DOI: 10.5281/zenodo.5045023
title: Structural basis for the specificity of PPM1H phosphatase for Rab GTPases

description: 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 and closely related PPM1J exhibits no activity toward substrates such as Rab8a phosphorylated at Thr72 (pThr72). Here we have identified the structural determinant of PPM1H specificity for Rabs. The crystal structure of PPM1H reveals that it is a dimer, confirmed by biophysical analysis, and possesses a conserved catalytic domain 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 <em>in vitro</em> and in cellular assays. Therefore, PPM1H has acquired a Rab-specific interaction domain within a conserved phosphatase fold. <strong>FILE DECRIPTIONS</strong> <strong>Figure 1D Rab8A.tif:</strong> Scans of Coomassie stained gels shown in Fig.1D:<br> Left:<br> Coomassie stained Phos-tag gel of PPM1H with Rab8A<br> Right:<br> Coomassie stained Phos-tag gel of PPM1J with Rab8A <strong>Figure 1D Rab10.tif:</strong> Scans of Coomassie stained gels shown in Fig.1D:<br> Upper gel:<br> Coomassie stained Phos-tag gel of PPM1H with Rab10<br> Lower gel:<br> Coomassie stained Phos-tag gel of PPM1J with Rab10 <strong>Figure 2A gel.tif:</strong> Coomassie-stained SDS-PAGE gel showing the result of crosslinking of PPM1H D288A with phospho-Rab8A. <strong>Figure 2BC. List of cross-links with distances.xlsx:</strong> List of crosslinks identified between PPM1H D288A with phospho-Rab8A shown in Fig.2B and Fig.2C. In green crosslinks ≤32 Å, in red crosslinks &gt;32 Å, in grey crosslinks mapping to the flexible loops missing from the crystal structure. <strong>Figure 2DEF Malachite.xlsx:</strong> Numerical data for the charts shown in Fig.2D, Fig.2E and Fig.2F. <strong>Figure 3B-WT.afe7</strong> Astra data file with data shown in Fig.3B (left) <strong>Figure 3B-2Glu.afe7</strong> Astra data file with data shown in Fig.3B (right) <strong>Figure 3C gel.tif:</strong><br> Coomassie stained gel of recombinant PPM1H WT and mutant proteins shown in Fig.3C <strong>Figure 3D Malachite.xlsx:</strong> Numerical data for the charts shown in Fig.3D <strong>Figure 4A - sequences of the chimaeras.txt:</strong> Aminoacid sequences of the chimaeric proteins used in the assays <strong>Figure 4B.tif:</strong><br> Coomassie stained gel of recombinant PPM phosphatases shown in Fig.4B. Last lane containing phosphatase PPM1A not shown in figure. <strong>Figure 4CD Malachite.xlsx:</strong> Numerical data for the charts shown in Fig.4C and Fig.4D. <strong>Figure 4E 700.tif:</strong><br> Top to bottom: Total LRRK2 blot shown in Fig.4E HA blot for PPM phosphatases (PPM constructs) shown In Fig.4E GAPDH loading controls for blots in Fig.4E HA blot for Rab8A shown in Fig.4E <strong>Figure 4E 800.tif:</strong> Phospho-Rab8A Thr72 blot shown in Fig.4E <strong>Figure 4F 700.tif:</strong><br> Top to bottom: Total LRRK2 blot shown in Fig.4F HA blot for PPM phosphatases (PPM constructs). HA blot was not used for Fig.4F. Membrane was reblotted, shown in <strong>tif file Figure 4F 700-2</strong> GAPDH loading controls for blots in Fig.4F HA blot for Rab10. HA blot was not used for Fig.4F. Membrane was reblotted, shown in <strong>tif file Figure 4F 700-2</strong> <strong>Figure 4F 700-2.tif:</strong><br> Top to bottom: HA blot for PPM phosphatases (PPM constructs) shown In Fig.4F GAPDH loading controls for blots in Fig.4F HA blot for Rab10 shown in Fig.4F <strong>Figure 4F 800.tif:</strong> Phospho-Rab10 Thr73 blot shown in Fig.4F <strong>Figure 5BE Malachite.xlsx:</strong> Numerical data for the charts shown in Fig.5B and Fig.5E. <strong>Figure 5C.tif:</strong> Coomassie stained gel of recombinant PPM1H WT and mutant proteins shown in Fig.5C <strong>Figure 5D 700.png:</strong> Top to bottom: Total LRRK2 blot shown in Fig.5D HA blot for PPM phosphatases (PPM constructs) shown in Fig.5D GAPDH loading control for blots in Fig.5D HA blot for Rab10 shown in Fig.5D <strong>Figure 5D 800.png:</strong> Phospho-Rab10 pThr73 blot shown in Fig.5D <strong>Figure 5E.tif:</strong> Coomassie stained gel of recombinant PPM1H WT and mutant proteins shown in Fig.5E <strong>Figure 5F 700 Rab8A.png:</strong> Top to bottom (Rab8A) Total LRRK2 blot shown in Fig.5F HA blot for PPM phosphatases (PPM constructs) shown in Fig.5F GAPDH loading control for blots in Fig.5F HA blot for Rab8A shown in Fig.5F <strong>Figure 5F 800 Rab8A.png:</strong> Phospho-Rab8A pThr72 blot shown in Fig.5F <strong>Figure 5F 700 Rab10.png:</strong> Top to bottom (Rab10) Total LRRK2 blot shown in Fig.5F HA blot for PPM phosphatases (PPM constructs) shown in Fig.5F GAPDH loading control for blots in Fig.5F HA blot for Rab10 shown in Fig.5F <strong>Figure 5F 800 Rab10.png:</strong> Phospho-Rab10 pThr73 blot shown in Fig.5F <strong>Figure 6B 700.tif:</strong> Top to bottom: Total LRRK2 blot shown in Fig.6B HA blot for PPM phosphatases (PPM constructs) shown In Fig.6B GAPDH loading controls for blots in Fig.6B HA blot for Rab10 shown in Fig.6B <strong>Figure 6B 800.tif:</strong> Phospho-Rab10 Thr73 blot shown in Fig.6B <strong>Figure EV1A 700.tif:</strong><br> Top to bottom: Total LRRK2 blot shown in Fig.EV1A GAPDH loading controls for blots in Fig.EV1A HA blot for Rab10 shown in Fig.EV1A <strong>Figure EV1A 800.tif:</strong> HA blot for PPM phosphatases (PPM constructs) shown In Fig.EV1A Phospho-Rab10 Thr72 blot shown in Fig.EV1A <strong>Figure EV1B.tif:</strong> Scans of Coomassie stained gels shown in Fig.EV1B <strong>Figure EV2C Malachite.xlsx:</strong> Numerical data for the chart shown in Fig.EV2C