Cryo-EM structures of PP2A:B55-FAM122A and PP2A:B55-ARPP19.

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation1. Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases2, whereas mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B553. Although the role of kinases in mitotic entry is well established, recent data have shown that mitosis is only successfully initiated when the counterbalancing phosphatases are also inhibited4. Inhibition of PP2A:B55 is achieved by the intrinsically disordered proteins ARPP195,6 and FAM122A7. Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the single-particle cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies, both intrinsically disordered proteins bind PP2A:B55, but do so in highly distinct manners, leveraging multiple distinct binding sites on B55. Our extensive structural, biophysical and biochemical data explain how substrates and inhibitors are recruited to PP2A:B55 and provide a molecular roadmap for the development of therapeutic interventions for PP2A:B55-related diseases.

The SDS22:PP1:I3 complex: SDS22 binding to PP1 loosens the active site metal to prime metal exchange.

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 ∼10-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.

A novel gain-of-function phosphorylation site modulates PTPN22 inhibition of TCR signaling.

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 a 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 is involved in complex regulation of TCR signaling and provides details that might inform the future development of allosteric modulators of PTPN22.

Conformational Rigidity and Protein Dynamics at Distinct Timescales Regulate PTP1B Activity and Allostery.

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales, and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in Protein Tyrosine Phosphatase 1B (PTP1B), a drug target for diabetes and cancer that catalyzes the dephosphorylation of numerous substrates in essential signaling pathways. By combining new experimental and computational data on WT-PTP1B and ≥10 PTP1B variants in multiple states, we discovered a fundamental and evolutionarily conserved CH/π switch that is critical for positioning the catalytically important WPD loop. Furthermore, our data show that PTP1B uses conformational and dynamic allostery to regulate its activity. This shows that both conformational rigidity and dynamics are essential for controlling protein activity. This connection between rigidity and dynamics at different timescales is likely a hallmark of all enzyme function.

MqsR is a noncanonical microbial RNase toxin that is inhibited by antitoxin MqsA via steric blockage of substrate binding.

The MqsRA toxin-antitoxin system is a component of the Escherichia coli stress response. Free MqsR, a ribonuclease, cleaves mRNAs containing a 5'-GC-3' sequence causing a global shutdown of translation and the cell to enter a state of dormancy. Despite a general understanding of MqsR function, the molecular mechanism(s) by which MqsR binds and cleaves RNA and how one or more of these activities is inhibited by its cognate antitoxin MqsA is still poorly understood. Here, we used NMR spectroscopy coupled with mRNA cleavage assays to identify the molecular mechanism of MqsR substrate recognition and the MqsR residues that are essential for its catalytic activity. We show that MqsR preferentially binds substrates that contain purines in the -2 and -1 position relative to the MqsR consensus cleavage sequence and that two residues of MqsR, Tyr81, and Lys56 are strictly required for mRNA cleavage. We also show that MqsA inhibits MqsR activity by sterically blocking mRNA substrates from binding while leaving the active site fully accessible to mononucleotides. Together, these data identify the residues of MqsR that mediate RNA cleavage and reveal a novel mechanism that regulates MqsR substrate specificity.

Degradation of the E. coli antitoxin MqsA by the proteolytic complex ClpXP is regulated by zinc occupancy and oxidation.

It is well established that the antitoxins of toxin-antitoxin (TA) systems are selectively degraded by bacterial proteases in response to stress. However, how distinct stressors result in the selective degradation of specific antitoxins remain unanswered. MqsRA is a TA system activated by various stresses, including oxidation. Here, we reconstituted the Escherichia coli ClpXP proteolytic machinery in vitro to monitor degradation of MqsRA TA components. We show that the MqsA antitoxin is a ClpXP proteolysis substrate, and that its degradation is regulated by both zinc occupancy in MqsA and MqsR toxin binding. Using NMR chemical shift perturbation mapping, we show that MqsA is targeted directly to ClpXP via the ClpX substrate targeting N-domain, and ClpX mutations that disrupt N-domain binding inhibit ClpXP-mediated degradation in vitro. Finally, we discovered that MqsA contains a cryptic N-domain recognition sequence that is accessible only in the absence of zinc and MqsR toxin, both of which stabilize the MqsA fold. This recognition sequence is transplantable and sufficient to target a fusion protein for degradation in vitro and in vivo. Based on these results, we propose a model in which stress selectively targets nascent and zinc-free MqsA, resulting in exposure of the ClpX recognition motif for ClpXP-mediated degradation.

Penicillin-Binding Proteins and Alternative Dual-Beta-Lactam Combinations for Serious Enterococcus faecalis Infections with Elevated Penicillin MICs.

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. We assessed the affinity of PBP2B from elevated-MIC strain E. faecalis LS4828 compared to type strain JH2-2 using the fluorescent beta-lactam Bocillin FL. We also characterized pbp4 and pbpA structures and PBP4 and PBP2B expression and used deletion and complementation studies to assess the impact of PBP2B on the levels of resistance. We tested penicillin-susceptible and -resistant E. faecalis isolates against ceftriaxone or ceftaroline combinations with other beta-lactams in 24-h time-kill studies. Two penicillin-susceptible strains (JH2-2 and L2052) had identical pbp sequences and similar PBP expression levels. One reduced-penicillin-susceptibility strain (L2068) had pbp sequences identical to those of the susceptible strains but expressed more PBP4. The second decreased-penicillin-susceptibility strain (LS4828) had amino acid substitutions in both PBP4 and PBP2B and expressed increased quantities of both proteins. PBP2B did not appear to contribute significantly to the elevated beta-lactam MICs. No synergy was demonstrable against the strains with both mutated PBPs and increased expression (L2068 and LS4828). Meropenem plus ceftriaxone or ertapenem plus ceftriaxone demonstrated the most consistent synergistic activity. PBP2B of strain LS4828 does not contribute significantly to reduced penicillin susceptibility. Neither the MIC nor the level of PBP expression correlated directly with the identified synergistic combinations when tested at static subinhibitory concentrations.

EDC3 phosphorylation regulates growth and invasion through controlling P-body formation and dynamics.

Regulation of mRNA stability and translation plays a critical role in determining protein abundance within cells. Processing bodies (P-bodies) are critical regulators of these processes. Here, we report that the Pim1 and 3 protein kinases bind to the P-body protein enhancer of mRNA decapping 3 (EDC3) and phosphorylate EDC3 on serine (S)161, thereby modifying P-body assembly. EDC3 phosphorylation is highly elevated in many tumor types, is reduced upon treatment of cells with kinase inhibitors, and blocks the localization of EDC3 to P-bodies. Prostate cancer cells harboring an EDC3 S161A mutation show markedly decreased growth, migration, and invasion in tissue culture and in xenograft models. Consistent with these phenotypic changes, the expression of integrin ß1 and α6 mRNA and protein is reduced in these mutated cells. These results demonstrate that EDC3 phosphorylation regulates multiple cancer-relevant functions and suggest that modulation of P-body activity may represent a new paradigm for cancer treatment.

SDS22 selectively recognizes and traps metal-deficient inactive PP1.

The metalloenzyme protein phosphatase 1 (PP1), which is responsible for ≥50% of all dephosphorylation reactions, is regulated by scores of regulatory proteins, including the highly conserved SDS22 protein. SDS22 has numerous diverse functions, surprisingly acting as both a PP1 inhibitor and as an activator. Here, we integrate cellular, biophysical, and crystallographic studies to address this conundrum. We discovered that SDS22 selectively binds a unique conformation of PP1 that contains a single metal (M2) at its active site, i.e., SDS22 traps metal-deficient inactive PP1. Furthermore, we showed that SDS22 dissociation is accompanied by a second metal (M1) being loaded into PP1, as free metal cannot dissociate the complex and M1-deficient mutants remain constitutively trapped by SDS22. Together, our findings reveal that M1 metal loading and loss are essential for PP1 regulation in cells, which has broad implications for PP1 maturation, activity, and holoenzyme subunit exchange.

Crystal Structure of TCPTP Unravels an Allosteric Regulatory Role of Helix α7 in Phosphatase Activity.

The T-cell protein tyrosine phosphatase (TCPTP/PTPN2) targets a broad variety of substrates across different subcellular compartments. In spite of that, the structural basis for the regulation of TCPTP's activity remains elusive. Here, we investigated whether the activity of TCPTP is regulated by a potential allosteric site in a comparable manner to its most similar PTP family member (PTP1B/PTPN1). We determined two crystal structures of TCPTP at 1.7 and 1.9 Å resolutions that include helix α7 at the TCPTP C-terminus. Helix α7 has been functionally characterized in PTP1B and was identified as its allosteric switch. However, its function is unknown in TCPTP. Here, we demonstrate that truncation or deletion of helix α7 reduced the catalytic efficiency of TCPTP by ∼4-fold. Collectively, our data supports an allosteric role of helix α7 in regulation of TCPTP's activity, similar to its function in PTP1B, and highlights that the coordination of helix α7 with the core catalytic domain is essential for the efficient catalytic function of TCPTP.

Cooperative dynamics across distinct structural elements regulate PTP1B activity.

Protein-tyrosine phosphatase 1B (PTP1B) is the canonical enzyme for investigating how distinct structural elements influence enzyme catalytic activity. Although it is recognized that dynamics are essential for PTP1B function, the data collected thus far have not resolved whether distinct elements are dynamically coordinated or, alternatively, whether they fulfill their respective functions independently. To answer this question, we performed a comprehensive 13C-methyl relaxation study of Ile, Leu, and Val (ILV) residues of PTP1B, which, because of its substantially increased sensitivity, provides a comprehensive understanding of the influence of protein motions on different time scales for enzyme function. We discovered that PTP1B exhibits dynamics at three distinct time scales. First, it undergoes a distinctive slow motion that allows for the dynamic binding and release of its two most N-terminal helices from the catalytic core. Second, we showed that PTP1B 13C-methyl group side chain fast time-scale dynamics and 15N backbone fast time-scale dynamics are fully consistent, demonstrating that fast fluctuations are essential for the allosteric control of PTP1B activity. Third, and most importantly, using 13C ILV constant-time Carr-Purcell-Meiboom-Gill relaxation measurements experiments, we demonstrated that all four catalytically important loops-the WPD, Q, E, and substrate-binding loops-work in dynamic unity throughout the catalytic cycle of PTP1B. Thus, these data show that PTP1B activity is not controlled by a single functional element, but instead all key elements are dynamically coordinated. Together, these data provide the first fully comprehensive picture on how the validated drug target PTP1B functions.

NMR Based SARS-CoV-2 Antibody Screening.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry into cells is a complex process that involves (1) recognition of the host entry receptor, angiotensin-converting enzyme 2 (ACE2), by the SARS-CoV-2 spike protein receptor binding domain (RBD), and (2) the subsequent fusion of the viral and cell membranes. Our long-term immune-defense is the production of antibodies (Abs) that recognize the SARS-CoV-2 RBD and successfully block viral infection. Thus, to understand immunity against SARS-CoV-2, a comprehensive molecular understanding of how human SARS-CoV-2 Abs recognize the RBD is needed. Here, we report the sequence-specific backbone assignment of the SARS-CoV-2 RBD and, furthermore, demonstrate that biomolecular NMR spectroscopy chemical shift perturbation (CSP) mapping successfully and rapidly identifies the molecular epitopes of RBD-specific mAbs. By incorporating NMR-based CSP mapping with other molecular techniques to define RBD-mAb interactions and then correlating these data with neutralization efficacy, structure-based approaches for developing improved vaccines and COVID-19 mAb-based therapies will be greatly accelerated.

Design, synthesis, kinetic, molecular dynamics, and hypoglycemic effect characterization of new and potential selective benzimidazole derivatives as Protein Tyrosine Phosphatase 1B inhibitors.

Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling pathway and has been validated as a therapeutic target for type 2 diabetes. A wide variety of scaffolds have been included in the structure of PTP1B inhibitors, one of them is the benzimidazole nucleus. Here, we report the design and synthesis of a new series of di- and tri- substituted benzimidazole derivatives including their kinetic and structural characterization as PTP1B inhibitors and hypoglycemic activity. Results show that compounds 43, 44, 45, and 46 are complete mixed type inhibitors with a Ki of 12.6 µM for the most potent (46). SAR type analysis indicates that a chloro substituent at position 6(5), a ß-naphthyloxy at position 5(6), and a p-benzoic acid attached to the linker 2-thioacetamido at position 2 of the benzimidazole nucleus, was the best combination for PTP1B inhibition and hypoglycemic activity. In addition, molecular dynamics studies suggest that these compounds could be potential selective inhibitors from other PTPs such as its closest homologous TCPTP, SHP-1, SHP-2 and CDC25B. Therefore, the compounds reported here are good hits that provide structural, kinetic, and biological information that can be used to develop novel and selective PTP1B inhibitors based on benzimidazole scaffold.

Dynamic activation and regulation of the mitogen-activated protein kinase p38.

Mitogen-activated protein kinases, which include p38, are essential for cell differentiation and autophagy. The current model for p38 activation involves activation-loop phosphorylation with subsequent substrate binding leading to substrate phosphorylation. Despite extensive efforts, the molecular mechanism of activation remains unclear. Here, using NMR spectroscopy, we show how the modulation of protein dynamics across timescales activates p38. We find that activation-loop phosphorylation does not change the average conformation of p38; rather it quenches the loop ps-ns dynamics. We then show that substrate binding to nonphosphorylated and phosphorylated p38 results in uniform µs-ms backbone dynamics at catalytically essential regions and across the entire molecule, respectively. Together, these results show that phosphorylation and substrate binding flatten the energy landscape of the protein, making essential elements of allostery and activation dynamically accessible. The high degree of structural conservation among ser/thr kinases suggests that elements of this mechanism may be conserved across the kinase family.

A Quantitative Chemical Proteomic Strategy for Profiling Phosphoprotein Phosphatases from Yeast to Humans.

A "tug-of-war" between kinases and phosphatases establishes the phosphorylation states of proteins. While serine and threonine phosphorylation can be catalyzed by more than 400 protein kinases, the majority of serine and threonine dephosphorylation is carried out by seven phosphoprotein phosphatases (PPPs). The PPP family consists of protein phosphatases 1 (PP1), 2A (PP2A), 2B (PP2B), 4 (PP4), 5 (PP5), 6 (PP6), and 7 (PP7). The imbalance in numbers between serine- and threonine-directed kinases and phosphatases led to the early belief that PPPs are unspecific and that kinases are the primary determinants of protein phosphorylation. However, it is now clear that PPPs achieve specificity through association with noncatalytic subunits to form multimeric holoenzymes, which expands the number of functionally distinct signaling entities to several hundred. Although there has been great progress in deciphering signaling by kinases, much less is known about phosphatases.We have developed a chemical proteomic strategy for the systematic interrogation of endogenous PPP catalytic subunits and their interacting proteins, including regulatory and scaffolding subunits (the "PPPome"). PP1, PP2A, PP4, PP5, and PP6 were captured using an immobilized, specific but nonselective PPP inhibitor microcystin-LR (MCLR), followed by protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a single analysis. Here, we combine this approach of phosphatase inhibitor bead profiling and mass spectrometry (PIB-MS) with label-free and tandem mass tag (TMT) quantification to map the PPPome in human cancer cell lines, mouse tissues, and yeast species, through which we identify cell- and tissue-type-specific PPP expression patterns and discover new PPP interacting proteins.

A peculiar IclR family transcription factor regulates para-hydroxybenzoate catabolism in Streptomyces coelicolor.

In Streptomyces coelicolor, we identified a para-hydroxybenzoate (PHB) hydroxylase, encoded by gene pobA (SCO3084), which is responsible for conversion of PHB into PCA (protocatechuic acid), a substrate of the ß-ketoadipate pathway which yields intermediates of the Krebs cycle. We also found that the transcription of pobA is induced by PHB and is negatively regulated by the product of SCO3209, which we named PobR. The product of this gene is highly unusual in that it is the apparent fusion of two IclR family transcription factors. Bioinformatic analyses, in vivo transcriptional assays, electrophoretic mobility shift assays (EMSAs), DNase I footprinting, and isothermal calorimetry (ITC) were used to elucidate the regulatory mechanism of PobR. We found that PobR loses its high affinity for DNA (i.e., the pobA operator) in the presence of PHB, the inducer of pobA transcription. PHB binds to PobR with a KD of 5.8 µM. Size-exclusion chromatography revealed that PobR is a dimer in the absence of PHB and a monomer in the presence of PHB. The crystal structure of PobR in complex with PHB showed that only one of the two IclR ligand binding domains was occupied, and defined how the N-terminal ligand binding domain engages the effector ligand.

Retinoic Acid Binding Leads to CRABP2 Rigidification and Dimerization.

Cellular retinoic acid-binding protein 2 (CRABP2) delivers all-trans retinoic acid (atRA) to retinoic acid receptors (RARs), allowing for the activation of specific gene transcription. The structural similarities between free and atRA-bound CRABP2 raise the questions of how atRA binding occurs and how the atRA:CRABP2 complex is recognized by downstream binding partners. Thus, to gain insights into these questions, we conducted a detailed atRA-CRABP2 interaction study using nuclear magnetic resonance spectroscopy. The data showed that free CRABP2 displays widespread intermediate-time scale dynamics that is effectively suppressed upon atRA binding. This effect is mirrored by the fast-time scale dynamics of CRABP2. Unexpectedly, CRABP2 rigidification in response to atRA binding leads to the stabilization of a homodimerization interface, which encompasses residues located on helix α2 and the ßC-ßD loop as well as residues on strands ßI-ßA and the ßH-ßI loop. Critically, this rigidification also affects CRABP2's nuclear localization signal and RAR-binding motif, suggesting that the loss of conformational entropy upon atRA binding may be the key for the diverse cellular functions of CRABP2.

A Sephin1-insensitive tripartite holophosphatase dephosphorylates translation initiation factor 2α.

The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative small-molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2αP dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.

The structures of penicillin-binding protein 4 (PBP4) and PBP5 from Enterococci provide structural insights into ß-lactam resistance.

The final steps of cell-wall biosynthesis in bacteria are carried out by penicillin-binding proteins (PBPs), whose transpeptidase domains form the cross-links in peptidoglycan chains that define the bacterial cell wall. These enzymes are the targets of ß-lactam antibiotics, as their inhibition reduces the structural integrity of the cell wall. Bacterial resistance to antibiotics is a rapidly growing concern; however, the structural underpinnings of PBP-derived antibiotic resistance are poorly understood. PBP4 and PBP5 are low-affinity, class B transpeptidases that confer antibiotic resistance to Enterococcus faecalis and Enterococcus faecium, respectively. Here, we report the crystal structures of PBP4 (1.8 Å) and PBP5 (2.7 Å) in their apo and acyl-enzyme complexes with the ß-lactams benzylpenicillin, imipenem, and ceftaroline. We found that, although these three ß-lactams adopt geometries similar to those observed in other class B PBP structures, there are small, but significant, differences that likely decrease antibiotic efficacy. Further, we also discovered that the N-terminal domain extensions in this class of PBPs undergo large rigid-body rotations without impacting the structure of the catalytic transpeptidase domain. Together, our findings are defining the subtle functional and structural differences in the Enterococcus PBPs that allow them to support transpeptidase activity while also conferring bacterial resistance to antibiotics that function as substrate mimics.

The KIM-family protein-tyrosine phosphatases use distinct reversible oxidation intermediates: Intramolecular or intermolecular disulfide bond formation.

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.