Structural basis for the specificity of PPM1H phosphatase for Rab GTPases
2021; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês
10.15252/embr.202152675
ISSN1469-3178
AutoresDieter Waschbüsch, Kerryn Berndsen, Paweł Lis, Axel Knebel, Yuko P. Y. Lam, Dario R. Alessi, Amir R. Khan,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoArticle28 September 2021Open Access Transparent process Structural basis for the specificity of PPM1H phosphatase for Rab GTPases Dieter Waschbüsch orcid.org/0000-0002-8953-2585 School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland These authors contributed equally to this work Search for more papers by this author Kerryn Berndsen orcid.org/0000-0002-9353-7565 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA These authors contributed equally to this work Search for more papers by this author Pawel Lis orcid.org/0000-0002-4978-7671 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA Search for more papers by this author Axel Knebel MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Yuko PY Lam orcid.org/0000-0001-5486-091X MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA Search for more papers by this author Dario R Alessi Corresponding Author [email protected] orcid.org/0000-0002-2140-9185 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA Search for more papers by this author Amir R Khan Corresponding Author [email protected] orcid.org/0000-0003-1176-6952 School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland Division of Newborn Medicine, Boston Children's Hospital, Boston, MA, USA Search for more papers by this author Dieter Waschbüsch orcid.org/0000-0002-8953-2585 School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland These authors contributed equally to this work Search for more papers by this author Kerryn Berndsen orcid.org/0000-0002-9353-7565 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA These authors contributed equally to this work Search for more papers by this author Pawel Lis orcid.org/0000-0002-4978-7671 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA Search for more papers by this author Axel Knebel MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Search for more papers by this author Yuko PY Lam orcid.org/0000-0001-5486-091X MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA Search for more papers by this author Dario R Alessi Corresponding Author [email protected] orcid.org/0000-0002-2140-9185 MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA Search for more papers by this author Amir R Khan Corresponding Author [email protected] orcid.org/0000-0003-1176-6952 School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland Division of Newborn Medicine, Boston Children's Hospital, Boston, MA, USA Search for more papers by this author Author Information Dieter Waschbüsch1, Kerryn Berndsen2,3, Pawel Lis2,3, Axel Knebel2, Yuko PY Lam2,3, Dario R Alessi *,2,3 and Amir R Khan *,1,4 1School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland 2MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, USA 4Division of Newborn Medicine, Boston Children's Hospital, Boston, MA, USA *Corresponding author. Tel: +44 1382 385 602; E-mail: [email protected] *Corresponding author. Tel: +1 617 713 8225; E-mail: [email protected] EMBO Rep (2021)22:e52675https://doi.org/10.15252/embr.202152675 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract LRRK2 serine/threonine kinase is associated with inherited Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases within their switch 2 motif to control their interactions with effectors. Recent work has shown that the metal-dependent protein phosphatase PPM1H counteracts LRRK2 by dephosphorylating Rabs. PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity towards substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here, we have identified the molecular determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals a structurally conserved phosphatase fold that strikingly has evolved a 110-residue flap domain adjacent to the active site. The flap domain distantly resembles tudor domains that interact with histones in the context of epigenetics. Cellular assays, crosslinking and 3-D modelling suggest that the flap domain encodes the docking motif for phosphorylated Rabs. Consistent with this hypothesis, a PPM1J chimaera with the PPM1H flap domain dephosphorylates pThr72 of Rab8a both in vitro and in cellular assays. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold. Synopsis PPM1H phosphatase belongs to the PPM (PP2C) family of enzymes that have a conserved Mg2+/Mn2+-dependent catalytic domain. PPM1H counters the LRRK2 kinase pathway by dephosphorylating a subset of Rab GTPases. Through X-ray, biochemical and cellular studies, the molecular basis for specificity can be attributed to a novel flap domain that has evolved to recognize Rab GTPases. Mutations in the flap domain reduce the ability of PPM1H to hydrolyze a phosphorylated threonine in the switch 2 helix of Rab8a and Rab10 Grafting of the PPM1H flap domain onto PPM1J phosphatase is sufficient to enable PPM1J to dephosphorylate Rab8a and Rab10 both in vitro and in cellular assays A hydrophobic N-terminal anchor that is conserved in PPM1H, PPM1J and PPM1M contributes to folding of their catalytic domains Introduction Metal-dependent Ser/Thr phosphatases (PPMs) have a structurally conserved catalytic domain that adopts a β-sandwich fold with Mg2+/Mn2+ ions at the active site. Among the family of human enzymes is PPM1A (formerly PP2Cα), which reverses stress-mediated protein kinase cascades (Moore et al, 1991; Maeda et al, 1994; Chen et al, 2017), PHLPP1/2, which regulates AGC kinases and cellular homeostasis (Grzechnik & Newton, 2016), and pyruvate dehydrogenase phosphatase (PDP1) that is expressed in the mitochondrial matrix and regulates the activity of pyruvate dehydrogenase in metabolism (Linn et al, 1969; Vassylyev & Symersky, 2007). The core PPM fold, first identified by the structure of PPM1A (Das et al, 1996), consists of an 11-stranded β-sandwich flanked on both sides by α-helices (for recent reviews, see Kamada et al (2020) and Gao et al (2021)). The catalytic cleft is formed on one side of the β-sandwich and comprises a binuclear Mg2+/Mn2+ metal centre that is coordinated by conserved aspartate residues. The 250-residue PPM fold is better conserved in structure rather than sequence (20–50% identities) across the mammalian enzymes. There is also considerable diversity among PPMs involving the incorporation of sequence elements outside of the catalytic domain. For example, PPM1A has a C-terminal α-helical domain that is not required for catalysis but may contribute to substrate specificity (Das et al, 1996; Stern et al, 2007; Debnath et al, 2018). Several enzymes including PPM1A, PPM1B, PPM1K and PDP1 also have a short 50-residue insertion termed the "flap" subdomain that is poorly conserved in sequence and structure. This region is predicted to contribute to substrate specificity, although chimeric enzymes involving grafts of the flap have not been successful in transferring substrate preference (Su & Forchhammer, 2013). Studies of bacterial enzymes have proposed a third metal-binding site that contributes to catalysis via coordination with a conserved aspartate residue (Pullen et al, 2004; Rantanen et al, 2007; Schlicker et al, 2008). Mutation of the equivalent residue in human PPM1A to glutamate (D146E) enabled crystallization of a complex of PPM1A with a cyclic phosphopeptide and subsequent structure determination (Debnath et al, 2018). Recently, PPM1H phosphatase has been identified as the enzyme that counteracts the LRRK2 signalling cascade by dephosphorylating Rab GTPases (Berndsen et al, 2019). A subset of at least 7 Rabs are physiological substrates of LRRK2 (Steger et al, 2016, 2017) a Ser/Thr kinase that is associated with inherited and sporadic forms of Parkinson's disease (PD) (Alessi & Sammler, 2018; Di Maio et al, 2018). Rabs are members of the Ras superfamily of molecular switches that regulate membrane trafficking in eukaryotes. Rabs oscillate between a membrane-bound GTP form and cytosolic GDP form that is distinguished by local conformational changes in nucleotide-sensitive switch 1 and switch 2 motifs (Hutagalung & Novick, 2011). LRRK2 phosphorylates Rab8a and Rab10 at a conserved threonine residue in their switch 2 α-helix (pThr72 in Rab8a and pThr73 in Rab10). Phosphorylated Rab8a/10 (pRab8a/10) recruit phospho-specific effectors RILPL1 and RILPL2 (Rab-interacting lysosomal protein-like 1 and 2) to subcellular compartments, downstream of LRRK2 activation. Autosomal dominant PD mutations that activate LRRK2 kinase interfere with the formation of primary cilia through a pathway involving pRab8a/10 binding to RILPL1 (Dhekne et al, 2018). LRRK2 kinase inhibitors are currently in phase 1 and 2 clinical trials with PD patients (Azeggagh & Berwick, 2021), while alternative strategies to antagonize LRRK2 signalling could be beneficial for future therapeutics. Here, we describe the crystal structure of PPM1H, a phosphatase that counteracts the LRRK2 pathway. The structure reveals novel features that have been incorporated into the core catalytic domain. The first is a 110-residue "flap domain" that is positioned next to the catalytic cleft. This domain is an expansion of a 50-residue flap that is found in other members of the PPM family. The PPM1H flap domain adopts an α/β fold resembling tudor domains that regulate histone functions in an epigenetic context. On the opposite face of the cleft, a 3-stranded β-sheet motif (β-motif) is also inserted into the core PPM fold. Thirdly, PPM1H has an N-terminal extension that winds behind the active site and inserts into the hydrophobic core of the β-sandwich. This anchor-like interaction has apparently evolved in the PPM1H/J/M subfamily of phosphatases as a motif that contributes to folding of the enzymes. Mutagenesis, cellular assays, crosslinking and modelling studies of a phospho-Rab substrate into the active site suggest that the PPM1H flap domain forms a docking site for phosphorylated Rab GTPases. In support of this hypothesis, transfer of the flap domain of PPM1H onto PPM1J is sufficient to convert the PPM1J chimaera into an active phospho-Rab8a phosphatase. Therefore, PPM1H phosphatase has evolved substrate specificity through the acquisition of a Rab-specific flap domain within the framework of a conserved catalytic domain. Results Overall structure of PPM1H phosphatase Full-length PPM1H expressed in E. coli cells was unstable and prone to degradation, thus difficult to crystallize. To design a crystallisable protein, the N-terminal residues 1-32 were eliminated due to predicted flexibility (Fig 1A). We also introduced a D288A mutation (PPM1HDA) that destabilizes a third metal ion (Mg2+/Mn2+) at the active site. Previous studies of PPM1A (D146A) showed that this variant acts as a substrate-trapping mutant (Debnath et al, 2018). Crystals were grown of PPM1HDA that diffracted to 3.1 Å resolution (Table 1), but no crystals grew of the WT enzyme. To grow WT crystals and improve diffraction, a "loop deletion" variant of PPM1H (PPM1H-LD) was designed to eliminate a flexible and non-conserved loop (188-226) that was predicted to be distant from the active site. WT and D288A variants of PPM1H (PPM1HWT-LD, PPM1HDA-LD) diffracted to 2.5 Å and 2.6 Å resolution, respectively. The variants PPM1HDA, PPM1HWT-LD, and PPM1HDA-LD have identical 3-D structures with two Mg2+ ions at the active site. In addition to the Mg2+ complexes, a structure of PPM1HWT (33-514, loopDEL) with 3 Mn2+ ions was also determined at 2.2 Å resolution (MnPPM1HWT-LD). The 2.5 Å model of PPM1HWT-LD will be used for ensuing discussions (Fig 1), except for 3-D docking analyses with model substrates in which we used the MnPPM1HWT-LD variant. However, all of the structures are identical with only minor differences arising from flexible loops and the presence or absence of the third metal ion at the active site. Deletion of the loop 188-226 and the N-terminus (1–32) does not affect the ability of PPM1HWT-LD to dephosphorylate pRab8a relative to the full-length enzyme in vitro and in cells (Fig EV1A and B). Statistics of data collection and refinement for all structures are shown in Table 1. Figure 1. Structure of Rab-specific PPM1H phosphatase Domain organization of PPM1H, PPM1J and PPM1A. The annotation of regions (anchor, flap domain) is discussed in the text. The loop deletion (188–226) that was engineered to improve diffraction is indicated. Ribbon model of the enzyme with a view to the catalytic cleft that contains two Mg2+ ions (cyan spheres). The N-terminal region is magenta (33–71), the flap domain is a wheat colour, and the β-sheet motif is green. The loop deletion (188–226) connects α1/α2 on the opposite face relative to the active site. The back view of the enzyme is also shown with a 180° rotation around the axis indicated. Parts of the anchor (RPxFL motif, magenta) that interact with the globular core are shown as stick models, and discussed in the text. The β1 strand is orange to emphasize its non-canonical conformation due to the presence of the preceding anchor. Comparisons of the flap domain of PPM1H with PPM1A (left) and the tudor domain (right). Apart from a conserved loop (dotted circle) which forms an interface with the catalytic domain, the sequences and structures of flaps are diverse among the PPM family. The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72-phosphorylated Rab8a (left) or pThr73-phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS sample buffer and analysed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A substrate-trapping (inactive) variant of PPM1H was used as a control. Download figure Download PowerPoint Table 1. PPM1H crystallographic data and refinement statistics. PPM1HDA PPM1HWT-LD PPM1HDA-LD MnPPM1HWT-LD Residues 33-514 33-514 (∆188-226) 33-514 (∆188-226) 33-514 (∆188-226) Crystallization 10% isopropanol, 5% PEG4000, 0.05 M MgCl2 0.1 M imidazole pH 7, 20% Jeffamine ED-2001 30% PEG1500, 0.1 M MES pH 6.5 100mM Tris-Cl pH 8, 15% reagent alcohol, 10mM MnCl2 Beamline NSLSII FMX NSLSII FMX NSLSII FMX APS 24-ID-E Wavelength (Å) 0.9789 0.9789 0.9789 0.97918 Space group P 21 21 21 P 21 21 21 P 21 21 21 P 21 21 21 Cell a, b, c, (Å) 69.68, 102.16, 148.70 71.31, 102.4, 148.72 70.69, 101.11, 148.97 70.24, 101.04, 149.36 Resolution (Å) 28.55–3.09 (3.20-3.09) 28.97–2.45 (2.54–2.45) 28.97–2.58 (2.68-2.58) 63.93-2.194 (2.272-2.194) Unique reflections 19,927 (1,853) 39,279 (3,324) 34,031 (3,228) 54,665 (4,758) Completeness (%) 99.25 (95.02) 98.28 (84.34) 99.42 (96.04) 98.42 (87.21) 19.83 (2.34) 14.6 (2.3) 16.79 (0.35) 12.72 (1.63) Multiplicity 6.7 (6.3) 6.7 (6.7) 6.6 (6.5) 6.7 (6.7) R-merge 0.070 (0.77) 0.08 (0.78) 0.0897 (0.673) 0.08947 (0.9865) R-meas 0.076 (0.84) 0.086 (0.855) 0.097 (0.731) 0.09714 (1.068) CC1/2 0.999 (0.855) 0.998 (0.779) 0.998 (0.847) 0.998 (0.764) Refinement Protein residues/waters/ions 794/14/4 793/115/4 794/91/4 785/211/8 No. reflections for R-free 1,003 (85) 1,968 (166) 1,708 (171) 2,485 (223) R-work 0.1878 (0.3349) 0.1824 (0.2684) 0.1897 (0.2441) 0.1952 (0.2848) R-free 0.2148 (0.4032) 0.2389 (0.3428) 0.2359 (0.2908) 0.2316 (0.3296) RMSD bond lengths (Å) 0.005 0.009 0.004 0.003 RMSD bond angles (°) 0.96 1.07 0.78 0.64 Average overall B-factor 98.78 61.03 57.65 57.06 Mean B-factors (Å2) protein/waters/ions 98.83/86.73/81.95 61.15/54.83/44.09 57.67/56.62/56.31 57.22/52.5/48.74 Ramachandran analysis favoured/allowed (%) 94.63/4.48 95.5/3.86 96.53/3.08 97.41/2.33 PDB accession code 7kpr 7l4j 7l4i 7n0z Values in parentheses correspond to the statistics in the highest resolution bin. RMSD, root mean square deviation. LD corresponds to the "loop deletion" variant of PPM1H. R-merge = Σhkl Σj∣Ihkl,j- ∣/Σhkl Σjhkl,j. R-work = Σhkl∣Fo,hkl – Fc,hkl∣/ΣhklFo,hkl. Click here to expand this figure. Figure EV1. The "loop deletion" variant of PPM1H is active in vitro and in cells HEK293 cells overexpressing indicated constructs were treated ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration, and membranes were analysed using the OdysseyClx Western blot imaging system. Each lane represents cell extract obtained from a different dish of cells (two replicates per condition without MLi-2 treatment, one replicate per condition with MLi-2 treatment). The "loopDEL" variant is the segment 33–514 with the region 188-226 replaced by the sequence "GSGS". In vitro assay of PPM1H activity using a PhosTag gel. Substrate pRab8a (GTP form, 10 μg) was incubated with WT PPM1H ± loopDEL for 15 min at room temperature. The full-length variant was fused to maltose-binding protein (MBP). The "loopDEL" variant was from 33 to 514 and was used for crystallization studies. A conventional 12% SDS–PAGE is shown in parallel lanes below the PhosTag gel. Structure-based sequence alignment of human PPMs from their associated PDB files using Chimera software (Pettersen et al, 2004). The PDB codes are 4ra2 (PPM1A; (Pan et al, 2015)), 2p8e (PPM1B; (Almo et al, 2007)) and 2iq1 (PPM1K; (Almo et al, 2007)). Residue His31 of PPM1H is in brackets since the first two residues (His-Met) arise from a cloning artefact. Secondary structures of PPM1H and PPM1A are above and below the sequences, respectively. Colours of secondary structures correspond to the scheme in Fig 1. The black triangle is a loop in PPM1H (188-226) that has been removed to simplify the alignment. The C-terminal α-helical domain of PPM1A is blue. Red circles are aspartate residues that directly coordinate metal ions. Download figure Download PowerPoint The structure of PPM1H adopts a conserved PPM fold consisting of 10 β-strands organized into a 5 × 5 β-sandwich (Fig 1B). The first β-sheet comprises β-strands β2, β7, β8, β10 and β11. The second β-sheet consists of β-strands β3, β4, β5, β6 and β9. Two α-helices pack against each of the two concave surfaces of the β-sandwich. The long and curved α-helices α1 and α2 are oriented in an anti-parallel manner and pack against the second β-sheet. The shorter helices α4 and α5 are also oriented in an anti-parallel fashion and pack against the first β-sheet. The active site cleft is formed by loops, including a short α-helical loop (α4, residues 443–447), that connect β-strands on one edge of the β-sandwich. Three conserved aspartate residues—D151, D437 and D498—form direct contacts with two Mg2+ ions (M1 and M2) at the active site. M1 is coordinated by D437 and D498, while D151 coordinates both metal sites. The metal binding includes water molecules, and the geometry is approximately octahedral for both metal sites (M1 and M2). A structure-based sequence alignment of PPM1H against human enzymes with known 3-D structures is shown to emphasize common and divergent features of the PPM family (Fig EV1C). One of the distinctive features of PPM1H is a 110-residue flap domain that adopts an α/β fold (Fig 1C). All known structures of human PPMs (PPM1A, PPM1B, PPM1K and PDP1) have a shorter 50-residue flap that is inserted between the terminal β-strands of the two β-sheets (β8 and β9 in PPM1H). In PPM1H, this domain comprises an α-helix that stacks against a highly twisted β-sheet. This extended flap domain effectively creates a surface adjacent to the active site for potential substrate recognition. Matching of the flap domain to structures in the DALI server (Holm, 2020) reveals a resemblance to the tudor domain despite the absence of sequence similarities (Fig 1C). Tudor domains are also composed of an α-helix that stacks against a highly twisted anti-parallel β-sheet. These domains are involved in the recognition of methylated lysine or arginine residues of histones via an aromatic cage. Although roughly similar in appearance, the α/β flap domain of PPM1H has a different topology and lacks characteristic structural motifs such as the aromatic cage that are common to tudor domains. Despite flap sequence and structural diversity among the PPM family, a short loop 386-396 that packs against the catalytic domain is highly conserved in sequence and conformation (dotted circle, Fig 1C). This conserved loop is investigated in more detail below. Compared to structures of other known enzymes, PPM1H has two additional structural elements that are novel. The N-terminal residues 33-79 follow an irregular path behind the active site that spans the two β-sheets of the core catalytic domain (Fig 1B). This region is termed an "anchor" due to a short 310 helix (residues 43-47) that caps the hydrophobic core of the β-barrel. The second feature is a β-sheet motif (β-motif; residues 480-496) that consists of 3 short anti-parallel β-strands. These novel features of PPM1H, along with an expanded flap domain, are shared by the PPM1H/J/M subfamily of phosphatases. The specificity of PPM1H against PPM1J was compared over a 20-min reaction at room temperature using catalytic amounts of enzyme. These in vitro assays used Phos-tag gels (Ito et al, 2016) to assess dephosphorylation of phospho-Rab8a (pRab8a) and phospho-Rab10 (pRab10) substrates (Fig 1D). Despite sharing common domains, PPM1J displays no activity, while PPM1H completely dephosphorylates pRab8a and pRab10 under these conditions. Crosslinking and 3-D docking suggest that the PPM1H flap is a pRab recognition domain Purified complexes of pRab8a and the substrate-trapping D288A variant of PPM1H were incubated together in the presence of DSBU (disuccinimidyl dibutyric urea), a mass spectrometry cleavable amine reactive crosslinker that is widely used to identify and map sites of protein–protein interactions (Pan et al, 2018). This reagent crosslinks Lys residues to acidic and hydroxyl amino acids located within 32 Å (Götze et al, 2019). In the presence of DSBU, pRab8a and PPM1H formed a stoichiometrically heavier band on Coomassie-stained gels migrating at ˜140 kDa (Fig 2A). The crosslinking was dependent on phosphorylation of Thr72, since the heavier band failed to form with PPM1H/Rab8a or PPM1H alone incubations. The size of the crosslinked species implied 2 molecules of PPM1H (50 kDa each) and 2 molecules of pRab8a (20 kDa each) in solution. Consistent with dimeric complexes of the enzyme, crosslinked PPM1H alone migrated as a dimer (100 kDa) and further information below is supportive of PPM1H being a dimer. Crosslinked samples were digested under 3 conditions (trypsin, trypsin/AspN and trypsin/GluC). In addition, SCX cartridge purification was applied in one of the tryptic digested samples to further enrich the crosslinked peptides. PPM1H and pRab8a crosslinked peptides were identified using meroX software (Götze et al, 2012). Potential crosslinked peptides with score higher than 50, as well as false discovery rate (FDR) < 5%, were manually inspected to confirm only a single crosslinked site was proposed from each peptide (Iacobucci et al, 2018). This protocol identified numerous residues within the flap domain of PPM1H that were crosslinked to pRab8a (Fig 2B; also see "Data Availability" section). Interestingly, other regions in PPM1H that formed significant crosslinks with pRab8a were situated in likely more flexible regions namely the N-terminal non-catalytic region, and within the 104-142 and 183-235 flexible loops (Fig 2B). These data are consistent with the flap domain of PPM1H functioning as a major substrate recognition site for pRab8a. Figure 2. Crosslinking and docking analysis suggest that the flap domain binds to pRab8a SDS–PAGE analysis of the PPM1H(D288A): pRab8a complex in the presence of DSBU crosslinker. Control migration of proteins is on the left. NP, non-phosphorylated Rab8a; P, phosphorylated Rab8a. The migration of control and crosslinked proteins is marked on the right. Crosslinked peptides from PPM1H and pRab8a are mapped onto the sequence. The flap domain forms extensive crosslinks with pRab8a. In addition, two flexible loops (104–142, 188–226) also have multiple crosslinks with the substrate. Ribbon model of pRab8a (left) and the switch 2 phosphopeptide (right) docked onto the active site of PPM1H. The crosslinks shown between PPM1H and pRab8a are within accepted distance constraints (32 Å) for DSBU (He et al, 2015). Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72-phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72-phosphorylated Rab8a [GTP-bound conformation] (blue) and pThr72-phosphorylated Rab8a [GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panels were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean. Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72-Rab8a phosphopeptide (blue) and pThr73-Rab10 phosphopeptide (red). Each experiment was performed twice (individual data points shown). Side-by-side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72-phosphorylated protein (blue) or Rab8a pThr72-phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Download figure Download PowerPoint In order to further understand the mode of pRab8a interactions with PPM1H, docking analyses were performed. Phosphorylated Rab8a (pRab8a, GTP-bound) from the recent structure of the pRab8a:RILPL2 complex (Waschbüsch et al, 2020) was docked onto PPM1H using Haddock software (Dominguez et al, 2003; van Zundert et al, 2016). The structure of MnPPM1HWT-LD with 3 Mn2+ ions was used for docking with distance restraints applied between pThr72 of pRab8a and the metal ions of PPM1H. The structures of PPM1H with two Mg2+ ions failed to dock with pRab8a at the active site, presumably due to the density of negative charges in the absence of a third metal ion. The top pRab8a docking solution is shown along with the experimentally determined crosslinks from the flap domain (Fig 2C). The model reveals that the extended active site cleft involving the flap domain forms multiple interactions with pRab8A within the PPM1H active site. Overall, the crosslinking data and docking model are consistent with pRab8a recognition by the flap domain with phosphorylated Thr72 in the switch II motif oriented towards the active site for dephosphorylation. We also performed docking of the switch 2 α-helical peptide from pRab8a with MnPPM1HWT-LD. The 15 residue fragment from 65 to 79 was extracted from the structure of the pRab8a complex, and the N/C termini were capped (acetyl/amide) to eliminate charges. The docking solutions were more heterogeneous and many poses would be sterically incompatible with the active site i
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