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

Abstract

Many prokaryotic and eukaryotic dsDNA viruses use a scaffolding protein to assemble their capsid. Assembly of the dsDNA 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 and double cysteine variants of scaffolding protein were generated for use in fluorescence resonance energy transfer and crosslinking experiments designed to probe the conformation of scaffolding protein in solution and within procapsids. We showed that the N- and C-termini 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, which indicated 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 to one another with associated C-termini.

Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

“…In solution, the N-terminal and the C-terminal domains seem to be in close proximity, which is not true for the scaffolding protein population present inside procapsids (17,25). Interestingly, the N-terminal domain of scaffolding protein, to a lesser extent, also affects the interaction of scaffolding protein and coat protein, possibly acting as a modulator to reduce the affinity between these two proteins (25)(26)(27). Recently, we determined the specific sites of interaction of scaffolding protein with coat protein.…”

“…The arginine is crucial for scaffolding protein-coat protein interactions, whereas lysine 296 is a secondary binding site (26). The specific orientation of the HTH is also important for the proper interaction with coat protein (25). In the present study, we sought to identify, through site-directed mutagenesis and biochemical studies, the residues in coat protein involved in interactions with scaffolding protein and, therefore, procapsid assembly.…”

Decoding bacteriophage P22 assembly: Identification of two charged residues in scaffolding protein responsible for coat protein interaction

“…Kinetic analysis of assembly by light scattering measurements at 45 mM NaCl gave similar results with a few quantitative differences; the K286A, D288A, and E290A proteins had half-times for assembly completion of about 5 minutes (the T 1/2 of his-tagged WT protein is 3 minutes), while the K294A protein had a significantly longer lag phase and a half time for its elongation phase of approximately 8 minutes (Figure 4B, C). These kinetics suggest that K294A may have a specific effect on the nucleation step, perhaps by affecting scaffolding protein dimerization (Padilla-Meier and Teschke, 2011). Finally, no assembly was observed by light scattering measurements with the R293A or R293E proteins (Figure 4D).…”

“…Biochemical and genetic data indicate that scaffolding protein may be folded upon itself as a U-shaped molecule, allowing the N-terminal region to be in contact with the C-terminal region (Padilla-Meier and Teschke, 2011; Tuma et al, 1998; Weigele et al, 2005). Addition of positive N-terminal charges may create an imbalance in charge distribution on the protein surface.…”

“…Scaffolding protein has been suggested to bind to coat protein at the capsid lattice 3-fold axis of symmetry (‘trimer tips’), represented by the gray region in Figure 8 (Chen et al, 2011; Padilla-Meier and Teschke, 2011; Parent et al, 2010; Thuman-Commike et al, 2000; Thuman-Commike et al, 1996). To date, our results cannot determine whether scaffolding protein interacts at the 3-fold axis of symmetry as a monomer or a dimer.…”