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.

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.

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

Progression through the cell cycle is controlled by regulated and abrupt changes in phosphorylation.1 Mitotic entry is initiated by increased phosphorylation of mitotic proteins, a process driven by kinases,2 while mitotic exit is achieved by counteracting dephosphorylation, a process driven by phosphatases, especially PP2A:B55.3 While 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 inhibited.4 For PP2A:B55, inhibition is achieved by the two intrinsically disordered proteins (IDPs), ARPP19 (phosphorylation-dependent)6,7 and FAM122A5 (inhibition is phosphorylation-independent). Despite their critical roles in mitosis, the mechanisms by which they achieve PP2A:B55 inhibition is unknown. Here, we report the cryo-electron microscopy structures of PP2A:B55 bound to phosphorylated ARPP19 and FAM122A. Consistent with our complementary NMR spectroscopy studies both IDPs bind PP2A:B55, but do so in highly distinct manners, unexpectedly 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 provides a molecular roadmap for the development of therapeutic interventions for PP2A:B55 related diseases.

Molecular basis of ß-lactam antibiotic resistance of ESKAPE bacterium E. faecium Penicillin Binding Protein PBP5.

Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of ß-lactam antibiotics. In Enterococcus faecium, high levels of resistance to ß-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 - due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased ß-lactam antibiotic turnover, facilitates ß-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family.

Inhibitor-3 inhibits Protein Phosphatase 1 via a metal binding dynamic protein-protein interaction.

To achieve substrate specificity, protein phosphate 1 (PP1) forms holoenzymes with hundreds of regulatory and inhibitory proteins. Inhibitor-3 (I3) is an ancient inhibitor of PP1 with putative roles in PP1 maturation and the regulation of PP1 activity. Here, we show that I3 residues 27-68 are necessary and sufficient for PP1 binding and inhibition. In addition to a canonical RVxF motif, which is shared by nearly all PP1 regulators and inhibitors, and a non-canonical SILK motif, I3 also binds PP1 via multiple basic residues that bind directly in the PP1 acidic substrate binding groove, an interaction that provides a blueprint for how substrates bind this groove for dephosphorylation. Unexpectedly, this interaction positions a CCC (cys-cys-cys) motif to bind directly across the PP1 active site. Using biophysical and inhibition assays, we show that the I3 CCC motif binds and inhibits PP1 in an unexpected dynamic, fuzzy manner, via transient engagement of the PP1 active site metals. Together, these data not only provide fundamental insights into the mechanisms by which IDP protein regulators of PP1 achieve inhibition, but also shows that fuzzy interactions between IDPs and their folded binding partners, in addition to enhancing binding affinity, can also directly regulate enzyme activity.

Structural mechanism for inhibition of PP2A-B56α and oncogenicity by CIP2A.

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.

The ribosomal RNA processing 1B:protein phosphatase 1 holoenzyme reveals non-canonical PP1 interaction motifs.

The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of substrates by associating with >200 regulatory proteins to form specific holoenzymes. The major PP1 targeting protein in the nucleolus is RRP1B (ribosomal RNA processing 1B). In addition to selectively recruiting PP1ß/PP1γ to the nucleolus, RRP1B also has a key role in ribosome biogenesis, among other functions. How RRP1B binds PP1 and regulates nucleolar phosphorylation signaling is not yet known. Here, we show that RRP1B recruits PP1 via established (RVxF/SILK/ΦΦ) and non-canonical motifs. These atypical interaction sites, the PP1ß/γ specificity, and N-terminal AF-binding pockets rely on hydrophobic interactions that contribute to binding and, via phosphorylation, regulate complex formation. This work advances our understanding of PP1 isoform selectivity, reveals key roles of N-terminal PP1 residues in regulator binding, and suggests that additional PP1 interaction sites have yet to be identified, all of which are necessary for a systems biology understanding of PP1 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.

Conserved conformational dynamics determine enzyme activity.

Homologous enzymes often exhibit different catalytic rates despite a fully conserved active site. The canonical view is that an enzyme sequence defines its structure and function and, more recently, that intrinsic protein dynamics at different time scales enable and/or promote catalytic activity. Here, we show that, using the protein tyrosine phosphatase PTP1B, residues surrounding the PTP1B active site promote dynamically coordinated chemistry necessary for PTP1B function. However, residues distant to the active site also undergo distinct intermediate time scale dynamics and these dynamics are correlated with its catalytic activity and thus allow for different catalytic rates in this enzyme family. We identify these previously undetected motions using coevolutionary coupling analysis and nuclear magnetic resonance spectroscopy. Our findings strongly indicate that conserved dynamics drives the enzymatic activity of the PTP family. Characterization of these conserved dynamics allows for the identification of novel regulatory elements (therapeutic binding pockets) that can be leveraged for the control of enzymes.

Denosumab treatment is associated with decreased cortical porosity and increased bone density and strength at the proximal humerus of ovariectomized cynomolgus monkeys.

Upper extremity fractures, including those at the humerus, are common among women with postmenopausal osteoporosis. Denosumab was shown to reduce humeral fractures in this population; however, no clinical or preclinical studies have quantified the effects of denosumab on humerus bone mineral density or bone microarchitecture changes. This study used micro-computed tomography (µCT) and computed tomography (CT), alongside image-based finite element (FE) models derived from both modalities, to quantify the effects of denosomab (DMAb) and alendronate (ALN) on humeral bone from acutely ovariectomized (OVX) cynomolgus monkeys. Animals were treated with 12 monthly injections of s.c. vehicle (VEH; n = 10), s.c. denosumab (DMAb; 25 mg/kg, n = 9), or i.v. alendronate (ALN; 50 µg/kg, n = 10). Two more groups received 6 months of VEH followed by 6 months of DMAb (VEH-DMAb; n = 7) or 6 months of ALN followed by 6 months of DMAb (ALN-DMAb; n = 9). After treatment, humeri were harvested and µCT was used to quantify tissue mineral density, trabecular morphology, and cortical porosity at the humeral head. Clinical CT imaging was also used to quantify trabecular and cortical bone mineral density (BMD) at the ultra-proximal, proximal, 1/5 proximal and midshaft of the bone. Finally, µCT-based FE models in compression, and CT-based FE models in compression, torsion, and bending, were developed to estimate differences in strength. Compared to VEH, groups that received DMAb at any time demonstrated lower cortical porosity and/or higher tissue mineral density via µCT; no effects on trabecular morphology were observed. FE estimated strength based on µCT was higher after 12-months DMAb (p = 0.020) and ALN-DMAb (p = 0.024) vs. VEH; respectively, FE predicted mean (SD) strength was 4649.88 (710.58) N, and 4621.10 (1050.16) N vs. 3309.4 (876.09) N. All antiresorptive treatments were associated with higher cortical BMD via CT at the 1/5 proximal and midshaft of the humerus; however, no differences in CT-based FE predicted strength were observed. Overall, these results help to explain the observed reductions in humeral fracture rate following DMAb treatment in women with postmenopausal osteoporosis.

Oxidative stress promotes fibrosis in systemic sclerosis through stabilization of a kinase-phosphatase complex.

Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.

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.

The catalytic activity of TCPTP is auto-regulated by its intrinsically disordered tail and activated by Integrin alpha-1.

T-Cell Protein Tyrosine Phosphatase (TCPTP, PTPN2) is a non-receptor type protein tyrosine phosphatase that is ubiquitously expressed in human cells. TCPTP is a critical component of a variety of key signaling pathways that are directly associated with the formation of cancer and inflammation. Thus, understanding the molecular mechanism of TCPTP activation and regulation is essential for the development of TCPTP therapeutics. Under basal conditions, TCPTP is largely inactive, although how this is achieved is poorly understood. By combining biomolecular nuclear magnetic resonance spectroscopy, small-angle X-ray scattering, and chemical cross-linking coupled with mass spectrometry, we show that the C-terminal intrinsically disordered tail of TCPTP functions as an intramolecular autoinhibitory element that controls the TCPTP catalytic activity. Activation of TCPTP is achieved by cellular competition, i.e., the intrinsically disordered cytosolic tail of Integrin-α1 displaces the TCPTP autoinhibitory tail, allowing for the full activation of TCPTP. This work not only defines the mechanism by which TCPTP is regulated but also reveals that the intrinsically disordered tails of two of the most closely related PTPs (PTP1B and TCPTP) autoregulate the activity of their cognate PTPs via completely different mechanisms.

PP2A/B55α substrate recruitment as defined by the retinoblastoma-related protein p107.

Protein phosphorylation is a reversible post-translation modification essential in cell signaling. This study addresses a long-standing question as to how the most abundant serine/threonine protein phosphatase 2 (PP2A) holoenzyme, PP2A/B55α, specifically recognizes substrates and presents them to the enzyme active site. Here, we show how the PP2A regulatory subunit B55α recruits p107, a pRB-related tumor suppressor and B55α substrate. Using molecular and cellular approaches, we identified a conserved region 1 (R1, residues 615-626) encompassing the strongest p107 binding site. This enabled us to identify an 'HxRVxxV619-625' short linear motif (SLiM) in p107 as necessary for B55α binding and dephosphorylation of the proximal pSer-615 in vitro and in cells. Numerous B55α/PP2A substrates, including TAU, contain a related SLiM C-terminal from a proximal phosphosite, 'p[ST]-P-x(4,10)-[RK]-V-x-x-[VI]-R.' Mutation of conserved SLiM residues in TAU dramatically inhibits dephosphorylation by PP2A/B55α, validating its generality. A data-guided computational model details the interaction of residues from the conserved p107 SLiM, the B55α groove, and phosphosite presentation. Altogether, these data provide key insights into PP2A/B55α's mechanisms of substrate recruitment and active site engagement, and also facilitate identification and validation of new substrates, a key step towards understanding PP2A/B55α's role in multiple cellular processes.

1H, 15N and 13C sequence specific backbone assignment of the MAP kinase binding domain of the dual specificity phosphatase 1 and its interaction with the MAPK p38.

The sequence-specific backbone assignment of the mitogen-activated protein kinase (MAPK) binding domain of the dual-specificity phosphatase 1 (DUSP1) has been accomplished using a uniformly [13C, 15N]-labeled protein. These assignments will facilitate further studies of DUSP1 in the presence of inhibitors/ligands to target MAPK associated diseases and provide further insights into the function of dual-specificity phosphatase 1 in MAPK regulation.

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.

Mechanical fatigue of whole rabbit-tibiae under combined compression-torsional loading is better explained by strained volume than peak strain magnitude.

The mechanical fatigue behavior of whole bone is poorly defined, particularly for the combined loading modes that occur in vivo. The purpose of this study was to quantify the fatigue life of whole rabbit-tibiae under cyclic uniaxial compression and biaxial (compression and torsion) loading, and to explore the relationship between fatigue life and specimen-specific finite element (FE) predictions of stress/strain. Twelve tibiae were tested cyclically until failure across a range of uniaxial-compressive loads. Another twenty-two tibiae were separated into three groups and loaded biaxially; peak compressive load was constant in all three groups (50% ultimate force) but torsion was varied (0%, 25%, or 50% of ultimate torque). FE models with heterogeneous linear-elastic material properties were developed from computed tomography. We assessed peak stress/strain and stressed/strained volume based on principal stress/strain, as well as von Mises and pressure modified von Mises criteria. A logarithmic (r2 = 0.68; p < 0.001) relationship was observed between uniaxial-compressive load and fatigue life. Biaxial tests demonstrated that fatigue life decreased with superposed torsion (p = 0.034). Strained volume, based on a maximum principal strain or pressure modified von Mises strain criteria, were strong predictors of fatigue life under both uniaxial (r2 = 0.73-0.82) and biaxial (r2 = 0.59-0.60) loads, and these outperformed equivalent peak stress- and strain-based measures. Our findings highlight the importance of evaluating strain distributions, rather than peak stress or strain, to predict the fatigue behavior or whole bone, which has important implications for the study of stress fracture.

The interaction of p38 with its upstream kinase MKK6.

Mitogen-activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.

The mode of action of the Protein tyrosine phosphatase 1B inhibitor Ertiprotafib.

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for the treatment of diabetes and obesity. Ertiprotafib is a PTP1B inhibitor that reached the clinical trial stage for the treatment of diabetes. Interestingly, Ertiprotafib reduces the melting temperature of PTP1B in differential scanning fluorimetry (DSF) assays, different from most drugs that increase the stability of their target upon binding. No molecular data on how Ertiprotafib functions has been published. Thus, to gain molecular insights into the mode of action of Ertiprotafib, we used biomolecular NMR spectroscopy to characterize the molecular details of the PTP1B:Ertiprotafib interaction. Our results show that Ertiprotafib induces aggregation of PTP1B in a concentration dependent manner. This shows that the insufficient clinical efficacy and adverse effects caused by Ertiprotafib is due to its tendency to cause aggregation of PTP1B.

The structure of the RCAN1:CN complex explains the inhibition of and substrate recruitment by calcineurin.

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the Ser/Thr phosphatase calcineurin (CN). It has been shown that excessive inhibition of CN is a critical factor for Down syndrome and Alzheimer's disease. Here, we determined RCAN1's mode of action. Using a combination of structural, biophysical, and biochemical studies, we show that RCAN1 inhibits CN via multiple routes: first, by blocking essential substrate recruitment sites and, second, by blocking the CN active site using two distinct mechanisms. We also show that phosphorylation either inhibits RCAN1-CN assembly or converts RCAN1 into a weak inhibitor, which can be reversed by CN via dephosphorylation. This highlights the interplay between posttranslational modifications in regulating CN activity. Last, this work advances our understanding of how active site inhibition of CN can be achieved in a highly specific manner. Together, these data provide the necessary road map for targeting multiple neurological disorders.