Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and decoration proteins in the development of robust nanoscale display vehicles

Abstract

A stepwise addition protocol was developed to display cargo using bacteriophage P22 capsids and the phage decorator (Dec) protein. Three-dimensional image reconstructions of frozen-hydrated samples of P22 particles with nanogold-labeled Dec bound to them revealed the locations of the N- and C- termini of Dec. Each terminus is readily accessible for molecular display through affinity tags such as nickel-nitrilotriacetic acid, providing a total of 240 cargo-binding sites. Dec was shown by circular dichroism to be a β–sheet rich protein, and fluorescence anisotropy binding experiments demonstrated that Dec binds to P22 heads with high (~110 nM) affinity. Dec also binds to P22 nanotubes, which are helically symmetric assemblies that form when the P22 coat protein contains the F170A amino acid substitution. Several classes of tubes with Dec bound to them were visualized by cryo-electron microscopy and their three-dimensional structures were determined by helical reconstruction methods. In all instances, Dec trimers bound to P22 capsids and nanotubes at positions where three neighboring capsomers (oligomers of six coat protein subunits) lie in close proximity to one another. Stable interactions between Dec and P22 allow for the development of robust, nanoscale size, display vehicles.

Multiple Functional Roles of the Accessory I-Domain of Bacteriophage P22 Coat Protein Revealed by NMR Structure and CryoEM Modeling

“…While the HK97-core of these coat protein models is similar, the non-conserved I-domains show marked differences making it difficult to reconcile biochemical and genetic data with the structures. As P22 capsids are often used as a platform for nanotechnology and display (Parent et al, 2012a; Patterson et al, 2014), fully understanding the structure of coat protein is crucial.…”

Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

“…The strategy used to process the CUS-3 virion film images and compute an asymmetric reconstruction was similar to that used to derive an asymmetric reconstruction of the P22 (Lander et al , 2006) and Sf6 (Parent et al , 2012a) virions. First, all digitized micrographs of the Sf6 virion samples were binned 2X, which generated a pixel size of 3.28 Å.…”

“…The CPs of different phages employ different “accessory” domains that embellish the core structure of the HK97-like fold. For example, P22 and Sf6 carry an external domain that is not present in the other tailed phage coat proteins whose structures are known (Parent et al , 2012a; Parent et al , 2012b). This domain was previously called “telokin-like” based on structural similarity, but recent high-resolution NMR data has led to a change in nomenclature (Rizzo et al , 2014), and as such we will refer to this domain as the “I-domain”.…”

Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology

“…Similarly Dec has been shown to bind to the P22 VLP at the quasi threefold axes of the VLP as a trimer with the C-terminus of each monomer projected away from the capsid (Parent et al, 2012). Douglas and coworkers demonstrated effective presentation of either monomeric or C3-symmetric protein cargo on Dec as well as the effective combination of this exterior display technique with the previously discussed P22 genetic encapsulation strategy (Schwarz et al, 2015).…”