An encephalitozoon cuniculi ortholog of the RNA polymerase II carboxyl-terminal domain (CTD) serine phosphatase Fcp1

Fcp1 is an essential protein serine phosphatase that dephosphorylates Ser2 or Ser5 of the RNA polymerase II carboxyl-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). The CTD of the microsporidian parasite Encephalitozoon cuniculi consists of 15 heptad repeats, which approximates the minimal CTD length requirement for cell viability in yeast. Here we show that E. cuniculi encodes a minimized 411-aa Fcp1-like protein (EcFcp1), which consists of a DxDx(T/V) phosphatase domain and a BRCA1 carboxyl terminus (BRCT) domain but lacks the large N- and C-terminal domains found in fungal and metazoan Fcp1 enzymes. Nonetheless, EcFcp1 can function in lieu of Saccharomyces cerevisiae Fcp1 to sustain yeast cell growth. Recombinant EcFcp1 is a monomeric enzyme with intrinsic phosphatase activity against nonspecific (p-nitrophenyl phosphate) and specific (CTD-PO(4)) substrates. EcFcp1 dephosphorylates CTD positions Ser2 and Ser5 with similar efficacy in vitro. We exploit synthetic CTD Ser2-PO(4) and Ser5-PO(4) peptides to define minimized substrates for EcFcp1 and to illuminate the importance of CTD primary structure in Ser2 and Ser5 phosphatase activity.     

Pin1 modulates the dephosphorylation of the RNA polymerase II C-terminal domain by yeast Fcp1

The reversible phosphorylation of serine and threonine residues N-terminal to proline (pSer/Thr-Pro) is an important signaling mechanism in the cell. The pSer/Thr-Pro moiety exists in the two distinct cis and trans conformations, whose conversion is catalyzed by the peptidyl-prolyl isomerase (PPIase) Pin1. Among others, Pin1 binds to the phosphorylated C-terminal domain (CTD) of the largest subunit of the RNA polymerase II, but the biochemical and functional relevance of this interaction is unknown. Here we confirm that the CTD phosphatase Fcp1 can suppress a Pin1 mutation in yeast. Furthermore, this genetic interaction requires the phosphatase domain as well as the BRCT domain of Fcp1, suggesting a critical role of the Fcp1 localization. Based on these observations, we developed a new in vitro assay to analyze the CTD dephosphorylation by Fcp1 that uses only recombinant proteins and mimics the in vivo situation. This assay allows us to present strong evidence that Pin1 is able to stimulate CTD dephosphorylation by Fcp1 in vitro, and that this stimulation depends on Pin1's PPIase activity. Finally, Pin1 significantly increased the dephosphorylation of the CTD on the Ser(5)-Pro motif, but not on Ser(2)-Pro in yeast, which can be explained with Pin1's substrate specificity. Together, our results indicate a new role for Pin1 in the regulation of CTD phosphorylation and present a further example for prolyl isomerization-dependent protein dephosphorylation.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Schizosaccharomyces pombe carboxyl-terminal domain (CTD) phosphatase Fcp1: distributive mechanism, minimal CTD substrate, and active site mapping

Schizosaccharomyces pombe Fcp1 is an essential protein serine phosphatase that preferentially dephosphorylates Ser(2) of the RNA polymerase II C-terminal domain (CTD) heptad repeat Y(1)S(2)P(3)T(4)S(5)P(6)S(7). Here we show that: (i) Fcp1 acts distributively during the hydrolysis of substrates containing tandem Ser(2)-PO(4) heptads; (ii) the minimal optimal CTD substrate for Fcp1 is a single heptad of phasing S(5)P(6)S(7)Y(1)S(2)P(3)T(4); and (iii) single alanine mutations of flanking residues Tyr(1) or Pro(3) result in 6-fold decrements in CTD phosphatase activity. Fcp1 belongs to the DXDX(T/V) family of phosphotransferases that act via an acyl-phosphoenzyme intermediate. An alanine scan of 11 conserved positions of S. pombe Fcp1 identifies Thr(174), Tyr(237), Thr(243), and Tyr(249) as important for phosphatase activity. Structure-activity relationships at these positions were determined by introducing conservative substitutions. Our results, together with previous mutational studies, highlight a constellation of 11 amino acids that are conserved in all Fcp1 orthologs and likely comprise the active site.     

Defining the active site of Schizosaccharomyces pombe C-terminal domain phosphatase Fcp1

Fcp1 is an essential protein serine phosphatase that dephosphorylates the C-terminal domain (CTD) of RNA polymerase II. By testing the effects of serial N- and C-terminal deletions of the 723-amino acid Schizosaccharomyces pombe Fcp1, we defined a minimal phosphatase domain spanning amino acids 156-580. We employed site-directed mutagenesis (introducing 24 mutations at 14 conserved positions) to locate candidate catalytic residues. We found that alanine substitutions for Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298) abrogated the phosphatase activity with either p-nitrophenyl phosphate or CTD-PO(4) as substrates. Structure-activity relationships were determined by introducing conservative substitutions at each essential position. Our results, together with previous mutational studies, highlight a constellation of seven amino acids (Asp(170), Asp(172), Arg(223), Asp(258), Lys(280), Asp(297), and Asp(298)) that are conserved in all Fcp1 orthologs and likely comprise the active site. Five of these residues (Asp(170), Asp(172), Lys(280), Asp(297), and Asp(298)) are conserved at the active site of T4 polynucleotide 3'-phosphatase, suggesting that Fcp1 and T4 phosphatase are structurally and mechanistically related members of the DXD phosphotransferase superfamily.