676bc22f9e2f413abf3878815cd83089 pmc_35163251.pdf 7bd2092d70e5c2b90474cbdbf60f9b13f58f7d5d pmc_35163251.pdf 99960663769e5a10472a2ec8fb579a3dcca6e57c304d20270a54835229f791e8 pmc_35163251.pdf Title: Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation Subject: Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi. Keywords: protein phosphatase; Ppz1; PP1c; sequence comparisons; cross-linking proteomics; enzyme inhibition; fungi Author: Antonio Casamayor, Diego Velázquez, Carlos Santolaria, Marcel Albacar, Morten I. Rasmussen, Peter Højrup and Joaquín Ariño Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Jan 31 14:03:59 2022 CET ModDate: Mon Jan 31 14:06:24 2022 CET Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 3221091 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AHRQMW+VnURWPalladioL Type 1 Custom yes yes no 10 0 DPPIRN+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 IMPRVO+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 FIBMRR+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 28 0 SSNIUI+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 40 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 44 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes 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yes yes yes 177 0 GIGKNE+PalatinoLinotype TrueType WinAnsi yes yes no 183 0 GIGKNG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 186 0 ZZFUXD+PazoMath Type 1 Builtin yes yes no 193 0 KCNJHC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 207 0 KCNJHE+PalatinoLinotype TrueType WinAnsi yes yes no 210 0 KCNJIF+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 213 0 JMADHF+CMSY10 Type 1 Builtin yes yes no 220 0 GIGKNC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 231 0 GIGKNE+PalatinoLinotype TrueType WinAnsi yes yes no 234 0 GIGKNG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 237 0 ILXAXG+MSAM10 Type 1 Builtin yes yes no 247 0 RAMSXI+wasy9 Type 1 Builtin yes yes no 251 0 GIHAPD+Symbol Type 1C Custom yes yes yes 264 0 GIHBDE+TimesNewRoman CID TrueType Identity-H yes yes yes 269 0 GIGKNC+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 275 0 GIGKND+PalatinoLinotype CID TrueType Identity-H yes yes yes 278 0 GIGKNE+PalatinoLinotype TrueType WinAnsi yes yes no 284 0 GIGKNF+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 287 0 GIGKNG+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 293 0 GIHAPE+Tahoma CID TrueType Identity-H yes yes yes 296 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-07-01 02:26:30 CEST RepresentationInformation: pmc_35163251.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-02-27 18:40:58 CET Size: 3221091 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 566 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4923ab24 Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Children: Item: Title: Sequence Comparison of Catalytic Domains across Fungi Identifies Conserved Ppz-Specific Residues Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: The PP1 Structural Features Required for Modulation by Regulatory Subunits Appear Largely Conserved in Fungi Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: The C-Terminal Extra Alpha-Helix of CaPpz1 Is Likely Present in Most Ppz Enzymes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Chemical Cross-Link of Co-Purified Ppz1 and Hal3 from S. cerevisiae Points to Possible Relevant Ppz-Specific Conserved Residues Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Mutagenesis Analysis Reveals a Key Role of Conserved D615 in Ppz1 Regulation and Function Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Children: Item: Title: Yeast Strains Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Plasmid Construction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Bioinformatics Analyses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Chemical Cross-Linking of the Ppz1 Catalytic Domain and Hal3 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: LC-MS/MS Analysis of Cross-Linked Proteins Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Preparation of Yeast Extracts and Immunoblotting Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Hal3 Pull down Experiments Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Ppz1 Inhibition Assays Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Info: Title: Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation Author: Antonio Casamayor, Diego Velázquez, Carlos Santolaria, Marcel Albacar, Morten I. Rasmussen, Peter Højrup and Joaquín Ariño Subject: Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi. 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676bc22f9e2f413abf3878815cd83089 Type: MD5 Checksum: 7bd2092d70e5c2b90474cbdbf60f9b13f58f7d5d Type: SHA-1