The molecular structure of Schistosoma mansoni PNP isoform 2 provides insights into the nucleoside selectivity of PNPs

Purine nucleoside phosphorylases (PNPs) play an important role in the blood fluke parasite Schistosoma mansoni as a key enzyme of the purine salvage pathway. Here we present the structural and kinetic characterization of a new PNP isoform from S. mansoni, SmPNP2. Thermofluorescence screening of different ligands suggested cytidine and cytosine are potential ligands. The binding of cytosine and cytidine were confirmed by isothermal titration calorimetry, with a KD of 27 μM for cytosine, and a KM of 76.3 μM for cytidine. SmPNP2 also displays catalytic activity against inosine and adenosine, making it the first described PNP with robust catalytic activity towards both pyrimidines and purines. Crystal structures of SmPNP2 with different ligands were obtained and comparison of these structures with the previously described S. mansoni PNP (SmPNP1) provided clues for the unique capacity of SmPNP2 to bind pyrimidines. When compared with the structure of SmPNP1, substitutions in the vicinity of SmPNP2 active site alter the architecture of the nucleoside base binding site thus permitting an alternative binding mode for nucleosides, with a 180° rotation from the canonical binding mode. The remarkable plasticity of this binding site enhances our understanding of the correlation between structure and nucleotide selectivity, thus suggesting new ways to analyse PNP activity.

1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 2.10 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based.

Metric
Whole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) R f ree 111664 4608 (2.10-2.10) Clashscore 122126 5109 (2.10-2.10) Ramachandran outliers 120053 5059 (2.10-2.10) Sidechain outliers 120020 5060 (2.10-2.10) RSRZ outliers 108989 4497 (2.10-2.10) The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain 1 A 287 2 Entry composition i ○ There are 4 unique types of molecules in this entry. The entry contains 2418 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein called Purine nucleoside phosphorylase. 3 Residue-property plots i ○ These plots are drawn for all protein, RNA and DNA chains in the entry. The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometryand electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

Mol Chain Residues
• Molecule 1: Purine nucleoside phosphorylase Chain A: Xtriage's analysis on translational NCS is as follows: The largest off-origin peak in the Patterson function is 3.93% of the height of the origin peak. No significant pseudotranslation is detected.
5 Model quality i ○

Standard geometry i ○
Bond lengths and bond angles in the following residue types are not validated in this section: HPA, DMS The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no bond angle outliers.

Mol Chain
There are no chirality outliers.
There are no planarity outliers. The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 1.

Too-close contacts
All (6) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude. There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues. There are no Ramachandran outliers to report.

Protein sidechains i ○
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. All (1) residues with a non-rotameric sidechain are listed below:

Mol Chain
Mol Chain Res Type 1 A 58 PHE Some sidechains can be flipped to improve hydrogen bonding and reduce clashes. There are no such sidechains identified.

RNA i ○
There are no RNA molecules in this entry.

5.4
Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no carbohydrates in this entry.

5.6
Ligand geometry i ○ 4 ligands are modelled in this entry.
In the following table, the Counts columns list the number of bonds (or angles) for which Mogul statistics could be retrieved, the number of bonds (or angles) that are observed in the model and the number of bonds (or angles) that are defined in the Chemical Component Dictionary. The Link column lists molecule types, if any, to which the group is linked. The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 2 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no chirality outliers.

Mol Type Chain Res Link
There are no torsion outliers.
There are no ring outliers.
No monomer is involved in short contacts.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry. 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9. 6.2 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no carbohydrates in this entry.

Ligands i ○
In the following table, the Atoms column lists the number of modelled atoms in the group and the number defined in the chemical component dictionary. The B-factors column lists the minimum, median, 95 th percentile and maximum values of B factors of atoms in the group. The column labelled 'Q< 0.9' lists the number of atoms with occupancy less than 0.9.