Human microtubule affinity-regulating kinase 4 is stable at extremes of pH

Abstract

MAP/microtubule affinity-regulating kinase 4 (MARK4) is a member of adenosine monophosphate-activated protein kinases, directly associated with cancer and neurodegenerative diseases. Here, we have cloned, expressed, and purified two variants of MARK4 [the kinase domain (MARK4-F2), and kinase domain along with 59 N-terminal residues (MARK4-F1)] and compared their stability at varying pH range. Structural and functional changes were observed by incubating both forms of MARK4 in buffers of different pH. We measured the secondary structure of MARK4 using circular dichroism and tertiary structure by measuring intrinsic fluorescence and absorbance properties along with the size of proteins by dynamic light scattering. We observed that at extremes of pH (below pH 3.5 and above pH 9.0), MARK4 is quite stable. However, a remarkable aggregate formation was observed at intermediate pH (between pH 3.5 and 9.0). To further validate this result, we have modeled both forms of MARK4 and performed molecular dynamics simulation for 15 ns. The spectroscopic observations are in excellent agreement with the findings of molecular dynamics simulation. We also performed ATPase activity at varying pH and found a significant correlation of structure of MARK4 with its enzyme activity. It is interesting to note that both forms of MARK4 are showing a similar pattern of structure changes with reference to pH.

Keywords:

• microtubule affinity-regulating kinase 4
• microtubule dynamics
• effect of pH
• kinase domain
• protein aggregation
• molecular dynamics simulation

Acknowledgements

FN acknowledge the Council of Scientific and Industrial Research for the award of fellowship. Authors thank Department of Science and Technology, Government of India for FIST support. We sincerely thank Harvard Medical School for providing MARK4 cDNA. FA and MIH are thankful to the Department of Science and Technology for funding.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Department of Science and Technology, Ministry of Science and Technology.