Solution structures of Mengovirus Leader protein, its phosphorylated derivatives, and in complex with nuclear transport regulatory protein, RanGTPase

Abstract

Cardiovirus Leader (L) proteins induce potent antihost inhibition of active cellular nucleocytoplasmic trafficking by triggering aberrant hyperphosphorylation of nuclear pore proteins (Nup). To achieve this, L binds protein RanGTPase (Ran), a key trafficking regulator, and diverts it into tertiary or quaternary complexes with required kinases. The activity of L is regulated by two phosphorylation events not required for Ran binding. Matched NMR studies on the unphosphorylated, singly, and doubly phosphorylated variants of Mengovirus L (L M ) show both modifications act together to partially stabilize a short internal α-helix comprising L M residues 43-46. This motif implies that ionic and Van der Waals forces contributed by phosphorylation help organize downstream residues 48-67 into a new interface. The full structure of L M as bound to Ran (unlabeled) and Ran (216 aa

Binding Interactions between the Encephalomyocarditis Virus Leader and Protein 2A

“…Interestingly, L E -2A interactions also clearly masked L E residue Y 41 , one of two crucial phosphorylation sites for the activity of L E . Phosphorylation is not required for L E interactions with Ran, but without these modifications, the subsequent complex cannot proceed to ternary or quaternary reactions required to trigger the Nup phosphorylation cascade (9). Therefore, logically, 2A cannot remain perpetually bound to L E during the normal course of events during infection.…”

“…The control hGB1 alone did not allow this masking, either on the wild-type GST-L E or with the phosphomimetic bait, T 47 E. Therefore, Y 41 , which lies to the C-terminal side of the L E hinge domain, is among the likely contact sites for 2A binding. This site and T 47 were found to be solvent exposed when L E binds Ran (9).…”

“…The Saffold (L S [71 aa]) and TMEV (L T [76 aa]) L proteins are slightly longer. The solution structure of the L protein (L M ) of mengovirus (a subtype of EMCV) has been determined in free form and as bound to Ran GTPase, a key cellular participant in L-dependent activities (9). The conformation is primarily random coil, except for an amino-proximal CHCC zinc finger motif (aa 10 to 22).…”

“…The consequence is induced hyperphosphorylation of Phe/Gly-containing nuclear pore proteins (nucleoporins [Nups]) by cellular kinases in the p38 and Erk1/2 pathways (8,14,15) and subsequent cessation of active NCT. It has been proposed that L M :Ran complexes achieve this by trapping exportin-bound activated kinases within the nuclear pores (nuclear pore complexes [NPC]) (9). Since Ran-dependent NCT is essentially shut down, the movement of cellular proteins and RNA through the NPC is reduced to that permitted by diffusion alone.…”

The Language of Life

“…Eventually the active players (p38 and ERK-1/2) were identified for those Nup phosphorylation events, and for required modifications of the L protein itself (CK2 and Syk). Fitting the pieces together though, required NMR of the L:Ran complex (41) a superb technical feat by Valjean Bacot-Davis, a microbiology student who self-taught himself structural biology, just for this project. The structure(s) showed that L hijacks Ran, locking it into an irreversible GTP conformer.…”