A structural exposé of noncanonical molecular reactivity within the protein tyrosine phosphatase WPD loop

ARTICLE https://doi.org/10.1038/s41467-022-29673-y OPEN A structural exposé of noncanonical molecular reactivity within the protein tyrosine phosphatase WPD loop 1234567890():,; Huanchen Wang1 ✉, Lalith Perera 2, Nikolaus Jork 3, Guangning Zong Barry V. L. Potter 4, Henning J. Jessen3 & Stephen B. Shears 1 ✉ 1, Andrew M. Riley 4, Structural snapshots of protein/ligand complexes are a prerequisite for gaining atomic level insight into enzymatic reaction mechanisms. An important group of enzymes has been deprived of this analytical privilege: members of the protein tyrosine phosphatase (PTP) superfamily with catalytic WPD-loops lacking the indispensable general-acid/base within a tryptophan-proline-aspartate/glutamate context. Here, we provide the ligand/enzyme crystal complexes for one such PTP outlier: Arabidopsis thaliana Plant and Fungi Atypical Dual Specificity Phosphatase 1 (AtPFA-DSP1), herein unveiled as a regioselective and efficient phosphatase towards inositol pyrophosphate (PP-InsP) signaling molecules. Although the WPD loop is missing its canonical tripeptide motif, this structural element contributes to catalysis by assisting PP-InsP delivery into the catalytic pocket, for a choreographed exchange with phosphate reaction product. Subsequently, an intramolecular proton donation by PPInsP substrate is posited to substitute functionally for the absent aspartate/glutamate general-acid. Overall, we expand mechanistic insight into adaptability of the conserved PTP structural elements. 1 Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. 2 Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. 3 Institute of Organic Chemistry, and CIBSS - the Center for Integrative Biological Signaling Studies, University of Freiburg, 79104 Freiburg, Germany. 4 Drug Discovery and Medicinal Chemistry, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford OX1 3QT, UK. ✉email: ; NATURE COMMUNICATIONS | (2022)13:2231 | https://doi.org/10.1038/s41467-022-29673-y | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-29673-y C onsiderable efforts continue to be made to understand the molecular basis of enzyme-catalyzed hydrolysis of phosphate esters and anhydrides1. A particular challenge for this objective is presented by the protein tyrosine phosphatase (PTP) family, in no small part because phosphotyrosine phosphatase activity is not the only function for this family of enzymes2. A significant number of PTPs dephosphorylate alternate substrates such as RNA, phosphatidylglycerophosphate, inositol phospholipids, and a specialized class of signaling molecules known as diphospho-myo-inositol polyphosphates (inositol pyrophosphates, or PP-InsPs; Fig. 1a, b)2–8. Despite the evolution of these catalytic differences, there has been a high degree of conservation of key structural features of the PTP active site (Supplementary Fig. 1)2–5,9. One of these prominent structural elements is a flexible loop named WPD after its three most highly conserved residues, which includes an Asp (or occasionally Glu) that is typically described as an indispensable proton-donor to the leaving group (Supplementary Figs. 1, 2a). This catalytic acid is inserted into the active site by the closure of the WPD loop. Much attention is being devoted to determining how differences in conformational dynamics of this loop can contribute to catalytic versatility within the PTP family9,10. Nevertheless, there are examples of PTPs that show intriguing departures from the canonical aspects of the WPD loop. For example, the human DUSP23 gene encodes a VH1-like member Z (VHZ) protein in which the WPD-loop catalytic-acid, Asp65, is subservient to a remote Glu134 fulfilling the primary general acid function11. The ability of substrate to enter the catalytic pocket in either of two binding modes allows Asp65 to substitute as the catalytic acid when Glu134 is mutated11. Other similar dual general acid PTPs have been identified (e.g., TkPtp expressed by the hyperthermophilic archaeon Thermococcus kodakaraensis KOD112). Furthermore, there are some important PTPs in which the WPD motif is entirely absent from the host loop (Supplementary Fig. 2a). These enzymes include human CDC25 phosphatase, which supervises cell-cycle checkpoints13, the phosphoinositide/protein phosphatase PTEN14,15, MCE1, an mRNA capping enzyme that is essential for mRNA processing16, Baculovirus RNA 5ˈ-phosphatase8 and its human ortholog, DUSP11/PIR17, which participates in innate immune responses a b 6 2 8 9 1 Activity (nmol min-1 mg -1) 5 3 Km = 60 µM 9 9 9 3 3 3 4 Km = 44 µM 4 4 4 4 4 Activity (nmol min-1 mg -1) d 6 6 36 6 6 f 3 6 6 6 6 g h Activity (nmol min-1 mg -1) e Activity (nmol min-1 mg -1) c Activity (nmol min-1 mg -1) Activity (nmol min-1 mg -1) 4 33 3 Km = 78 µM 13 5 4 5 5 2-InsP7 4 j i 4 1-InsP7 3 3 3 3 3-InsP7 3 6 2 1 InsP6 Fig. 1 Structures of ligands used in this study and their rates of hydrolysis by AtPFA-DSP1. Michaelis–Menten kinetic plots are shown for the phosphatase activities of AtPFA-DSP1 towards: (a), 5-InsP7, (b), 1,5-InsP8, (c), 6-InsP7, (d), 5-PP-InsP4 and (e), 4-InsP7. Activity data (circles, some overlapping) are from each independent experiment at which the indicated substrate concentration was tested; the total number of such experiments is given above each data set in blue font. Km values were calculated when statistically appropriate. The insets in panels (a–e) depict chair conformations of each substrate; the positions of each β-phosphate are emphasized in red. In panel (f), vertical bars represent mean values of activities against the weakest substrates when all were assayed at 10 µM concentrations. Activity data (circles, some overlapping) are from each independent experiment; the total number of such experiments is given above each data set in blue font. Phenylphosphate is abbreviated as Phenyl-P. Structures of the inositol phosphates are given as chair conformations in panels (g) (1-InsP7), (h) (2-InsP7, (i) (3-InsP7) and (j) (InsP6). Locants (using standard nomenclature for myo-inositol) are provided with the structures of 5-InsP7 and InsP6. Source data are provided as a Source Data file. 2 NATURE COMMUNICATIONS | (2022)13:2231 | https://doi.org/10.1038/s41467-022-29673-y | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-29673-y to viral infection17, and Siw14, a PTP-type PP-InsP phosphatase in Saccharomyces cerevisiae6 (Supplementary Fig. 2a,b). There has been only limited speculation in the literature as to how PTP reactions might proceed in the complete absence of a classical WPD-loop catalytic acid7,8,13,14,18,19. Prac (...truncated)