Recently, de novo heterozygous variants of Calcineurin (CN) were reported as the cause of a neurodevelopmental disorder that presents with epileptic encephalopathy and dysmorphism (DEE91), with the largest group of patients harboring the CN missense mutation E282K (glutamate → lysine). Here, we use molecular and cellular techniques to define how this mutation alters CN activity. We discover that basophilic substrates use an arginine residue to bind to CN via an acidic substrate recruitment pocket adjacent to the CN active site, the E282 pocket. Furthermore, we show that basic residues in the i-1 position of the substrate relative to the substrate phosphosite enhance CN-mediated dephosphorylation. While the CNE282K structure shows that the overall conformation is unchanged, the E282 pocket transforms from acidic to basic, with pocket access blocked by the formation of a E282K-E237 salt bridge. Finally, in vitro assays and in cell phosphoproteomics show that CNE282K shifts CN substrate dephosphorylation profiles from basic to acidic, thereby altering CN-mediated dephosphorylation signaling. Together, these data define the molecular impact of the CNE282K variant in cells and development, providing a key step for developing strategies to treat this disorder and its accompanying complications.

The protein-tyrosine phosphatase SHP2 ( PTPN11 ) regulates growth factor- and cytokine-induced RAS/ERK MAP kinase (MAPK) pathway activation, and aberrant SHP2 function causes developmental disorders and cancer 1–5 . It is widely believed that the catalytic activity of SHP2 is essential for pathway activation 1,4,6–8 . This view has shaped our interpretation of how germline PTPN11 mutations cause Noonan Syndrome (NS) and NS with Multiple Lentigines (NS-ML) 2,9 and how somatic mutations contribute to myeloproliferative neoplasms and solid tumors 1 . Here we identify a previously undetected, protein-tyrosine phosphatase (PTP) activity-independent mechanism that revises our understanding of how SHP2 promotes RAS/ERK activation. We find that certain mutations of the nucleophilic cysteine that abolish catalytic activity still promote RAS/ERK pathway activation in normal and neoplastic mammalian cells, zebrafish embryos, and mice. Structural studies show that the SHP2 PTP domain binds directly to the Son of Sevenless 1 (SOS1) Dbl homology (DH) domain. Proximity labeling and super-resolution microscopy demonstrate that SHP2/SOS1 interaction occurs in cells and facilitates SOS1 translocation to the plasma membrane to form clusters. Our results overturn decades of dogma on SHP2 regulation of the RAS/ERK pathway and provide new insights into the mechanism of action of disease-associated PTPN11 mutations.

Phosphoprotein phosphatase 1 (PP1) forms holoenzymes composed of a catalytic subunit (PP1c) and one or two of over 200 regulatory subunits (PP1Rs). Humans express four conserved PP1c isoforms: PP1cα, PP1cβ/δ, and splice variants PP1cγ1 and PP1cγ2. To systematically characterize PP1c isoform-specific interactions, we employed mass spectrometry to identify PP1cα, PP1cβ, and PP1cγ interacting proteins, determine their isoform specificity, and assess and quantify their abundance within the PP1 holoenzyme pool. Our data show that PP1c forms hundreds of dimeric and trimeric holoenzymes, but the 10 most abundant PP1Rs make up 74% of PP1 holoenzymes, and they are highly uniform among PP1c isoforms. A key exception is myosin phosphatase N-terminal element (MyPhoNE)-containing PP1Rs, which form abundant holoenzyme complexes exclusively with PP1cβ. To define the determinants of MYPT1-PP1cβ specificity, we systematically assessed the contributions of MYPT1-PP1cβ interactions. First, we generated PP1cβ-PP1cγ chimeras and PP1cβ Tyr 305/Tyr307 point mutations to test the contribution of the PP1 C-terminal residues, and secondly, we used PP1cβ Thr197Gln (T197Q) mutation to test the effect of the MYPT1:MyPhoNE-specific interaction. Using genome editing, we demonstrate that PP1cβ T197Q-expressing cells exhibit altered PP1 holoenzyme composition and phosphorylation signaling, including increased phosphorylation of the Polo-like kinase 1 (Plk1) activation loop. Our studies further the understanding of the PP1c isoform-specific preference and demonstrate how a single amino acid change can alter PP1 holoenzyme composition and phosphorylation signaling, potentially explaining how recently discovered PP1cβ clinical variants impact PP1 biology.

Protein phosphatases are dynamic enzymes that exhibit complex regulatory mechanisms, with disruptions in these regulatory processes associated with disease. It is now clear that many phosphatases assemble into large macromolecular complexes via the interaction of phosphatase-specific regulatory proteins and substrates containing short linear motifs (SLiMs) or short helical motifs (SHelMs). Here, we review how cryo-electron microscopy (cryo-EM) integrated with orthogonal methods to study dynamic protein-protein interactions (NMR spectroscopy, hydrogen-deuterium exchange mass spectrometry, among others) is leading to new discoveries about the mechanisms controlling phosphatase assembly, substrate recruitment and dephosphorylation and, in turn, are providing novel strategies for targeting phosphatase-related diseases. This review focuses on the recently determined structures and regulation of the phosphoprotein phosphatase (PPP) family of ser/thr phosphatases-PP1, PP2A, Calcineurin and PP5.

The cell wall is essential for bacterial survival. Its core component is peptidoglycan (PG), a polymer comprised of disaccharide-peptides (stem peptides) that are cross-linked to one another via transpeptidation by penicillin-binding proteins (PBPs). While much is known about how PBPs are inactivated by β-lactam antibiotics, little is known about how PBPs bind and catalyze the transpeptidation of PG. Here we show how native PG and stem peptides are recruited to PBP5 of E. faecium, a critical ESKAPE pathogen. We discovered that PG binds PBP5 at the periphery of the PBP active site cleft, not the active site, and that the D-Ala leaving group contributes minimally to PBP binding. We show that β-lactam antibiotics and stem peptides can bind PBP5 simultaneously. We also show that only the single central residue of the stem peptide (L-Lys substituted by D-iAsn in E. faecium) is both necessary and sufficient for peptide recruitment. Finally, we translate our molecular findings by demonstrating that recruitment binding variants are unable to create a PG cell wall in E. faecium. Our studies define the key molecular interactions that govern bacterial cell wall formation and provide opportunities for the development of antibiotics that do not rely on PBP inactivation.

Phosphoprotein phosphatases (PPPs) achieve specificity by binding substrates and regulators using PPP-specific short motifs. Protein phosphatase 2A (PP2A) is a highly conserved phosphatase that regulates cell signaling and is a tumor suppressor. Here, we use cryo-electron microscopy and nuclear magnetic resonance (NMR) spectroscopy to investigate the mechanisms of human p107 substrate and Eya3 regulator recruitment to the PP2A:B55 holoenzyme. We show that, while they associate with B55 using a common set of interaction pockets, the mechanism of substrate and regulator binding differs and is distinct from that observed for PP2A:B56 and other PPPs. We also identify the core B55 recruitment motif in Eya3 proteins, a sequence conserved amongst the Eya family. Lastly, using NMR-based dephosphorylation assays, we demonstrate how B55 recruitment directs PP2A:B55 fidelity through the selective dephosphorylation of specific phosphosites. As PP2A:B55 orchestrates mitosis and DNA damage repair, these data provide a roadmap for pursuing new avenues to therapeutically target this complex by individually blocking a subset of regulators that use different B55 interaction sites.

Ser/Thr protein phosphatase 1 (PP1) forms a large nuclear holoenzyme (with PNUTS, WDR82, and Tox4) whose emerging role is to regulate transcription. However, the role of Tox4, and its interplay with the other phosphatase subunits in this complex, is poorly understood. Here, we combine biochemical, structural, cellular, and in vivo experiments to show that, while tox4 is dispensable for viability, it is essential for fertility, having both PNUTS-dependent and -independent roles in Drosophila germline development. We also show that Tox4 requires zinc for PNUTS TFIIS N-terminal domain (TND) binding, and that it binds the TND on a surface distinct from that used by established TND-interacting transcriptional regulators. We also show that selective disruption of the PNUTS-Tox4 and the PNUTS-PP1 interaction is critical for normal gene expression and chromosomal dispersal during oogenesis. Together, these data demonstrate how interactions within the PNUTS-Tox4-PP1 phosphatase combine to tune transcriptional outputs driving developmental transitions.

Aminoacyl-tRNA deacylases safeguard the accurate translation of the genetic code by hydrolyzing incorrectly synthesized aminoacyl-tRNAs. Canavanyl-tRNA deacylase (CtdA) was recently shown to protect cells against the toxicity of canavanine (Can), a nonproteinogenic amino acid synthesized and accumulated by leguminous plants. In most organisms, Can is ligated to tRNAArg, causing translation of arginine codons with Can. CtdA prevents Can toxicity by hydrolyzing canavanyl-tRNAArg. Here, we investigated the function, structure, substrate specificity, phylogenetic distribution, and evolution of CtdA. We show that CtdA is essential for preventing Can cytotoxicity in Salmonella enterica, and its heterologous expression can also protect Escherichia coli. By determining the structure of CtdA, we identified its putative binding pocket and residues that modulate enzymatic activity and specificity. We also found that CtdA displays robust specificity for the canavanyl moiety, a feature that contributes to maintaining arginyl-tRNAArg levels unaffected. Finally, we showed that despite their structural homology, CtdA and the aminoacyl-tRNA hydrolytic domain of phenylalanyl-tRNA synthetase are functionally and evolutionarily divergent. Collectively, these results substantially expand our understanding of the CtdA family, providing new insights into its structure, function, and evolution. This work also highlights the diverse mechanisms, unique to each organism, that ensure faithful translation of the genetic code.

Intrinsically disordered proteins/regions (IDPs/IDRs) frequently engage in dynamic charge:charge interactions, commonly referred to as 'fuzzy' interactions. These fuzzy interactions play critical roles in enzymatic regulation and substrate recruitment, especially for protein kinases and protein phosphatases. Here, we review recent advances that demonstrate how inter- and intramolecular fuzzy interactions among kinases and phosphatases and their cognate regulators and substrates allow for enzyme assembly, activation and substrate recruitment. We also highlight a unique mechanism of protein inhibition, where a protein phosphatase is inhibited by dynamic fuzzy interactions with its active site metals.

SDS22 and Inhibitor-3 (I3) are two ancient regulators of protein phosphatase 1 (PP1) that regulate multiple essential biological processes. Both SDS22 and I3 form stable dimeric complexes with PP1; however, and atypically for PP1 regulators, they also form a triple complex, where both proteins bind to PP1 simultaneously (SPI complex). Here we report the crystal structure of the SPI complex. While both regulators bind PP1 in conformations identical to those observed in their individual PP1 complexes, PP1 adopts the SDS22-bound conformation, which lacks its M1 metal. Unexpectedly, surface plasmon resonance (SPR) revealed that the affinity of I3 for the SDS22:PP1 complex is X10-fold lower than PP1 alone. We show that this change in binding affinity is solely due to the interaction of I3 with the PP1 active site, specifically PP1's M2 metal, demonstrating that SDS22 likely allows for PP1 M2 metal exchange and thus PP1 biogenesis.

The phosphoprotein phosphatase (PPP) family of ser/thr phosphatases are responsible for the majority of all ser/thr dephosphorylation in cells. However, unlike their kinase counterpart, they do not achieve specificity via phosphosite recognition sequences, but instead bind substrates and regulators using PPP-specific short linear and/or helical motifs (SLiMs, SHelMs). Protein phosphatase 2A (PP2A) is a highly conserved PPP that regulates cell signaling and is a tumor suppressor. Here, we investigate the mechanisms of substrate and regulator recruitment to the PP2A:B55 holoenzyme to define how substrates and regulators engage B55 and understand, in turn, how these interactions direct phosphosite dephosphorylation. Our cryo-EM structures of PP2A:B55 bound to p107 (substrate) and Eya3 (regulator), coupled with biochemical, biophysical and cell biology assays, show that while B55 associates using a common set of interaction pockets, the mechanisms of substrate and regulator binding can differ substantially. This shows that B55-mediated substrate recruitment is distinct from that observed for PP2A:B56 and other PPPs. It also allowed us to identify the core B55 recruitment motif in Eya3 proteins, a sequence we show is conserved amongst the Eya family. Finally, using NMR-based dephosphorylation assays, we also showed how B55 recruitment directs PP2A:B55 fidelity, via the selective dephosphorylation of specific phosphosites. Because of the key regulatory functions of PP2A:B55 in mitosis and DNA damage repair, these data provide a roadmap for pursuing new avenues to therapeutically target this complex by individually blocking a subset of regulators that use different B55 interaction sites. ### Competing Interest Statement The authors have declared no competing interest.

Protein tyrosine phosphatase nonreceptor type 22 (PTPN22) is encoded by a major autoimmunity gene and is a known inhibitor of T cell receptor (TCR) signaling and drug target for cancer immunotherapy. However, little is known about PTPN22 posttranslational regulation. Here, we characterize phosphorylation site at Ser325 situated C terminal to the catalytic domain of PTPN22 and its roles in altering protein function. In human T cells, Ser325 is phosphorylated by glycogen synthase kinase-3 (GSK3) following TCR stimulation, which promotes its TCR-inhibitory activity. Signaling through the major TCR-dependent pathway under PTPN22 control was enhanced by CRISPR/Cas9-mediated suppression of Ser325 phosphorylation and inhibited by mimicking it via glutamic acid substitution. Global phospho-mass spectrometry showed Ser325 phosphorylation state alters downstream transcriptional activity through enrichment of Swi3p, Rsc8p, and Moira domain binding proteins, and next-generation sequencing revealed it differentially regulates the expression of chemokines and T cell activation pathways. Moreover, in vitro kinetic data suggest the modulation of activity depends on a cellular context. Finally, we begin to address the structural and mechanistic basis for the influence of Ser325 phosphorylation on the protein's properties by deuterium exchange mass spectrometry and NMR spectroscopy. In conclusion, this study explores the function of a novel phosphorylation site of PTPN22 that involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.

Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (PhosTAC)-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, we show that, counter to our current understanding, many PP1 regulators are also substrates, that the number of residues between regulator PP1-binding and phosphosites vary significantly, and that PP1 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells.

Ampicillin-ceftriaxone has become a first-line therapy for Enterococcus faecalis endocarditis. We characterized the penicillin-binding protein (PBP) profiles of various E. faecalis strains and tested for synergy to better inform beta-lactam options for the treatment of E. faecalis infections.