Machine learning-based modulation of Ca 2+ -binding affinity in EF-hand proteins and comparative structural insights into site-specific cooperative binding.

Autor: Mazumder M; School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India; Pine Biotech, 1441 Canal Street, New Orleans, LA 70112, USA., Kumar S; School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India; Department of Biochemistry, Vanderbilt University School of Medicine, 2215 Garland Avenue, Nashville, TN 37232-0146, USA., Kumar D; School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India., Bhattacharya A; School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India; Ashoka University, Rajiv Gandhi Education City, Sonipat, Haryana 131029, India., Gourinath S; School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. Electronic address: sgourinath@mail.jnu.ac.in.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2023 Sep 01; Vol. 248, pp. 125866. Date of Electronic Publication: 2023 Jul 19.
DOI: 10.1016/j.ijbiomac.2023.125866
Abstrakt: Ca 2+ -binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we report two new scoring schemes enabling the user to estimate and manipulate the calcium binding affinities in EF hand containing proteins. To validate this, we designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues. The N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1) is used for site-directed mutagenesis to incorporate the designed loop sequence into the second EF-hand motif of this protein, referred as Nt-EhCaBP1-EF2 mutant. The binding isotherms calculated using ITC calorimetry showed that Nt-EhCaBP1-EF2 mutant site binds Ca 2+ with higher affinity than Wt-Nt-EhCaBP1, by ∼600 times. The crystal structure of the mutant displayed more compact Ca 2+ -coordination spheres in both of its EF loops than the structure of the wildtype protein. The compact coordination sphere of EF-2 causes the bend in the helix-3, which leads to the formation of unexpected hexamer of NtEhCaBP1-EF2 mutant structure. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in stronger coordination of Ca 2+ even in another EF-hand loop.
Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Samudrala Gourinath reports financial support was provided by India Ministry of Science & Technology Department of Biotechnology.
(Copyright © 2023 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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