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

Abstract

We analysed the tailspike from bacteriophage Sf6 in complex with the O-polysaccharide of the pathogen Shigella flexneri. The conformational space populated by the polyrhamnose backbone of the S. flexneri O-polysaccharide as studied by an octasaccharide in complex with Sf6TSP could be well described with 2D 1H,1H-trNOESY NMR, utilizing a combination of methine-methine and methine-methyl correlations. The results are in good agreement with the conformations obtained from molecular dynamics (MD) simulations. To examine the impact of amino acid exchanges in the glycan binding site of Sf6TSP, MD simulations were used to predict increased O-polysaccharide binding affinities. We used surface plasmon resonance on S. flexneri O-polysaccharide surfaces to measure affinity increases in the obtained mutants. <br>

Host range expansion of Shigella phage Sf6 evolves through the dual roles of its tailspike

“…Previous work using molecular dynamics demonstrated that mutations near the O-antigen binding site of the Sf6 tailspike can dramatically affect the flexibility of the site (Kunstmann, et al 2020).…”

“…These changes could allow for a better fit of the glucosylated 2a 2 O-antigen structure, which likely assumes a helical rather than a linear shape (West, et al 2005). Previous work using molecular dynamics demonstrated that mutations near the O-antigen binding site of the Sf6 tailspike can dramatically affect the flexibility of the site (Kunstmann, et al 2020). As part of their study, Kunstmann et al modeled a T443C substitution.…”

A perfect fit: Bacteriophage receptor-binding proteins for diagnostic and therapeutic applications

“…Experimental determination and verification of RBP function is still needed, and macromolecular (baseplate and tail) cryo-EM studies to decipher activation mechanisms of phage tails are highly desirable. Importantly, more and more RBP-ligand interactions are being systematically dissected on an atomic level to reveal residues and regions that are critical for ligand binding and which are amenable to mutation in order to manipulate RBP-binding affinities and modification of binding ranges [29][30][31]35,37,66,68,74,75]. Such studies are essential for further development of RBPs as bioprobes or depolymerase tools and provide the necessary atomic-resolution 'blueprints' for host range programming of whole, engineered phage therapeutics.…”