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.
Highlights
Metal-dependent Ser/Thr phosphatases (PPMs) have a structurally conserved catalytic domain that adopts a b-sandwich fold with Mg2+/Mn2+ ions at the active site
PPM1H is highly selective for LRRK2 phosphorylated Rabs, and closely related PPM1J exhibits no activity towards substrates such as Rab8a phosphorylated at Thr72
The flap is adjacent to the active site and poorly conserved in sequence and structure within the PPM family
Summary
Metal-dependent Ser/Thr phosphatases (PPMs) have a structurally conserved catalytic domain that adopts a b-sandwich fold with Mg2+/Mn2+ ions at the active site. Several enzymes including PPM1A, PPM1B, PPM1K and PDP1 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, 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)
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