Conformational Changes in Bacteriophage P22 Scaffolding Protein Induced by Interaction with Coat Protein

Many prokaryotic and eukaryotic double-stranded DNA viruses use a scaffolding protein to assemble their capsid. Assembly of the double-stranded DNA bacteriophage P22 procapsids requires the interaction of 415 molecules of coat protein and 60–300 molecules of scaffolding protein. Although the 303-amino-acid scaffolding protein is essential for proper assembly of procapsids, little is known about its structure beyond an NMR structure of the extreme C-terminus, which is known to interact with coat protein. Deletion mutagenesis indicates that other regions of scaffolding protein are involved in interactions with coat protein and other capsid proteins. Single-cysteine and double-cysteine variants of scaffolding protein were generated for use in fluorescence resonance energy transfer and cross-linking experiments designed to probe the conformation of scaffolding protein in solution and within procapsids. We showed that the N-terminus and the C-terminus are proximate in solution, and that the middle of the protein is near the N-terminus but not accessible to the C-terminus. In procapsids, the N-terminus was no longer accessible to the C-terminus, indicating that there is a conformational change in scaffolding protein upon assembly. In addition, our data are consistent with a model where scaffolding protein dimers are positioned parallel with one another with the associated C-termini.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (147 K)Download as PowerPoint slideResearch Highlights► The conformation of scaffolding protein was studied in solution and inside procapsids. ► Our study used fluorescence resonance energy transfer and cross-linking to examine the structure. ► The N-terminus is modeled in proximity to both the C-terminus and a middle domain. ► Inside procapsids, scaffolding protein rearranges to a more open conformation. ► Scaffolding proteins are arranged in parallel inside procapsids.