Mutations Of The Flo1 Flocculation Protein For Enhancing Oligosaccharide Binding:Molecular Dynamics Simulations

Cellular adhesion is a general biological process such as flocculation between yeast cells which enhances the efficiency of yeast in the food industry and biofuel production.The flocculation involves the binding of exopolysaccharides on the flocculation protein of neighboring cel.In the flocculation protein family from Saccharomyces cerevisiae,the first member Flo1 p is the best flocculation protein.The binding of oligomannose on the N-terminus of this protein(N-Flo1p)directly impacts the flocculation efficiency.In this work,we will use several computational methods including molecular dynamics simulation and molecular docking,to determine proper N-Flo1 p mutants for enhancing the oligomannose binding,in order to provide a theoretical guide to modulating the cellular adhesion process.1.By calculating the binding free energy of the complex structure(crystal structure)of N-Flo1 p and mannose before and after amino acid residue mutation,it can be found that the binding free energy of the mutant complex are not significantly improved compared with wild type complex.In addition,mutations in some residues can cause a decrease in the ability of NFlo1 p to bind to mannose.2.The coordinate center where the mannose is bound to the N-Flo1 p active site in the crystal structure is used as the reference coordinate for molecular docking.The mannodiose,mannosetriose and mannotetraose constructed with GLYCAM-06 j force field are docked to the active pocket of N-Flo1 p.To determine how many mannose molecules the flocculent's active pocket can hold.At the same time,amino acid residue mutation studies are performed on the complex of N-Flo1 p bind mannobiose.Several proper residue mutation sites are identified by alanine scanning mutagenesis,including Q98,Q117 and K194.Then,by comparing the binding free energy of the complex systems in which the mutant flocculins and wild type flocculin bind to mannobiose respectively,it can be found that two mutants Q117 N and Q117 R can cause the binding free energy of the complex system to decrease from-22.63 kcal/mol to-32.44 kcal/mol and-35.86 kcal/mol,can significantly improve the binding effect of N-Flo1 p to mannobiose.And by analyzing the strength of the hydrogen bond of the complex systems,the decompositio n of the binding free energy and the change of the active pocket volume,the influence of residue mutations on the binding of flocculent to mannobiose is further determined.3.Mannotriose consists of three mannose residues can be used to evaluate the binding strength of oligomannose.Through detailed analysis of the trajectories obtained from the molecular dynamics simulations of mutant and wild-type complexes,RMSD fluctuat io n,conformational stability,shape changes and the hydrogen bond occupancy rate in the complex systems,to help understand the interactions between the oligomannose residues and the flocculation protein.This study not only recommends two mutants Q117 N and Q117 R for the improvement of flocculation protein N-Flo1 p to oligomannose,but also provides a computational approach way to evaluate oligosaccharide bindings which can be extended to other relevant biologic a l processes.