3), with pertinent files to visualize and analyze the MD (topolology, coordinates, and DSSP output). DSSP output (XPM file) is only included for *wt* MDs (DCLK1.1, DCLK1.2). **File Types:** \&gt; PDB = structure/topology file \&gt; XTC = GROMACS coordinates file \&gt; XPM = GROMACS DSSP image output of secondary structure **File Structure**: * DCLK1\_MDs.zip/ * DCLK1.1/ * rep1/ * PDB File * XTC File * XPM File * rep2/ * rep3/ * DCLK1.2/ * DCLKpT688/ * rep1/ * PDB File * XTC File **Visualization of MD files:** 1. Download and install VMD (Visual Molecular Dynamics) 2. Navigate to a replicate (rep) folder 3. **Example usage in terminal**: VMD md\_frame0.pdb md\_trajectory.xtc ## Code/Software PDB constructs were generated by retrieving structural models from RCSB and the ΑlphaFold2 database. Post-translational modifications were performed in PyMOL using the PyTMs plugin. All structures were solvated using the TIP3P water model. Energy minimization was run for a maximum of 10,000 steps, performed using the steepest-descent algorithm, followed by the conjugate-gradient algorithm. The system was heated from 0K to a temperature of 300K. After two equilibration steps that each lasted 20 picoseconds, 1 microsecond long simulations were run at a two femtosecond timestep. Long-range electrostatics were calculated via particle mesh Ewald (PME) algorithms using the GROMACS MD engine. We utilized the CHARMM36 force field. The resulting output was visualized using VMD 1.9.3. All molecular dynamics analysis was conducted using scripts coded in Python using the MDAnalysis module.