The crystal structures of Thermus thermophilus CMP kinase complexed with a phosphoryl group acceptor and donor

Nucleoside monophosphate kinases play crucial roles in biosynthesis and regeneration of nucleotides. These are bi-substrate enzymes that catalyze reversible transfers of a phosphoryl group between ATP and nucleoside monophosphate. These enzymes are comprised of the CORE domain, the NMP-binding domain, and the LID domain. Large conformational rearrangement of the three domains occurs during the catalytic cycle. Although many structures of CMP kinase have been determined, only limited structural information has been available on the conformational changes along the reaction pathway. We determined five crystal structures of CMP kinase of Thermus thermophilus HB8 in ligand-free form and the CMP "open", CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms at resolutions of 1.7, 2.2, 1.5, 1.6, and 1.7 Å, respectively. The ligand-free form was in an open conformation, whereas the structures of the CMP "closed", ADP-CDP-Gd3+-, and CDP-bound forms were in a closed conformation, in which the shift of the NMP-binding domain and LID domain caused closure of the substrate-binding cleft. Interestingly, the CMP "open" form was in an open conformation even with CMP bound, implying intrinsic conformational fluctuation. The structure of the ADP-CDP complex is the first structure of CMP kinase with a phosphoryl group donor and an acceptor. Upon simultaneous binding of ADP and CDP, the side chains of several residues in the LID domain moved toward the nucleotides without global open−closed conformational changes compared to those in the CMP "closed" and CDP complexes. These global and local conformational changes may be crucial for the substrate recognition and catalysis. The terminal phosphate groups of ADP and CDP had similar geometry to those of two ADP in AMP kinase, suggesting common catalytic mechanisms to other nucleoside monophosphate kinases. Our findings are expected to contribute to detailed understanding of the reaction mechanism of CMP kinase.

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 208 2 Entry composition i ○ There are 5 unique types of molecules in this entry. The entry contains 1760 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 Cytidylate kinase. 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: Cytidylate kinase Xtriage's analysis on translational NCS is as follows: The largest off-origin peak in the Patterson function is 4.18% 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: GD, CDP, ADP 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).

Mol Chain Bond lengths Bond angles
There are no bond length outliers.
There are no bond angle outliers.
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
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.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
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 8.
All (26)  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. 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.

Mol Chain
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues. 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.

Ligand geometry i ○
Of 3 ligands modelled in this entry, 1 is monoatomic -leaving 2 for Mogul analysis.
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. There are no such residues in this entry.