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The Impact of Mutation L138F/L210F on the Orai Channel: A Molecular Dynamics Simulation Study

ABSTRACT: The calcium release-activated calcium channel, composed of the Orai channel and the STIM protein, plays a crucial role in maintaining the Ca2+ concentration in cells. Previous studies showed that the L138F mutation in the human Orai1 creates a constitutively open channel independent of STIM, causing severe myopathy, but how the L138F mutation activates Orai1 is still unclear. Here, based on the crystal structure of Drosophila melanogaster Orai (dOrai), molecular dynamics simulations for the wild-type (WT) and the L210F (corresponding to L138F in the human Orai1) mutant were conducted to investigate their structural and dynamical properties. The results showed that the L210F dOrai mutant tends to have a more hydrated hydrophobic region (V174 to F171), as well as more dilated basic region (K163 to R155) and selectivity filter (E178). Sodium ions were located deeper in the mutant than in the wild-type. Further analysis revealed two local but essential conformational changes that may be the key to the activation. A rotation of F210, a previously unobserved feature, was found to result in the opening of the K163 gate through hydrophobic interactions. At the same time, a counter-clockwise rotation of F171 occurred more frequently in the mutant, resulting in a wider hydrophobic gate with more hydration. Ultimately, the opening of the two gates may facilitate the opening of the Orai channel independent of STIM.

SUBMITTER: Zhang X

PROVIDER: S-EPMC8592927 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Several natural mutants of the human G6PD enzyme exist and have been reported. Because the enzymatic activities of many mutants are different from that of the wildtype, the genetic polymorphism of G6PD plays an important role in the synthesis of nucleic acids via ribulose-5-phosphate and formation of reduced NADP in response to oxidative stress. G6PD mutations leading to its deficiency result in the neonatal jaundice and acute hemolytic anemia in human. Herein, we demonstrate the molecular dynamics simulations of the wildtype G6PD and its three mutants to monitor the effect of mutations on dynamics and stability of the protein. These mutants are Chatham (A335T), Nashville (R393H), Alhambra (V394L), among which R393H and V394L lie closer to binding site of structural NADP+. MD analysis including RMSD, RMSF and protein secondary structure revealed that decrease in the stability of mutants is key factor for loss of their activity. The results demonstrated that mutations in the G6PD sequence resulted in altered structural stability and hence functional changes in enzymes. Also, the binding site, of structural NADP+, which is far away from the catalytic site plays an important role in protein stability and folding. Mutation at this site causes changes in structural stability and hence functional deviations in enzyme structure reflecting the importance of structural NADP+ binding site. The calculation of binding free energy by post processing end state method of Molecular Mechanics Poisson Boltzmann SurfaceArea (MM-PBSA) has inferred that ligand binding in wildtype is favorable as compared to mutants which represent destabilised protein structure due to mutation that in turn may hinder the normal physiological function. Exploring individual components of free energy revealed that the van der Waals energy component representing non-polar/hydrophobic energy contribution act as a dominant factor in case of ligand binding. Our study also provides an insight in identifying the key inhibitory site in G6PD and its mutants which can be exploited to use them as a target for developing new inhibitors in rational drug design.

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The Impact of Mutation L138F/L210F on the Orai Channel: A Molecular Dynamics Simulation Study

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