The Bacteriophage Lambda gpNu3 Scaffolding Protein Is an Intrinsically Disordered and Biologically Functional Procapsid Assembly Catalyst

Abstract

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell. The scaffolding proteins are essential to procapsid assembly. Here, we describe the solution-based biophysical and functional characterization of the bacteriophage lambda (λ) scaffolding protein gpNu3. The purified protein possesses significant α-helical structure and appears to be partially disordered. Thermally induced denaturation studies indicate that secondary structures are lost in a cooperative, apparent two-state transition (Tm =40.6±0.3 °C) and that unfolding is, at least in part, reversible. Analysis of the purified protein by size-exclusion chromatography suggests that gpNu3 is highly asymmetric, which contributes to an abnormally large Stokes radius. The size-exclusion chromatography data further indicate that the protein self-associates in a concentration-dependent manner. This was confirmed by analytical ultracentrifugation studies, which reveal a monomer–dimer equilibrium (Kd,app ~50 μM) and an asymmetric protein structure at biologically relevant concentrations. Purified gpNu3 promotes the polymerization of gpE, the λ major capsid protein, into virus-like particles that possess a native-like procapsid morphology. The relevance of this work with respect to procapsid assembly in the complex double-stranded DNA viruses is discussed.

Discovery of a new motion mechanism of biomotors similar to the earth revolving around the sun without rotation

“…This model explains how the motor can transport dsDNA without involving rotation, coiling, or a torsional force. Revolution mechanism might reconcile the stoichiometric inconsistency among many bacteriophages whose ATPases have been reported to be tetrameric ( Chang et al, 2012 ; Medina et al, 2011 ; Fuller et al, 2007 ; Ortega and Catalano, 2006 ), pentameric (see above), hexameric ( Guo et al, 1998 ; Zhang et al, 1998 ; Hendrix, 1998 ; Shu et al, 2007 ; Xiao et al, 2008 ; Moll and Guo, 2007 ; Shu et al, 2007 ; Xiao et al, 2010 ; Zhang et al, 2012 ), and nonameric ( Roy et al, 2011 ).…”

Thermodynamic Characterization of Viral Procapsid Expansion into a Functional Capsid Shell

“…[12][13][14][15] This nucleates polymerization of the major capsid protein (gpE) into an icosahedral shell, chaperoned by co-polymerization with the scaffolding protein (gpNu3). [16][17][18][19] A limited number of viral protease proteins (gpC) are also incorporated into the nascent procapsid interior, which auto digests, degrades the scaffold protein, and removes 20 residues from the N-terminus of roughly half of the portal proteins. 13,14 The proteolysis products exit the structure to afford the mature procapsid composed of a portal ring situated at a unique vertex of the icosahedral shell; this portal vertex provides a hole through which viral DNA can enter during packaging and exit during infection.…”

Bacteriophage lambda: The path from biology to theranostic agent

“…of the 140 threefold axes of the expanded shell lattice (420 copies total, Figures 4 and 5), a reaction that can be performed in vitro under defined conditions (Lambert et al, 2017;Medina et al, 2012;Yang et al, 2008). Structural studies reveal that the base of the gpD trimer is highly hydrophobic (Yang et al, 2000), and our current model is that the hydrophobic patches exposed during procapsid expansion reside at the threefold axes of the shell, providing a nucleation site for spike assembly (see Box 1) (Lambert et al, 2017;Medina, Andrews, Nakatani, & Catalano, 2011). Capsid decoration is essentially irreversible and is required to stabilize the shell, both from the internal stress of the packaged DNA and from environmental insult (Hernando-Pérez et al, 2014;Q.…”

“…Yang et al, 2008). Importantly, the expanded, decorated shells are functional and can be packaged with viral and exogenous DNA in vitro (Lambert et al, 2017;M. M. Medina et al, 2011).…”