4wyw

From Proteopedia

Mutant K20E of 3D polymerase from Foot-and-Mouth Disease Virus

Structural highlights

4wyw is a 1 chain structure with sequence from Foot-and-mouth disease virus (strain C1-Santa Pau). Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.8Å
Ligands:	, ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

POLG_FMDVS The leader protease autocatalytically cleaves itself from the polyprotein at the L/VP0 junction. It cleaves the host translation initiation factors EIF4G1 and EIF4G3, in order to shut down the capped cellular mRNA transcription (By similarity). Capsid proteins VP1, VP2, VP3 and VP4 form a closed capsid enclosing the viral positive strand RNA genome. VP4 lies on the inner surface of the protein shell formed by VP1, VP2 and VP3. All the three latter proteins contain a beta-sheet structure called beta-barrel jelly roll. Together they form an icosahedral capsid (T=3) composed of 60 copies of each VP1, VP2, and VP3, with a diameter of approximately 300 Angstroms. VP1 is situated at the 12 fivefold axes, whereas VP2 and VP3 are located at the quasi-sixfold axes. The capsid interacts with host heparan sulfate and various integrins (alphavbeta1, alphavbeta3, alpha5beta1, alphavbeta6, alphavbeta8) to provide virion attachment to target Attachment via host integrins induces virion internalization predominantly through clathrin-mediated endocytosis (By similarity). Protein VP0: VP0 precursor is a component of immature procapsids (By similarity). Protein 2B: Affects membrane integrity and cause an increase in membrane permeability (By similarity). Protein 2C: Associates with and induces structural rearrangements of intracellular membranes. It displays RNA-binding, nucleotide binding and NTPase activities (By similarity). Protein 3A, via its hydrophobic domain, serves as membrane anchor (By similarity). Protein 3B-1, 3B-2 and 3B-3 are covalently linked to the 5'-end of both the positive-strand and negative-strand genomic RNAs. They acts as a genome-linked replication primer (By similarity). Protease 3C: cysteine protease that generates mature viral proteins from the precursor polyprotein. In addition to its proteolytic activity, it binds to viral RNA, and thus influences viral genome replication. RNA and substrate bind cooperatively to the protease (By similarity). RNA-directed RNA polymerase 3D-POL replicates genomic and antigenomic RNA by recognizing replications specific signals (By similarity).

Publication Abstract from PubMed

The N-terminal region of the FMDV 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD, and severely impaired virus infectivity. These residues have also been implicated in template binding as seen in the crystal structures of different 3D-RNA elongation complexes. Here we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular K18E, K18A, K20E, K20A and the double mutant KAKA. All mutant enzymes exhibit low RNA binding activity, low processivity and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides, as compared with the wild type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both, global rearrangements of the closed hand architecture and local conformational changes at loop beta9-alpha11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate both RNA replication, processivity and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal and in template binding, is also involved in nucleotide recognition, and can affect the incorporation of nucleotide analogues. IMPORTANCE: The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside-monophosphate versus ribavirin-monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function, and for lethal mutagenesis mechanisms.

Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.,Ferrer-Orta C, de la Higuera I, Caridi F, Sanchez-Aparicio MT, Moreno E, Perales C, Singh K, Sarafianos SG, Sobrino F, Domingo E, Verdaguer N J Virol. 2015 Apr 22. pii: JVI.03283-14. PMID:25903341^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

References

↑ Ferrer-Orta C, de la Higuera I, Caridi F, Sanchez-Aparicio MT, Moreno E, Perales C, Singh K, Sarafianos SG, Sobrino F, Domingo E, Verdaguer N. Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition. J Virol. 2015 Apr 22. pii: JVI.03283-14. PMID:25903341 doi:http://dx.doi.org/10.1128/JVI.03283-14