Potato virus A genome-linked protein VPg is an intrinsically disordered molten globule-like protein with a hydrophobic core

Abstract

Genome-linked protein VPg of Potato virus A (PVA; genus Potyvirus) has essential functions in all critical steps of PVA infection, i.e. replication, movement, and virulence. Structural features of the recombinant PVA VPg were investigated with the aim to create an outline for structure-function relationships. Circular dichroism data of PVA VPg revealed a distinct near-UV spectrum indicating that the environment around its aromatic residues is structured but rather flexible, and a far-UV spectrum that was characterized by features typical for intrinsically disordered proteins. Temperature-induced denaturation followed a typical all-or-none transition whereas urea- and GdmHCl-induced denaturation proceeded via a route best described by a three-state-model. The conclusion drawn was that the overall structure of PVA VPg is significantly unstable even in the absence of denaturants. Acrylamide fluorescence quenching and 1-anilino-8-naphthalene sulfonate binding experiments together with 1D and 2D NMR data further verified that PVA VPg behaves as a partially folded species that contains a hydrophobic core domain. Regions predicted to be disordered in PVA VPg were the ones that were cut the fastest by trypsin whereas regions predicted to be structured and to contain the most conserved amino acids among potyvirus VPgs were trypsin-resistant. Amino acid composition analysis of potyvirus VPgs revealed a clear enrichment of disorder and depletion of structure-promoting residues. Taken together it seems that the native structure of PVA VPg, and probably that of potyviral VPg in general, resembles a partially disordered molten globule. Further experimentation is required to understand the functional regulation achieved via this property.

Structural Flexibility Allows the Functional Diversity of Potyvirus Genome-Linked Protein VPg

“…Disorder, however, is not a homogeneous state but comes in different structural flavors, ranging from random coil through premolten globule to a rather compact structural state denoted as molten globule (10,51). The native structure of PVA VPg is a partially disordered molten globule-like type (39,40). Our previous observations with circular dichroism (CD) spectroscopy, ANS (1-anilo-8-naphthalene sulfonate) binding, and disorder prediction are all consistent with this.…”

“…However, several recent studies have shown the intrinsic structural disorder of these proteins. The resulting structural flexibility has been proposed to enable VPg to carry out the variety of functions (16,20,40,44). The N terminus, NTP-binding region, and a short segment in the middle of the protein were speculated to be part of a natively unstructured stretch of amino acids, which may allow these regions to be multifunctional and structurally flexible and thereby facilitate participation of VPg in various interactions.…”

“…It is argued by Tokuriki et al that viral proteins have a lot of flexibility and a high level of disorder and that they apparently are rapidly evolving proteins, stabilized only in the presence of partners. Accordingly, structural disorder together with polyprotein intermediates and various posttranslational modifications of VPg (18) as well as formation of VPg dimers (16,31,40) give rise to a great variety of functions and complex targets for structure-function studies.…”

Control of Nuclear and Nucleolar Localization of Nuclear Inclusion Protein a of Picorna-LikePotato virus AinNicotianaSpecies

“…Substitution of this Tyr residue with other amino acids abolishes infectivity of both viruses (Murphy et al, 1996;Germundsson et al, 2007). The intrinsically disordered structure of VPg (Grzela et al, 2008;Rantalainen et al, 2008) provides flexibility for many types of interactions with viral and host proteins (Hong et al, 1995;Li et al, 1997;Wittmann et al, 1997;Schaad et al, 2000;Guo et al, 2001;Dunoyer et al, 2004;Lé onard et al, 2004;Thivierge et al, 2008). Interactions between eIF4E or eIF(iso)4E (Wittmann et al, 1997;Schaad et al, 2000;Robaglia and Caranta, 2006) and VPg or NIa are important for virus infectivity (Lé onard et al, 2000;Robaglia and Caranta, 2006).…”

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs

“…For Potato virus A (PVA), Potato virus Y, Lettuce mosaic virus, SeMV and RYMV an unfolded/disordered structure of VPg has been described previously (Grzela et al, 2008;Hébrard et al, 2009;Rantalainen et al, 2008;Satheshkumar et al, 2005). VPg proteins lack a unique 3D-structure and exist as a dynamic ensemble of conformations.…”