Structure and function of Campylobacter jejuni polynucleotide phosphorylase (PNPase): Insights into the role of this RNase in pathogenicity.

Bárria C, Athayde D, Hernandez G, Fonseca L, Casinhas J, Cordeiro TN, Archer M, Arraiano CM, Brito JA, Matos RG, Biochimie (2023) Europe PMC

SASDLE7 – Polyribonucleotide nucleotidyltransferase (PNPase): 2-GMP complex

Polyribonucleotide nucleotidyltransferase
MWI(0) 221 kDa
MWexpected 237 kDa
VPorod 312 nm3
log I(s) 2.10×10-1 2.10×10-2 2.10×10-3 2.10×10-4
Polyribonucleotide nucleotidyltransferase small angle scattering data  s, nm-1
ln I(s)
Polyribonucleotide nucleotidyltransferase Guinier plot ln 2.10×10-1 Rg: 3.9 nm 0 (3.9 nm)-2 s2
(sRg)2I(s)/I(0)
Polyribonucleotide nucleotidyltransferase Kratky plot 1.104 0 3 sRg
p(r)
Polyribonucleotide nucleotidyltransferase pair distance distribution function Rg: 3.8 nm 0 Dmax: 11 nm

Data validation


Fits and models


log I(s)
 s, nm-1
Polyribonucleotide nucleotidyltransferase DAMFILT model

Synchrotron SAXS data from PNPase:2-GMP complex in 20 mM Tris·HCl at pH 8.0, 10 mM NAH2PO4, 60 mM KCl, 1mM MgCl2 and 2mM DTT was collected on the B21-DLS beam-line at the Diamond Light Source (Didcot, UK) using a Pilatus 2M detector at a sample-detector distance of 3.988 m and at a wavelength of λ = 0.1024 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed using an Agilent 1200 HPLC System by injecting a 60 μl sample at 6.4 mg/ml of protein with 417μM of ligand under a flow rate of 0.075 ml/min onto Superdex 200 Increase 3.2/300 column (GE Healthcare) at 25°C collecting 915 successive three-second frames. The scattering intensities from PNPase:2-GMP complex elution peak region (46-frames) were integrated, buffer subtracted and averaged using the ScÅtter software (version 3Q) to produce the averaged SEC-SAXS profile. From this profile, the pair-wise distance distribution function, P(r), was obtained by indirect Fourier Transform with GNOM (version 5.0) using a momentum transfer range of 0.08990 < s <2.67956 nm-1. The Rg values were estimated by applying the Guinier approximation in the range s < 1.3/Rg. The displayed SAXS-generated ab initio molecular reconstruction of PNPase:2-GMP complex was obtained using DAMMIF (version 2.3, from ATSAS) - DAMFILT occupancy and volume-corrected bead model - by clustering and averaging dummy residue models from 20 independent runs, with an averaged Normalized Spatial Discrepancy (NSD) of 0.960.

Polyribonucleotide nucleotidyltransferase (PNPase:2-GMP)
Mol. type   Protein
Organism   Campylobacter jejuni subsp. jejuni serotype O:6 (strain 81116 / NCTC 11828)
Olig. state   Trimer
Mon. MW   79.0 kDa
 
UniProt   A8FMV9 (1-718)
Sequence   FASTA