Increasing the Affinity of an O-Antigen Polysaccharide Binding Site in Shigella Flexneri Bacteriophage Sf6 Tailspike Protein

Abstract

Broad and unspecific use of antibioticsa ccelerates spread of resistances. Sensitivea nd robust pathogen detection is thus important for am ore targeted application. Bacteriophages contain al arge repertoire of pathogen-binding proteins. Theset ailspike proteins (TSP) often bind surface glycansa nd represent ap romising design platform for specific pathogen sensors. We analysed bacteriophage Sf6 TSP that recognizest he O-polysaccharide of dysentery-causing Shigellaf lexneri to develop variants with increased sensitivity for sensor applications. Ligand polyrhamnoseb ackbonec on-formationswere obtained from 2D 1 H, 1 H-trNOESY NMR utilizing methine-methine and methine-methyl correlations. They agreedw ell with conformationsobtained from molecular dynamics (MD), validating the methodf or furtherp redictions.I naset of mutants, MD predicted ligand flexibilities that were in good correlationw ith binding strengtha sc onfirmedo ni mmobilized S. flexneri O-polysaccharide (PS) with surface plasmonr esonance. In silico approaches combined with rapid screening on PS surfaces hence provide valuable strategies for TSP-based pathogen sensor design.[a] Dr.

Resources and Methods for Engineering “Designer” Glycan-Binding Proteins

“…The most common and efficient way to target specific sites is by PCR—desired single point mutations are included in the primers that amplify the gene of interest. PCR based SDM is one of the most common SDM methods applied in protein engineering and variations of PCR site-directed mutagenesis have been successfully applied in engineering GBPs in several examples including altering the binding specificity of a fucose-binding lectin PA-IIL [ 75 ] and increasing the affinity of a tail spike protein Sf6 to the glycans comprising bacterial O-antigens [ 76 ]. SDM has also been used to inactivate a streptococcal endo- N -acetylglucosaminidase (EndoS) [ 77 ] and an E. coli K1 bacteriophage endosialidase (EndoNF) to dissociate binding from hydrolytic activity [ 43 , 44 ] and produce novel GBPs for specific glycan detection [ 45 , 46 ].…”

“…There are plenty of MD program packages available to use, with some of the most common being YASSA, MOE, Enlighten2, and GROMACS. In recent work on GBP engineering, Kunstmann et al used MD simulations generated with the GROMACS 4.5.5 package to accurately link mutations in the tail-spike protein of bacteriophage Sf6 to varying affinities towards glycans of the O-antigens on Shigella flexneri (which is host to the phage) [ 76 ]. Yet being able to make such predictions accurately is rare and it is more common to identify beneficial mutations through screening focused libraries.…”