Structural Analysis of Thymidylate Synthase from Kaposi’s Sarcoma-Associated Herpesvirus with the Anticancer Drug Raltitrexed

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a highly infectious human herpesvirus that causes Kaposi’s sarcoma. KSHV encodes functional thymidylate synthase, which is a target for anticancer drugs such as raltitrexed or 5-fluorouracil. Thymidylate synthase catalyzes the conversion of 2′-deoxyuridine-5′-monophosphate (dUMP) to thymidine-5′-monophosphate (dTMP) using 5,10-methylenetetrahydrofolate (mTHF) as a co-substrate. The crystal structures of thymidylate synthase from KSHV (apo), complexes with dUMP (binary), and complexes with both dUMP and raltitrexed (ternary) were determined at 1.7 Å, 2.0 Å, and 2.4 Å, respectively. While the ternary complex structures of human thymidylate synthase and E. coli thymidylate synthase had a closed conformation, the ternary complex structure of KSHV thymidylate synthase was observed in an open conformation, similar to that of rat thymidylate synthase. The complex structures of KSHV thymidylate synthase did not have a covalent bond between the sulfhydryl group of Cys219 and C6 atom of dUMP, unlike the human thymidylate synthase. The catalytic Cys residue demonstrated a dual conformation in the apo structure, and its sulfhydryl group was oriented toward the C6 atom of dUMP with no covalent bond upon ligand binding in the complex structures. These structural data provide the potential use of antifolates such as raltitrexed as a viral induced anticancer drug and structural basis to design drugs for targeting the thymidylate synthase of KSHV.

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

X-RAY DIFFRACTION
The reported resolution of this entry is 2.00 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 91344 6249 (2.00-2.00)Clashscore 102246 7340 (2.00-2.00)Ramachandran outliers 100387 7248 (2.00-2.00)Sidechain outliers 100360 7247 (2.00-2.00)RSRZ outliers 91569 6262 (2.00-2.00) The table below summarises the geometric issues observed across the polymeric chains and their t 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 t to the electron density.The numeric value is given above the bar.
Mol Chain Length Quality of chain 2 Entry composition i O There are 3 unique types of molecules in this entry.The entry contains 4942 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.

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These plots are drawn for all protein, RNA and DNA chains in the entry.The rst graphic for a chain summarises the proportions of errors displayed in the second graphic.The second graphic shows the sequence view annotated by issues in geometry and electron density.Residues are colorcoded 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 t 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.
There are no chirality outliers.
There are no planarity outliers.The all-atom clashscore is dened as the number of clashes found per 1000 atoms (including hydrogen atoms).The all-atom clashscore for this structure is 2.
All (19) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude. Atom

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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.
Mol Chain Analysed Favoured Allowed Outliers Percentiles There are no chirality outliers.
There are no torsion outliers.
There are no ring outliers.
No monomer is involved in short contacts.

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

Polymer linkage issues i O
There are no chain breaks in this entry.
6 Fit of model and data i O 6.1 Protein, DNA and RNA chains i O 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.

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There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i O
There are no carbohydrates in this entry.

Ligands i O
In the following table, the Atoms column lists the number of modelled atoms in the group and the number dened in the chemical component dictionary.LLDF column lists the quality of electron density of the group with respect to its neighbouring residues in protein, DNA or RNA chains.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.
Mol Type Chain Res Atoms RSCC RSR LLDF B-factors(Å 2 ) Q<0.9 Bond lengths and bond angles in the following residue types are not validated in this section: UMPThe 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).
Xtriage's analysis on translational NCS is as follows: The largest o-origin peak in the Patterson function is 6.87% of the height of the origin peak.No signicant pseudotranslation is detected.1Intensities estimated from amplitudes. 2 Theoretical values of < |L| >, < L 2 > for acentric reections are 0.5, 0.333 respectively for untwinned datasets, and 0.375, 0.2 for perfectly twinned datasets.
In the following table, the Non-H and H(model) columns list the number of non-hydrogen atoms and hydrogen atoms in the chain respectively.The H(added) column lists the number of hydrogen atoms added and optimized by MolProbity.The Clashes column lists the number of clashes within the asymmetric unit, whereas Symm-Clashes lists symmetry related clashes.
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 dened 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).In the following table, the Chirals column lists the number of chiral outliers, the number of chiral centers analysed, the number of these observed in the model and the number dened in the chemical component dictionary.Similar counts are reported in the Torsion and Rings columns.'-' means no outliers of that kind were identied.
Mol Chain Res Type Atoms Z Observed( o ) Ideal( o ) There are no such residues in this entry.