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• W2892393119 abstract "•SelO adopts a protein kinase fold with ATP flipped in the active site•SelO transfers AMP to Ser, Thr, and Tyr residues on protein substrates (AMPylation)•SelO AMPylates proteins involved in redox homeostasis•SelO protects cells from oxidative stress and regulates protein glutathionylation Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities. Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities. Protein kinases are an important class of enzymes that transfer phosphate from ATP to protein substrates, a process known as phosphorylation (Fischer, 2013Fischer E.H. Cellular regulation by protein phosphorylation.Biochem. Biophys. Res. Commun. 2013; 430: 865-867Crossref PubMed Scopus (44) Google Scholar). Virtually every cellular activity is regulated by protein kinases, and abnormal phosphorylation has been linked to numerous diseases. More than 500 human protein kinases have been identified and assembled into an evolutionary tree known as the human kinome (Manning et al., 2002Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6240) Google Scholar). However, research is largely biased toward kinases with well-established roles in disease; it has been estimated that the molecular functions of more than 50% of human kinases remain uncharacterized (Fedorov et al., 2010Fedorov O. Müller S. Knapp S. The (un)targeted cancer kinome.Nat. Chem. Biol. 2010; 6: 166-169Crossref PubMed Scopus (240) Google Scholar). Furthermore, several new kinase families have been identified that are so different they were not included on the human kinome tree. These include the Fam20 and Fam69 families of secretory pathway kinases (Dudkiewicz et al., 2013Dudkiewicz M. Lenart A. Pawłowski K. A novel predicted calcium-regulated kinase family implicated in neurological disorders.PLoS ONE. 2013; 8: e66427Crossref PubMed Scopus (31) Google Scholar, Tagliabracci et al., 2012Tagliabracci V.S. Engel J.L. Wen J. Wiley S.E. Worby C.A. Kinch L.N. Xiao J. Grishin N.V. Dixon J.E. Secreted kinase phosphorylates extracellular proteins that regulate biomineralization.Science. 2012; 336: 1150-1153Crossref PubMed Scopus (333) Google Scholar) and the selenocysteine (Sec)-containing protein selenoprotein-O (SelO) (Dudkiewicz et al., 2012Dudkiewicz M. Szczepińska T. Grynberg M. Pawłowski K. A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.PLoS ONE. 2012; 7: e32138Crossref PubMed Scopus (46) Google Scholar). About 10% of human protein kinases are predicted to be inactive and referred to as pseudokinases because they are missing residues located in highly conserved sequence motifs believed to be required for ATP binding and catalysis (Manning et al., 2002Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6240) Google Scholar). Pseudokinases serve a multitude of non-catalytic roles, such as allosteric regulators or scaffolding functions (Eyers and Murphy, 2013Eyers P.A. Murphy J.M. Dawn of the dead: protein pseudokinases signal new adventures in cell biology.Biochem. Soc. Trans. 2013; 41: 969-974Crossref PubMed Scopus (69) Google Scholar, Kung and Jura, 2016Kung J.E. Jura N. Structural Basis for the Non-catalytic Functions of Protein Kinases.Structure. 2016; 24: 7-24Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Taylor et al., 2013Taylor S.S. Shaw A. Hu J. Meharena H.S. Kornev A. Pseudokinases from a structural perspective.Biochem. Soc. Trans. 2013; 41: 981-986Crossref PubMed Scopus (33) Google Scholar, Zeqiraj and van Aalten, 2010Zeqiraj E. van Aalten D.M. Pseudokinases-remnants of evolution or key allosteric regulators?.Curr. Opin. Struct. Biol. 2010; 20: 772-781Crossref PubMed Scopus (111) Google Scholar). For example, the Fam20A pseudokinase binds to and increases the stability and activity of the secretory pathway kinase Fam20C, thus acting as an allosteric regulator (Cui et al., 2015Cui J. Xiao J. Tagliabracci V.S. Wen J. Rahdar M. Dixon J.E. A secretory kinase complex regulates extracellular protein phosphorylation.eLife. 2015; 4: e06120Crossref PubMed Scopus (84) Google Scholar). Similarly, the HER3 pseudokinase, although reported to have low catalytic activity, serves mostly as an allosteric activator for other members of the EGFR family of receptor kinases (Jura et al., 2009Jura N. Shan Y. Cao X. Shaw D.E. Kuriyan J. Structural analysis of the catalytically inactive kinase domain of the human EGF receptor 3.Proc. Natl. Acad. Sci. USA. 2009; 106: 21608-21613Crossref PubMed Scopus (248) Google Scholar, Shi et al., 2010Shi F. Telesco S.E. Liu Y. Radhakrishnan R. Lemmon M.A. ErbB3/HER3 intracellular domain is competent to bind ATP and catalyze autophosphorylation.Proc. Natl. Acad. Sci. USA. 2010; 107: 7692-7697Crossref PubMed Scopus (361) Google Scholar). These studies have highlighted the importance of pseudokinases in human biology, and their diverse signaling functions make them attractive drug targets (Bailey et al., 2015Bailey F.P. Byrne D.P. McSkimming D. Kannan N. Eyers P.A. 2) Going for broke: targeting the human cancer pseudokinome.Biochem. J. 2015; 466: 201Crossref PubMed Scopus (0) Google Scholar, Byrne et al., 2017Byrne D.P. Foulkes D.M. Eyers P.A. Pseudokinases: update on their functions and evaluation as new drug targets.Future Med. Chem. 2017; 9: 245-265Crossref PubMed Scopus (54) Google Scholar). Pseudokinases were initially predicted to be inactive if they were missing one or more of the three critical residues known to participate in phosphotransfer in active kinases (Manning et al., 2002Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6240) Google Scholar). These include (1) the VAIK motif in the β3-strand, where the Lys positions the α and β phosphates of ATP for catalysis (K72 using protein kinase A; PKA nomenclature); (2) the HRD motif located in the catalytic loop, where the Asp acts as the catalytic base (PKA; D166); and (3) the DFG motif; where the Asp binds the divalent cation to coordinate the β and γ phosphates of ATP (PKA; D184). However, some predicted pseudokinases have evolved compensatory mechanisms to catalyze phosphorylation by migration of active site residues including the WNK family of kinases (Min et al., 2004Min X. Lee B.H. Cobb M.H. Goldsmith E.J. Crystal structure of the kinase domain of WNK1, a kinase that causes a hereditary form of hypertension.Structure. 2004; 12: 1303-1311Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and the protein O-mannosyl kinase, SGK196 (Zhu et al., 2016Zhu Q. Venzke D. Walimbe A.S. Anderson M.E. Fu Q. Kinch L.N. Wang W. Chen X. Grishin N.V. Huang N. et al.Structure of protein O-mannose kinase reveals a unique active site architecture.eLife. 2016; 5: e22238Crossref PubMed Scopus (26) Google Scholar). Such compensatory mutations are often difficult to identify by primary amino acid sequence alone and have resulted in the wrongful annotation of some kinases as inactive. We previously predicted SelO to adopt a protein kinase fold (Dudkiewicz et al., 2012Dudkiewicz M. Szczepińska T. Grynberg M. Pawłowski K. A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.PLoS ONE. 2012; 7: e32138Crossref PubMed Scopus (46) Google Scholar). However, its sequence suggests that SelO would be an inactive pseudokinase because it lacks the catalytic Asp (PKA; D166) (Figures 1A and S1). Human SelO localizes to the mitochondria and incorporates the 21st genetically encoded amino acid Sec (Han et al., 2014Han S.J. Lee B.C. Yim S.H. Gladyshev V.N. Lee S.R. Characterization of mammalian selenoprotein o: a redox-active mitochondrial protein.PLoS ONE. 2014; 9: e95518Crossref PubMed Scopus (37) Google Scholar, Kryukov et al., 2003Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigó R. Gladyshev V.N. Characterization of mammalian selenoproteomes.Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1847) Google Scholar). Structurally, Sec is similar to Cys but contains a selenium atom in place of sulfur (Stadtman, 1974Stadtman T.C. Selenium biochemistry.Science. 1974; 183: 915-922Crossref PubMed Scopus (187) Google Scholar). The resulting selenol group has a lower pKa than the sulfur-containing thiol group and is deprotonated at physiological pH, resulting in higher nucleophilicity and oxidoreductase efficiency (Labunskyy et al., 2014Labunskyy V.M. Hatfield D.L. Gladyshev V.N. Selenoproteins: molecular pathways and physiological roles.Physiol. Rev. 2014; 94: 739-777Crossref PubMed Scopus (760) Google Scholar). 25 selenoproteins are encoded in the human genome, and many are involved in cellular redox homeostasis (Kryukov et al., 2003Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigó R. Gladyshev V.N. Characterization of mammalian selenoproteomes.Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1847) Google Scholar). In higher eukaryotes, most SelO homologs contain a single Sec near the carboxy terminus (Han et al., 2014Han S.J. Lee B.C. Yim S.H. Gladyshev V.N. Lee S.R. Characterization of mammalian selenoprotein o: a redox-active mitochondrial protein.PLoS ONE. 2014; 9: e95518Crossref PubMed Scopus (37) Google Scholar, Kryukov et al., 2003Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigó R. Gladyshev V.N. Characterization of mammalian selenoproteomes.Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1847) Google Scholar). In lower eukaryotes and all prokaryotes containing a SelO homolog, an invariant Cys occupies the equivalent position (Figure 1B). For simplicity, we will use the SelO name for the entire family regardless of whether or not the protein contains a Sec.Figure S1SelO Family Sequence Logo, Related to Figure 1Show full captionThe sequence of Pseudomonas syringae pv. tomato str. DC3000 SelO was subjected to 7 iterations of Jackhmmer search against Reference Proteomes in order to build a collection of homologs. The search yielded 3427 aligned protein sequences. The alignment was filtered so position numbers match P. syringae SelO residues and used as an input to the WebLogo server to generate the figure, which highlights conserved residues in the SelO family of pseudokinases. The height of the amino acid stack indicates the sequence conservation at that position. The position of the missing catalytic Asp (PKA; D166) is shaded and highlighted with an asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The sequence of Pseudomonas syringae pv. tomato str. DC3000 SelO was subjected to 7 iterations of Jackhmmer search against Reference Proteomes in order to build a collection of homologs. The search yielded 3427 aligned protein sequences. The alignment was filtered so position numbers match P. syringae SelO residues and used as an input to the WebLogo server to generate the figure, which highlights conserved residues in the SelO family of pseudokinases. The height of the amino acid stack indicates the sequence conservation at that position. The position of the missing catalytic Asp (PKA; D166) is shaded and highlighted with an asterisk. SelO is highly conserved, having homologs widespread among most eukaryotic taxa, and is also common in many major bacterial taxa (Dudkiewicz et al., 2012Dudkiewicz M. Szczepińska T. Grynberg M. Pawłowski K. A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.PLoS ONE. 2012; 7: e32138Crossref PubMed Scopus (46) Google Scholar). Despite this sequence-based indicator of a universal role across kingdoms, the molecular function of SelO is unknown. In fact, when prioritizing targets for experimental study, Koonin and colleagues listed SelO among the top ten most-attractive “unknown unknowns’’ because of phyletic spread and potential to reveal new and exciting biology (Galperin and Koonin, 2004Galperin M.Y. Koonin E.V. ‘Conserved hypothetical’ proteins: prioritization of targets for experimental study.Nucleic Acids Res. 2004; 32: 5452-5463Crossref PubMed Scopus (294) Google Scholar). Here, we report the crystal structure of a SelO homolog, which reveals a protein kinase-like fold. Remarkably, the ATP in the active site is flipped relative to the orientation of ATP in the active site of canonical kinases. Our structural studies led us to discover that the SelO pseudokinases are in fact active enzymes, yet transfer AMP, instead of a phosphate group, to Ser, Thr, and Tyr residues on protein substrates (AMPylation, aka adenylylation). Furthermore, we uncover that SelO plays an evolutionarily conserved role in the cellular response to oxidative stress by AMPylating proteins involved in redox homeostasis. We anticipate that the results of this work will have important implications for redox biology and may have the potential to define new paradigms of cellular regulation and signal transduction. To demonstrate the unique conservation of SelO, we performed basic local alignment search tool (BLAST) analyses against the Representative Proteomes RP55 database limited to bacteria using known human kinase domains and selenoproteins as queries in the search. We represent conservations of these different kinase and selenoprotein families as a plot of E value versus sequence identity of the top bacterial protein returned from the search (Figure 1C). Our results indicate that SelO is one of the most highly conserved members of either the human protein kinase families or the various selenoprotein families. Among bacteria, SelO is ubiquitous in Proteobacteria and Cyanobacteria, while in other phyla it is less frequent. In eukaryotes, most phyla contain on average 1 SelO gene per genome, while chordates and arthropods are exceptions, having an average of 2 or 0.14 genes per genome, respectively. To gain insight into the function of the SelO pseudokinases, we solved the crystal structure of the SelO homolog from the gram-negative plant pathogen Pseudomonas syringae bound to an ATP derivative AMP-PNP (Figures 2A and S2A; Table S1). Despite the unique sequence found in the SelO family, P. syringae SelO adopts a protein kinase-like fold consisting of 12 β strands and 22 α helices, with the kinase domain identifying a number of kinase structures as top hits using DALI or VAST structural homology search engines (i.e., aerobactin synthase IucA, the plant receptor kinase BRASSINOSTEROID INSENSITIVE 1, the human interleukin-1 receptor-associated kinase 4, and tyrosine kinase Syk). The kinase core (β4–α14) consists of a β strand-rich N lobe and an α-helical-rich C lobe connected by a flexible linker that can be superimposed onto protein kinase CK1 with a root-mean-square deviation (RMSD) of 3.5 Å over 164 Cα atoms (Figure S2B). The SelO N lobe includes the regulatory αC helix (Figure 2A, orange) packing against the core β sheet (Figure 2A, magenta), while the C lobe includes a pseudo-catalytic loop lacking HRD, followed by an apparent activation loop. An N-terminal extension (α1–α5, white) stabilizes the N lobe β sheet, and the unique C-terminal domains, CTD1 (α15–α19) and CTD2 (α20–α22), contact the C lobe and the αC helix, respectively. There is no clear electron density for the last 12 residues, including the C-terminal Cys, suggesting that this region is disordered.Figure S2Crystal Structure of P. syringae SelO Reveals an Atypical Protein Kinase Fold with a Unique Orientation of the Nucleotide in the Active Site, Related to Figure 2Show full caption(A) Amino acid sequence of P. syringae SelO depicting the secondary structural elements, color-coded as in Figure 2A.(B) Ribbon representation of protein kinase CK1 and P. syringae SelO. Color coding is shown as in Figure 2A.(C) Superposition of SelO with CK1 highlights flipped ATP binding mode. Stereo view (crosseye) of SelO pseudokinase (green) bound to AMP-PNP (green ball and stick) superimposed with CK1 (cyan) bound to ATP (cyan ball and stick) reveals flipped nucleotide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Amino acid sequence of P. syringae SelO depicting the secondary structural elements, color-coded as in Figure 2A. (B) Ribbon representation of protein kinase CK1 and P. syringae SelO. Color coding is shown as in Figure 2A. (C) Superposition of SelO with CK1 highlights flipped ATP binding mode. Stereo view (crosseye) of SelO pseudokinase (green) bound to AMP-PNP (green ball and stick) superimposed with CK1 (cyan) bound to ATP (cyan ball and stick) reveals flipped nucleotide. The SelO nucleotide sits in a cleft between the two lobes of the kinase domain. Remarkably, the AMP-PNP molecule is flipped in the active site when compared to canonical protein kinases. The γ-phosphate, which is normally transferred to protein in a kinase reaction, is buried in a pocket between the two lobes of the kinase domain (Figure 2B). In fact, after superposition of the kinase domains, the SelO α-, β-, and γ-phosphates occupy the positions of the typical γ-, β-, and α-phosphates of protein kinases, respectively (Figures 2B, 2C, and S2C). The binding site for the flipped nucleotide adenine base and sugar ribose is formed by unique insertions in two SelO loops: the β6–β7 (Gly-rich loop) and the β8-αC loop (colored white, Figures 2B and 2D). The flipped orientation of ATP in the active site led us to hypothesize that the SelO proteins could transfer adenosine monophosphate (AMP) to protein substrates (AMPylation) (Casey and Orth, 2018Casey A.K. Orth K. Enzymes Involved in AMPylation and deAMPylation.Chem. Rev. 2018; 118: 1199-1215Crossref PubMed Scopus (40) Google Scholar). We incubated recombinant E. coli (ydiU), S. cerevisiae (Fmp40), and H. sapiens SelO with [γ32P]ATP or [α32P]ATP of similar specific radioactivity and observed 32P-incorporation into the wild-type (WT) proteins only when [α32P]ATP was used as substrate (Figures 3A–3C). Mutation of the active site metal-binding DFG motif in the SelO proteins abolished 32P incorporation. E. coli and human SelO, but not the inactive mutants, were immunoreactive to an anti-Thr AMP antibody (Figures S3A and S3B) (Hao et al., 2011Hao Y.H. Chuang T. Ball H.L. Luong P. Li Y. Flores-Saaib R.D. Orth K. Characterization of a rabbit polyclonal antibody against threonine-AMPylation.J. Biotechnol. 2011; 151: 251-254Crossref PubMed Scopus (27) Google Scholar). Likewise, mass spectrometry (MS) analysis revealed peptides from WT SelO proteins but not the inactive mutants, with mass shifts of 329 Da, consistent with the covalent addition of AMP to Ser, Thr, and Tyr residues (Figures 3D and S3C). Furthermore, E. coli SelO, but not the inactive mutant, could AMPylate the generic protein kinase substrate myelin basic protein (MBP) in a time-dependent manner (Figure 3E). E. coli SelO prefers ATP over other nucleotides as a co-substrate (Figure S3D) and displayed a Km for ATP of ∼2.0 μM (Figure S3E). Thus, the SelO pseudokinases can AMPylate protein substrates.Figure S3SelO Pseudokinases AMPylate Protein Substrates, Related to Figure 3Show full caption(A and B) α-Thr AMP protein immunoblotting of E. coli SelO or the inactive D256A mutant (A) or human SelO (U667C) or the inactive D348A mutant (B). The SelO proteins were preincubated with or without Mg2+/ATP prior to SDS-PAGE and immunoblotting. The Ponceau stained membrane is shown as a loading control.(C) MS/MS data was searched using the Mascot search engine (Matrix Science) for peptide identification and determination of MS2 spectral counts of AMPylated peptide ions. The modification sites were localized to the residues shown in red. When the site could not be assigned to a single residue, all possible sites are shown in red. SelO proteins were digested with trypsin prior to LC-MS/MS.(D) SelO prefers ATP over other nucleotides as a cosubstrate. Autoradiograph depicting the incorporation of α-32P AMP from 100 μM [α-32P]ATP into E. coli glutaredoxin A (grxA) (See Figure 6) by E. coli SelO in the presence of 0, 0.1mM or 2mM unlabeled “cold” ATP, GTP, CTP or UTP. The reaction products were resolved by SDS-PAGE and visualized by Coomassie blue staining (lower) and autoradiography (upper).(E) Kinetic analysis depicting the concentration dependence of Mg2+/ATP on the rate of AMP incorporation into grxA (see Figure 6) by E. coli SelO. (Inset) Km for Mg2+/ATP is indicated. Reaction products were analyzed as in Figure 4B.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A and B) α-Thr AMP protein immunoblotting of E. coli SelO or the inactive D256A mutant (A) or human SelO (U667C) or the inactive D348A mutant (B). The SelO proteins were preincubated with or without Mg2+/ATP prior to SDS-PAGE and immunoblotting. The Ponceau stained membrane is shown as a loading control. (C) MS/MS data was searched using the Mascot search engine (Matrix Science) for peptide identification and determination of MS2 spectral counts of AMPylated peptide ions. The modification sites were localized to the residues shown in red. When the site could not be assigned to a single residue, all possible sites are shown in red. SelO proteins were digested with trypsin prior to LC-MS/MS. (D) SelO prefers ATP over other nucleotides as a cosubstrate. Autoradiograph depicting the incorporation of α-32P AMP from 100 μM [α-32P]ATP into E. coli glutaredoxin A (grxA) (See Figure 6) by E. coli SelO in the presence of 0, 0.1mM or 2mM unlabeled “cold” ATP, GTP, CTP or UTP. The reaction products were resolved by SDS-PAGE and visualized by Coomassie blue staining (lower) and autoradiography (upper). (E) Kinetic analysis depicting the concentration dependence of Mg2+/ATP on the rate of AMP incorporation into grxA (see Figure 6) by E. coli SelO. (Inset) Km for Mg2+/ATP is indicated. Reaction products were analyzed as in Figure 4B. Several interactions within the SelO active site contribute to the inverted orientation of the nucleotide, including K113 (PKA; K72) that coordinates the γ-phosphate of ATP (Figure 4A). K113 extends into the active site and is stabilized by E136 from the α6/αC helix. The formation of this ion pair, which typically positions the α-phosphate of ATP, is considered a hallmark of the activated state of a protein kinase (Taylor and Kornev, 2011Taylor S.S. Kornev A.P. Protein kinases: evolution of dynamic regulatory proteins.Trends Biochem. Sci. 2011; 36: 65-77Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar). Two invariant arginines (R176 and R183) also form interactions with the γ-phosphate. R176 extends into the active site from β12 and R183 lies in the flexible hinge region that connects the N lobe to the C lobe (Figure 4A). Mutations of K113, E136, R176, or R183 to Ala inactivate E. coli SelO (Figure 4B). Most kinases require a divalent cation to orient the phosphates of ATP. In the P. syringae SelO structure, Mg2+ and Ca2+ are bound to the α- and β-phosphates of AMP-PNP and are coordinated by N253 and D262 (PKA; N171 and D184). Mutations of these residues to Ala abolished E. coli SelO activity (Figure 4B). SelO was predicted to be a pseudokinase because it lacks the catalytic Asp (PKA; D166), which deprotonates the phosphoacceptor hydroxyl on the protein substrate. However, we anticipate that D252 in P. syringae SelO could fulfill this role because of its conservation and its proximity to the α-phosphate of ATP. Furthermore, its mutation to Ala inactives E. coli SelO (Figure 4B). Collectively, the active site of P. syringae SelO reveals evolutionarily conserved interactions that provide this family of kinases with the unique ability to transfer AMP to protein substrates. The N terminus of eukaryotic SelO proteins contains a predicted mitochondrial targeting peptide (mTP). When overexpressed in mammalian cells as a GFP-fusion protein, human SelO localizes to the mitochondria (Han et al., 2014Han S.J. Lee B.C. Yim S.H. Gladyshev V.N. Lee S.R. Characterization of mammalian selenoprotein o: a redox-active mitochondrial protein.PLoS ONE. 2014; 9: e95518Crossref PubMed Scopus (37) Google Scholar). We expressed S. cerevisiae SelO (official gene name Fmp40) in yeast as a C-terminally tagged GFP-fusion protein and observed co-localization with the mitochondrial resident protein citrate synthase (Figure S4A). Mitochondrial localization was dependent on the presence of the mTP (residues 1–23) because a truncated mutant of SelO failed to localize to the mitochondria (Figure S4A). Furthermore, we fractionated yeast extracts by sucrose-gradient centrifugation and detected endogenous SelO in fractions enriched for the mitochondrial resident protein porin (Figure S4B). Thus, S. cerevisiae SelO is a mitochondrial protein, and its localization depends on a functional mTP. Many mitochondrial proteins are subjected to redox regulation, including several selenoproteins. To test the redox function of SelO, we purified the E. coli protein under non-reducing conditions and observed a doublet when the protein was resolved by non-reducing SDS-PAGE (Figure 5A). The faster migrating species was converted to the slower migrating species upon treatment with the reducing agent dithiothreitol (DTT). Likewise, endogenous S. cerevisiae SelO migrated as two distinct species during non-reducing SDS-PAGE and was also sensitive to DTT (Figure 5B). MS analysis of E. coli SelO identified an intramolecular disulfide bond between Cys272 and Cys476 (Figure 5C). To confirm the sites of modification, we incubated E. coli SelO with the cysteine alkylating agent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS). AMS reacts with free thiols, resulting in a change in electrophoretic mobility that can be easily observed by SDS-PAGE. AMS reduced the electrophoretic mobility of WT SelO only in the presence of the reducing agent, Tris(2-carboxyethyl) phosphine (TCEP). However, AMS reduced the electrophoretic mobility of the C272A and C476A mutants in the absence of TCEP (Figure 5D). Collectively, these results suggest that E. coli SelO forms an intramolecular disulfide bond between a Cys in the activation loop and the Cys at the C terminus, the latter being replaced by a Sec in higher eukaryotes (Figures 1B and 5E). The formation of disulfide bonds occurs primarily in the oxidizing environment of the secretory pathway. However, some mitochondrial proteins can also form disulfide or selenyl-sulfide bonds as part of their catalytic mechanism (Collet and Messens, 2010Collet J.F. Messens J. Structure, function, and mechanism of thioredoxin proteins.Antioxid. Redox Signal. 2010; 13: 1205-1216Crossref PubMed Scopus (295) Google Scholar). To test whether the SelO disulfide bond regulates its activity, we incubated E. coli SelO purified under non-reducing conditions, with MBP and [α32P]ATP, and observed low levels of AMPylation (Figure 5F). However, addition of DTT or the thioredoxin system that uses reducing equivalents from nicotinamide adenine dinucleotide phosphate (NADPH) to reduce disulfides markedly increased SelO activity. Therefore, E. coli SelO activity is regulated by the formation of an intramolecular disulfide bridge. Based on the chemistry of the AMPylation reaction and the location of the adenine ring of ATP in the SelO crystal structure, we reasoned that using a biotinylated ATP analog would be an efficient strategy to identify proteins AMPylated by SelO. In this reaction, biotinylated AMP would be transferred to proteins, which would greatly faci" @default.
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• W2892393119 date "2018-10-01" @default.
• W2892393119 modified "2023-10-17" @default.
• W2892393119 title "Protein AMPylation by an Evolutionarily Conserved Pseudokinase" @default.
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