Structural insights to how mammalian capping enzyme reads the CTD code

Abstract

Physical interaction between the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD) and cellular capping enzymes is required for efficient formation of the 5' mRNA cap, the first modification of nascent mRNA. Here, we report the crystal structure of the RNA guanylyltransferase component of mammalian capping enzyme (Mce) bound to a CTD phosphopeptide. The CTD adopts an extended β-like conformation that docks Tyr1 and Ser5-PO(4) onto the Mce nucleotidyltransferase domain. Structure-guided mutational analysis verified that the Mce-CTD interface is a tunable determinant of CTD binding and stimulation of guanylyltransferase activity, and of Mce function in vivo. The location and composition of the CTD binding site on mammalian capping enzyme is distinct from that of a yeast capping enzyme that recognizes the same CTD primary structure. Thus, capping enzymes from different taxa have evolved different strategies to read the CTD code.

Figure1. Structure of the mammalian capping enzyme Mce in complex with phosphorylated Pol II CTD

A) A view of the Mce-CTD structure shown in ribbon representation with secondary structure elements indicated by arrows (β-strands) and ribbons (helices). A continuous thirteen amino acid CTD segment (in stick representation and colored green) interacts with two Mce protomers (protomer A and B). The NTase domains of the two protomers are labeled and colored in different shades of blue and the corresponding OB-fold domain elements that could be modeled are labeled and colored in yellow. N and C denote the location of the amino- and carboxy-terminus. The active site Lys294 and guanine base are labeled, shown in sticks and colored red and pale-yellow, respectively, to indicate the relative position of the Mce active site. All figures depicting structure were generated with PyMol (DeLano, 2002). B) A close-up view of the CTD associated with the two protomers of Mce. Mce is shown in ribbon representation with CTD residues labeled and represented by sticks. The distance between N and C termini of the CTD is denoted with an arrow. C) A close-up of Mce protomer A (surface representation colored by positive (blue) and negative (red) electrostatic potential). CTD residues are labeled and represented by sticks.

Figure 2. Capping enzyme interactions with phosphorylated CTD

A) Stereo diagram of interactions between Mce and CTD. Selected side chains and phosphoserine residues are shown in stick representation and labeled. Potential hydrogen bonds and van der Waals contacts are denoted with black and red dashed lines, respectively. Mce residues that interact with CTD Tyr1 and Ser5-PO₄ are labeled in blue and red, respectively. B) Stereo view of C. albicans guanylyltransferase Cgt1 (in light gray) bound to CTD (stick representation and colored cyan) depicting the same surface of Cgt1 as shown for Mce in panel A. Potential hydrogen bonds with Ser5-PO₄ are shown with black dashes, and residues that interact with Ser5-PO₄ are labeled in red.

Figure 3. Genetic analysis of Mce-CTD interactions

A) Serial 10-fold dilutions of S. cerevisiaeRPB1-WTceg1Δ and RPB1-11ceg1Δ strains bearing indicated MCE1 alleles spotted on YPAD agar grown at 23°C (left), 30°C (middle), and 37°C (right). B) S. cerevisiaeRPB1-WTceg1Δ and RPB1-11ceg1Δ strains bearing pYX-MCE1 plasmids containing wild-type or mutant alleles K294A, R330A/K331A/C383Y, and V372Y/C383Y were assessed for growth at 30°C on agar medium lacking (left) or containing (right) 5-FOA. C) C375R mutation in the Mce-CTD interface results in a gain of function. A C375R substitution rescues conditional lethality in yeast when S. cerevisiae YBS50 cet1Δceg1Δ strain is complemented with a centromeric pRS412 plasmid containing MCE1-C375R flanked by CET1 5' and 3' UTR elements but not when complemented with a similar plasmid bearing MCE1 wild-type (WT) or CET1(276–549)-linker-MCE1(227–567) (Experimental Procedures). Strains were streaked on agar medium lacking (left) or containing (right) 5-FOA.

Figure 4. Stimulation of Mce guanylyltransferase activity by the CTD

A) Stimulation of Mce wild-type, R330A/K331A/C383Y and C375R mutant proteins by increasing concentrations of CTD Ser5-PO₄ (Experimental Procedures). Error bars (one standard deviation) were calculated from three independent experiments performed in triplicate. B) The fold increase in guanylylation activity was calculated by comparing the amount of enzyme-guanylate at 0 and 8 μM CTD Ser5-PO₄ and depicted by bar graphs with error bars (one standard deviation). C) Data obtained from fluorescence polarization assays used to derive K_d values for Mce wild-type, R330A/K331A/C383Y and C375R mutant proteins at pH 7.0 (Experimental Procedures). Error bars (one standard deviation) obtained from three independent experiments performed in triplicate. D) Summary of dissociation constants in bar graph representation with error bars (one standard deviation) as determined by fluorescence polarization assays for Mce wild-type or mutant proteins and phosphorylated CTD. E) Displacement of fluorescein labeled CTD peptide (Fluor-S₂P₃T₄S_5PP₆S₇Y₁S_2PP₃T₄S₅) bound to Mce wild-type protein with increasing quantities of unlabeled peptide (S₂P₃T₄S_5PP₆S₇Y₁S_2PP₃T₄S₅) (Experimental Procedures). Fluorescence polarization was measured after incubating ternary complexes on ice for 20 minutes. Error bars (one standard deviation) were calculated from three independent experiments performed in triplicate. F) Mce1-CTD interaction depends on CTD phosphorylation at Ser2 and Ser5 positions. Fluorescein labeled Ser2 and Ser5 phosphorylated (Fluor-S₂P₃T₄S_5PP₆S₇Y₁S_2PP₃T₄S₅; filled circles) and labeled dephosphorylated CTD (Fluor-S₂P₃T₄S₅P₆S₇Y₁S_PP₃T₄S₅; open circles) peptides were incubated with increasing concentrations of Mce wild-type protein (Experimental Procedures).

Figure 5. CTD interactions with yeast and mammalian capping enzymes

A) A close-up view and surface representation of the NTase domain of Mce (colored light gray) centered at the CTD binding site. Fully or partially conserved amino acids are colored in red or pink, respectively. Sequence conservation for Mce was generated by structure based sequence alignment of the corresponding metazoan NTase domains from M. musculus (Mm), H. sapiens (Hs), X. laevis (Xl), C. elegans (Ce), and A. thaliana (At). CTD residues are labeled, shown in stick representation and colored green. B) A closeup view of C. albicans Cgt1 surface (colored light gray) centered at the CTD binding site. Conserved residues are color coded as described in panel A. Sequence conservation was mapped on Cgt1 surface by aligning sequences corresponding to the NTase domains of C. albicans (Ca), S. cerevisiae (Sc), C. glabrata (Cg), K. lactis (Kl), and Y. lipolytica (Yl). CTD residues are shown in sticks, colored cyan and select positions are labeled. Aligned primary amino acid sequences are shown in the right panel and shaded according to sequence conservation. Secondary structure elements of Mce and Cgt1 are shown above the alignments. Residues in Mce or Cgt1 that are involved in H-bond and hydrophobic interactions are specified by red and blue circles above the sequence alignments, respectively. C) Surfaces of the NTase domains of Mce (blue) and Cgt1 (light gray) were superimposed to illustrate that the respective CTD elements (in stick representation colored as in panels A and B) are mostly non-overlapping and involve distinct surfaces of the Mce or Cgt1 NTase domains.

Figure 6. Conformational plasticity in CTD structures and mode of CTD recognition

CTD heptad arrays extracted from structures of A) Mce (in green), B) Cgt1- CDS1 and CDS2 (in cyan; PDB: 1P16), C) Pin1 (in orange; PDB: 1F8A), and D) Scp1 (in gray; PDB: 1GHQ). CTD segments shown in ribbon (main chain) and stick representation (side chain) were aligned vertically based on the position of the Ser5-PO₄ Cα atom. Although approximate, the orientation of the CTD structures also places the respective binding partners below the CTD. Right panels indicate phased CTD heptad sequences in bar graph format to illustrate the number of contacts (magnitude of the bar height) between the respective binding partner and CTD side chains (above) and main chain (below).