MARK4 protein can explore the active-like conformations in its non-phosphorylated state

Sajjad Ahrari,Amirhossein Sakhteman,Fatemeh Khosravi,Younes Ghasemi,Ali Osouli,Amir Savardashtaki,Alireza Nematollahi

doi:10.1038/s41598-019-49337-0

Sajjad Ahrari, Amirhossein Sakhteman + Show 5 more

Open Access

https://doi.org/10.1038/s41598-019-49337-0

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Abstract

Microtubule affinity-regulating kinase 4 (MARK4) is a Ser/Thr protein kinase, best known for its role in phosphorylating microtubule associated proteins, causing their detachment from microtubules. In the current study, the non-phosphorylated conformation of the activation loop was modeled in a structure representing the enzymatically inactive form of this protein, and its dynamics were evaluated through a 100 ns initial all-atom simulation, which was prolonged by another 2 μs. Although the activation loop was folding on itself and was leaning toward ATP site in the initial modeled structure, soon after the initiating the simulation, this loop stretched away from the ATP binding site and stably settled in its new position for the rest of simulation time. A network of hydrogen bonds, mainly between the activation segment residues, αC-helix and the catalytic loop reinforced this conformation. Interestingly, several features of active kinase conformation such as formation of R-spine, Glu106-Lys88 salt-bridge, and DFG-In motif were observed during a considerable number of trajectory frames. However, they were not sustainably established during the simulation time, except for the DFG-In motif. Consequently, this study introduces a stable conformation of the non-phosphorylated form of MARK4 protein with a partially stretched activation loop conformation as well as partial formation of R-spine, closely resembling the active kinase.

Highlights

Microtubule affinity-regulating kinase 4 (MARK4) protein is a Serine/Threonine protein kinase, structurally related to the AMPK/Snf[1] subfamily of the CaMK group of kinases[1,2,3]
We modeled the conformation of activation loop in a crystal structure of MARK4 protein that was introduced as an inactive conformation of MARK4 protein[15] and evaluated its dynamics through an un-biased MD-simulation approach
We modeled the conformation of activation loop in a crystal structure of non-phosphorylated MARK4 protein (PDB ID: 5ES1)[15] and studied the kinase core dynamics in the presence of the modeled loop

Summary

Introduction

MARK4 protein is a Serine/Threonine protein kinase, structurally related to the AMPK/Snf[1] subfamily of the CaMK group of kinases[1,2,3]. The only available crystal structure of MARK4 represents the conformation of kinase core plus UBA domain. Like other MARK isoforms, the enzymatic core is folded into the so-called kinase bilobal structure, with the N-lobe being composed of five β-sheets and holding one α-helix (αC-helix) while the C-lobe comprises of six α-helices and an extended loop, known as the activation segment. This structure is followed by CD-like motif (a sequence of negatively charged residues), linker loop and UBA domain[16]. Upon phosphorylation of this loop on the conserved Thr residue (and the kinase activation), this loop assumes a stretched conformation which is much more stable in comparison to the non-phosphorylated state[5,15,16,20]

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