Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain.

Masoud Keighobadi,Reza Valadan,Saeed Emami,Mahdi Fakhar,Alireza Rafiei,Milad Lagzian

doi:10.3390/molecules23030696

Abstract

Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. Leishmania major is the most common cause of cutaneous leishmaniasis. In the Leishmania parasites, sterol 14α-demethylase (CYP51), which is involved in the biosynthesis of sterols, has been identified as an attractive target for development of new therapeutic agents. In this study, the sequence and structure of CYP51 in a laboratory strain (MRHO/IR/75/ER) of L. major were determined and compared to the wild-type strain. The results showed 19 mutations including seven non-synonymous and 12 synonymous ones in the CYP51 sequence of strain MRHO/IR/75/ER. Importantly, an arginine to lysine substitution at position of 474 resulted in destabilization of CYP51 (ΔΔG = 1.17 kcal/mol) in the laboratory strain; however, when the overall effects of all substitutions were evaluated by 100 ns molecular dynamics simulation, the final structure did not show any significant changes (p-value < 0.05) in stability parameter of the strain MRHO/IR/75/ER compared to the wild-type protein. The energy level for the CYP51 of wild-type and MRHO/IR/75/ER strain were −40,027.1 and −39,706.48 Kcal/mol respectively. The overall Root-mean-square deviation (RMSD) deviation between two proteins was less than 1 Å throughout the simulation and Root-mean-square fluctuation (RMSF) plot also showed no substantial differences between amino acids fluctuation of the both protein. The results also showed that, these mutations were located on the protein periphery that neither interferes with protein folding nor with substrate/inhibitor binding. Therefore, L. major strain MRHO/IR/75/ER is suggested as a suitable laboratory model for studying biological role of CYP51 and inhibitory effects of sterol 14α-demethylase inhibitors.

Highlights

Leishmaniasis is a major health problem in most countries that endangers more than 350 million people in the endemic area
The results revealed that these mutations were new in the sequence of L. major, these variations are naturally present these mutations were new in the sequence of L. major, these variations are naturally present in the in the sequence of CYP51 of other Leishmania spp. (Figure 4)
Using protein modeling and in silico mutagenesis studies, we showed that these seven mutations could not alter the protein stability and folding

Summary

Introduction

Leishmaniasis is a major health problem in most countries that endangers more than 350 million people in the endemic area. Leishmania major (L. major) the main cause of cutaneous leishmaniasis, is a major public health problem in the African and the Eastern Mediterranean regions. CL is four times more prevalent than the visceral form [4] the dermal form is very common in Iran, where about 37,000 cases were reported in 2012–2013. This represents an incidence of 25/100,000 people in a country where 72% of the population lived in endemic areas [1]

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