4X2B

K20A RNA dependent RNA polymerase mutant from Foot-and-Mouth disease Virus complexed with an RNA

Summary for 4X2B

• Entry DOI: 10.2210/pdb4x2b/pdb
• Related: 1U09 1WNE
• Descriptor: RNA dependent RNA polymerase, RNA template, RNA primer, ... (5 entities in total)
• Functional Keywords: nuclear signal localization, picornavirus, foot-and-mouth disease, virus, rna dependent rna polymerase, 3d polymerase, transferase
• Biological source: Foot-and-mouth disease virus (FMDV); More
• Cellular location: Protein VP2: Virion. Protein VP3: Virion. Protein VP1: Virion. Protein 2B: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Protein 2C: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Protein 3A: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side . Protein 3B-1: Virion . Protein 3B-2: Virion . Protein 3B-3: Virion . Picornain 3C: Host cytoplasm . RNA-directed RNA polymerase 3D-POL: Host cytoplasmic vesicle membrane ; Peripheral membrane protein ; Cytoplasmic side : P03311
• Total number of polymer chains: 3
• Total formula weight: 58219.14

Authors

Verdaguer, N.,Ferrer-Orta, C. (deposition date: 2014-11-26, release date: 2015-05-06, Last modification date: 2024-01-10)

Primary citation

Ferrer-Orta, C.,de la Higuera, I.,Caridi, F.,Sanchez-Aparicio, M.T.,Moreno, E.,Perales, C.,Singh, K.,Sarafianos, S.G.,Sobrino, F.,Domingo, E.,Verdaguer, N.
Multifunctionality of a picornavirus polymerase domain: nuclear localization signal and nucleotide recognition.
J.Virol., 89:6848-6859, 2015

PubMed Abstract: The N-terminal region of the foot-and-mouth disease virus (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 K18A K20A (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 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 β9-α11 (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 RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues.

PubMed: 25903341
DOI: 10.1128/JVI.03283-14

Experimental method

X-RAY DIFFRACTION (2.94 Å)