The first crystal structure of a dTTP-bound deoxycytidylate deaminase validates and details the allosteric-inhibitor binding site

Abstract

Deoxycytidylate deaminase is unique within the zinc-dependent cytidine deaminase family as being allosterically regulated, activated by dCTP, and inhibited by dTTP. Here we present the first crystal structure of a dTTP-bound deoxycytidylate deaminase from the bacteriophage S-TIM5, confirming that this inhibitor binds to the same site as the dCTP activator. The molecular details of this structure, complemented by structures apo- and dCMP-bound, provide insights into the allosteric mechanism. Although the positioning of the nucleoside moiety of dTTP is almost identical to that previously described for dCTP, protonation of N3 in deoxythymidine and not deoxycytidine would facilitate hydrogen bonding of dTTP but not dCTP and may result in a higher affinity of dTTP to the allosteric site conferring its inhibitory activity. Further the functional group on C4 (O in dTTP and NH2 in dCTP) makes interactions with nonconserved protein residues preceding the allosteric motif, and the relative strength of binding to these residues appears to correspond to the potency of dTTP inhibition. The active sites of these structures are also uniquely occupied by dTMP and dCMP resolving aspects of substrate specificity. The methyl group of dTMP apparently clashes with a highly conserved tyrosine residue, preventing the formation of a correct base stacking shown to be imperative for deamination activity. The relevance of these findings to the wider zinc-dependent cytidine deaminase family is also discussed.

Keywords: Allosteric Regulation; Cytidine Deaminase; Deoxycytidylate Deaminase; Protein Structure; S-TIM5; X-ray Crystallography; Zinc; dCMP Deaminase; dTTP.

FIGURE 1.

Functional characterization of S-TIM5-dCD. A, sequence alignment of S-TIM5-dCD with structurally characterized dCDs. T4 bact., T4 bacteriophage. Displayed are only sequences of loop 3, including the catalytic motif and the allosteric motif highlighting the distinct tryptophan of S-TIM5-dCD allosteric motif. B, deamination activity of S-TIM5-dCD shown in absorbance (290 nm) as a function of time. Protein incubated with dCMP, dCTP, and Mg²⁺ (line a); dCMP and Mg²⁺ (line b); dCTP and Mg²⁺ (line c); CMP, dCTP, and Mg²⁺ (line d); and dCMP, dCTP, dTTP, and Mg²⁺ (line e). The ratio of dCTP to dTTP is 1:1. C, relative dCMP deamination activity of S-TIM5-dCD as a function of pH. The error bars represent triplicates. D, relative dCMP deamination activity of S-TIM5-dCD WT and Trp⁴² mutants in the presence of dCTP and Mg²⁺. The error bars represent three samples each of three biological replicates.

FIGURE 2.

Overall structure of S-TIM5-dCD. A, superposition of the in crystallo hexameric forms (carried out in Coot). For clarity, every second protomer is shaded gray (S-TIM5-A in purple, S-TIM5-T in red, and S-TIM5-C in cyan). Blue arrowheads highlight interfaces between protomers. Inset, close-up of the middle region of the hexamer highlighting the greater compactness of the unbound S-TIM5-A structure (opposite protomers in the hexamer are ∼2.5 Å closer) as compared with the ligand-bound structures of S-TIM5-C and S-TIM5-T. B, superposition of the monomers from each structure. Zn²⁺ ions are displayed as spheres. C, secondary zinc binding motif in the S-TIM5-C (cyan) is substituted with a disulfide bond (represented by Cys²² and Cys⁵⁴ as sticks). Secondary zinc (Zn”, orange sphere) and its binding residues (orange lines, His⁹⁴ is labeled) from the structurally aligned T4 bacteriophage dCD (PDB code 1VQ2) are shown. Catalytic zinc ions are represented as overlapped cyan and orange spheres for S-TIM5-C and 1VQ2, respectively.

FIGURE 3.

dTTP bound at the allosteric binding site. A, cross-eyed stereo view of an F_o − F_c map (blue mesh) at 3 σ calculated with dTTP omitted from the S-TIM5-T structure. Trp⁴² is displayed as sticks, and the Mg²⁺ ion is a yellow sphere. B, structural alignment of the allosteric binding sites of S-TIM5-T (magenta) and 2HVW (yellow). Trp⁴² and dCMP from the S-TIM5-C structure are superimposed in cyan. The aromatic residue of the allosteric binding site and the bound nucleotide are represented as sticks. C, polar interactions made between dTTP (green) and the protein environment (magenta) are shown in black dashed lines. Interacting residues are labeled and represented with sticks, and the Mg²⁺ ion is a yellow sphere. D, MST measurements. S-TIM5-dCD binding affinities to dCTP and dTTP. Standard deviations were obtained from triplicate experiments. ΔF_Norm, normalized fluorescence units calculated as percentage of bound and unbound substrate.

FIGURE 4.

Monophosphate nucleotides bound at the catalytic sites. A, cross-eyed stereo view of the catalytic site of S-TIM5-C (cyan) with dUMP (green sticks) bound. Interacting residues (sticks), polar interactions (black dashed lines), and catalytic zinc ion (cyan sphere) are shown. Highlighted in blue sticks are the residues of the GWNG motif (with blue labels) separating the catalytic from allosteric sites, and shown with green lines is the dCMP bound at the allosteric site. B, deamination activity of S-TIM5-dCD containing substitutions to the conserved Tyr¹¹⁶, Asn⁴³, and nonconserved Thr⁶¹, which interact with the bound substrate. C, the highly conserved hydrogen bonding triad (black dashed lines) formed in dCDs by Ser²⁰, Val²⁹, and Asn⁴³ (shown as sticks) is highlighted on the cyan cartoon structure of S-TIM5-C. dUMP in the catalytic site is shown as green sticks, and the catalytic zinc is a gray sphere. D, cross-eyed view of superposition of CDAs with substrate/inhibitor-bound catalytic sites (PDB codes 1ZAB, 1JTK, 2FR6, 2FR5, 4EG2, 1ALN, and 1MQ0). Highlighted are the catalytic site zinc (green sphere), zinc-coordinating residues (gray), catalytic Glu (blue), substrate/inhibitor (magenta), the conserved phenylalanine from an adjacent subunit (yellow), and conserved asparagines (green). E, cross-eyed stereo view of superposition of dCDs with substrate/inhibitor-bound catalytic sites (S-TIM5-T in magenta sticks, S-TIM5-C in cyan sticks, 2HVW in pink lines, and 1VQ2 in orange lines). A CDA (2FR5 in green) from D is included for reference. Arrows and distances refer to relative movement of S-TIM5-T compared with S-TIM5-C. Residue numbering is for S-TIM5.