Spliceosomal Prp8 intein at the crossroads of protein and RNA splicing.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNAs. At its functional core lies the essential pre-mRNA processing factor 8 (Prp8) protein. Across diverse eukaryotes, this protein cofactor of RNA catalysis harbors a self-splicing element called an intein. Inteins in Prp8 are extremely pervasive and are found at 7 different sites in various species. Here, we focus on the Prp8 intein from Cryptococcus neoformans (Cne), a human fungal pathogen. We solved the crystal structure of this intein, revealing structural homology among protein splicing sequences in eukaryotes, including the Hedgehog C terminus. Working with the Cne Prp8 intein in a reporter assay, we find that the biologically relevant divalent metals copper and zinc inhibit intein splicing, albeit by 2 different mechanisms. Copper likely stimulates reversible modifications on a catalytically important cysteine, whereas zinc binds at the terminal asparagine and the same critical cysteine. Importantly, we also show that copper treatment inhibits Prp8 protein splicing in Cne. Lastly, an intein-containing Prp8 precursor model is presented, suggesting that metal-induced protein splicing inhibition would disturb function of both Prp8 and the spliceosome. These results indicate that Prp8 protein splicing can be modulated, with potential functional implications for the spliceosome.

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

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) as bound by L(M) (unlabeled) places L(M) into the BP1 binding site of Ran, wrapped by the conformational flexible COOH tail. The arrangement explains the tight KD for this complex and places the LM zinc finger and phosphorylation interface as surface exposed and available for subsequent reactions. The core structure of Ran, outside the COOH tail, is not altered by L(M) binding and remains accessible for canonical RanGTP partner interactions. Pull-down assays identify at least one putative Ran:L(M) partner as an exportin, Crm1, or CAS. A model of Ran:L(M):Crm1, based on the new structures suggests LM phosphorylation status may mediate Ran's selection of exportin(s) and cargo(s), perverting these native trafficking elements into the lethal antihost Nup phosphorylation pathways.

Encephalomyocarditis virus leader is phosphorylated by CK2 and syk as a requirement for subsequent phosphorylation of cellular nucleoporins.

Encephalomyocarditis virus and Theilovirus are species in the Cardiovirus genus of the Picornaviridae family. For all cardioviruses, the viral polyprotein is initiated with a short Leader (L) protein unique to this genus. The nuclear magnetic resonance (NMR) structure of LE from encephalomyocarditis virus (EMCV) has been determined. The protein has an NH2-proximal CHCC zinc finger, a central linker, and a contiguous, highly acidic motif. The theiloviruses encode the same domains, with one or two additional, COOH-proximal domains, characteristic of the human Saffold viruses (SafV) and Theiler's murine encephalomyelitis viruses (TMEV), respectively. The expression of a cardiovirus L, in recombinant form, or during infection/transfection, triggers an extensive, cell-dependent, antihost phosphorylation cascade, targeting nucleoporins (Nups) that form the hydrophobic core of nuclear pore complexes (NPC). The consequent inhibition of active nucleocytoplasmic trafficking is potent and prevents the host from mounting an effective antiviral response. For this inhibition, the L proteins themselves must be phosphorylated. In cells (extracts or recombinant form), LE was shown to be phosphorylated at Thr47 and Tyr41. The first reaction (Thr47), catalyzed by casein kinase 2 (CK2), is an obligatory precedent to the second event (Tyr41), catalyzed by spleen tyrosine kinase (Syk). Site mutations in LE, or kinase-specific inhibitors, prevented LE phosphorylation and subsequent Nup phosphorylation. Parallel experiments with LS (SafV-2) and LT (TMEV BeAn) proteins confirmed the general cardiovirus requirement for L phosphorylation, but CK2 was not the culpable kinase. It is likely that LS and LT are both activated by alternative kinases in different cell types, probably reactive within the Theilo-specific domains. IMPORTANCE An understanding of the diverse methods used by viruses to interfere with cellular processes is important because they can teach us how to control virus infections. This report shows how viruses in the same genus use different cellular enzymes to phosphorylate their proteins. If these processes are interfered with, the viruses are severely disabled.

Binding interactions between the encephalomyocarditis virus leader and protein 2A.

UNLABELLED: The leader (L) and 2A proteins of cardioviruses are the primary antihost agents produced during infection. For encephalomyocarditis virus (EMCV), the prototype of the genus Cardiovirus, these proteins interact independently with key cellular partners to bring about inhibition of active nucleocytoplasmic trafficking and cap-dependent translation, respectively. L and 2A also bind each other and require this cooperation to achieve their effects during infection. Recombinant L and 2A interact with 1:1 stoichiometry at a KD (equilibrium dissociation constant) of 1.5 µM. The mapped contact domains include the amino-proximal third of 2A (first 50 amino acids) and the central hinge region of L. This contact partially overlaps the L segment that makes subsequent contact with Ran GTPase in the nucleus, and Ran can displace 2A from L. The equivalent proteins from Theiler's murine encephalomyelitis virus (TMEV; BeAn) and Saffold virus interact similarly in any subtype combination, with various affinities. The data suggest a mechanism whereby L takes advantage of the nuclear localization signal in the COOH region of 2A to enhance its trafficking to the nucleus. Once there, it exchanges partners in favor of Ran. This required cooperation during infection explains many observed codependent phenotypes of L and 2A mutations. IMPORTANCE: Cardiovirus pathogenesis phenotypes vary dramatically, from asymptomatic, to mild gastrointestinal (GI) distress, to persistent demyelination and even encephalitic death. Leader and 2A are the primary viral determinants of pathogenesis, so understanding how these proteins cooperate to induce such a wide variety of outcomes for the host is of great important and interest to the field of virology, especially to those who use TMEV as a murine model for multiple sclerosis.

Effect of solvent and protein dynamics in ligand recognition and inhibition of aminoglycoside adenyltransferase 2″-Ia.

The aminoglycoside modifying enzyme (AME) ANT(2″)-Ia is a significant target for next generation antibiotic development. Structural studies of a related aminoglycoside-modifying enzyme, ANT(3″)(9), revealed this enzyme contains dynamic, disordered, and well-defined segments that modulate thermodynamically before and after antibiotic binding. Characterizing these structural dynamics is critical for in situ screening, design, and development of contemporary antibiotics that can be implemented in a clinical setting to treat potentially lethal, antibiotic resistant, human infections. Here, the first NMR structural ensembles of ANT(2″)-Ia are presented, and suggest that ATP-aminoglycoside binding repositions the nucleotidyltransferase (NT) and C-terminal domains for catalysis to efficiently occur. Residues involved in ligand recognition were assessed by site-directed mutagenesis. In vitro activity assays indicate a critical role for I129 toward aminoglycoside modification in addition to known catalytic D44, D46, and D48 residues. These observations support previous claims that ANT aminoglycoside sub-class promiscuity is not solely due to binding cleft size, or inherent partial disorder, but can be controlled by ligand modulation on distinct dynamic and thermodynamic properties of ANTs under cellular conditions. Hydrophobic interactions in the substrate binding cleft, as well as solution dynamics in the C-terminal tail of ANT(2″)-Ia, advocate toward design of kanamycin-derived cationic lipid aminoglycoside analogs, some of which have already shown antimicrobial activity in vivo against kanamycin and gentamicin-resistant P. aeruginosa. This data will drive additional in silico, next generation antibiotic development for future human use to combat increasingly prevalent antimicrobial resistance.

Encephalomyocarditis virus Leader protein hinge domain is responsible for interactions with Ran GTPase.

Encephalomyocarditis virus (EMCV), a Cardiovirus, initiates its polyprotein with a short 67 amino acid Leader (L) sequence. The protein acts as a unique pathogenicity factor, with anti-host activities which include the triggering of nuclear pore complex hyperphosphorylation and direct binding inhibition of the active cellular transport protein, Ran GTPase. Chemical modifications and protein mutagenesis now map the Ran binding domain to the L hinge-linker region, and in particular, to amino acids 35-40. Large deletions affecting this region were shown previously to diminish Ran binding. New point mutations, especially K35Q, D37A and W40A, preserve the intact L structure, abolish Ran binding and are deficient for nucleoporin (Nup) hyperphosphorylation. Ran itself morphs through multiple configurations, but reacts most effectively with L when in the GDP format, preferably with an empty nucleotide binding pocket. Therefore, L:Ran binding, mediated by the linker-hinge, is a required step in L-induced nuclear transport inhibition.