Dorothy Koveal

Targeting the disordered C terminus of PTP1B with an allosteric inhibitor.

PTP1B, a validated therapeutic target for diabetes and obesity, plays a critical positive role in HER2 signaling in breast tumorigenesis. Efforts to develop therapeutic inhibitors of PTP1B have been frustrated by the chemical properties of the active site. We defined a novel mechanism of allosteric inhibition that targets the C-terminal, non-catalytic segment of PTP1B. We present the first ensemble structure of PTP1B containing this intrinsically disordered segment, within which we identified a binding site for the small molecule inhibitor, MSI-1436. We demonstrate binding to a second site close to the catalytic domain, with cooperative effects between the two sites locking PTP1B in an inactive state. MSI-1436 antagonized HER2 signaling, inhibited tumorigenesis in xenografts and abrogated metastasis in the NDL2 mouse model of breast cancer, validating inhibition of PTP1B as a therapeutic strategy in breast cancer. This new approach to inhibition of PTP1B emphasizes the potential of disordered segments of proteins as specific binding sites for therapeutic small molecules.

Structural basis of p38α regulation by hematopoietic tyrosine phosphatase

MAP kinases regulate essential cellular events, including cell growth, differentiation and inflammation. The solution structure of a complete MAPK-MAPK-regulatory protein complex, p38α-HePTP, was determined, enabling a comprehensive investigation of the molecular basis of specificity and fidelity in MAPK regulation. Structure determination was achieved by combining NMR spectroscopy and small-angle X-ray scattering data with a new ensemble calculation-refinement procedure. We identified 25 residues outside of the HePTP kinase interaction motif necessary for p38α recognition. The complex adopts an extended conformation in solution and rarely samples the conformation necessary for kinase deactivation. Complex formation also does not affect the N-terminal lobe, the activation loop of p38α or the catalytic domain of HePTP. Together, these results show how the downstream tyrosine phosphatase HePTP regulates p38α and provide for fundamentally new insights into MAPK regulation and specificity.

Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake

Proper brain function requires a substantial energy supply, up to 20% of whole-body energy in humans, and brain activation produces large dynamic variations in energy demand. While local increases in cerebral blood flow are well known, the cellular responses to energy demand are controversial. During brain excitation, glycolysis of glucose to lactate temporarily exceeds the rate of mitochondrial fuel oxidation; although the increased energy demand occurs mainly within neurons, some have suggested this glycolysis occurs mainly in astrocytes, which then shuttle lactate to neurons as their primary fuel. Using metabolic biosensors in acute hippocampal slices and brains of awake mice, we find that neuronal metabolic responses to stimulation do not depend on astrocytic stimulation by glutamate release, nor do they require neuronal uptake of lactate; instead they reflect increased direct glucose consumption by neurons. Neuronal glycolysis temporarily outstrips oxidative metabolism, and provides a rapid response to increased energy demand.

A CC-SAM, for coiled coil-sterile α motif, domain targets the scaffold KSR-1 to specific sites in the plasma membrane.

A previously unknown module that mediates membrane binding is identified in the scaffold KSR. Another Helping Hand to the Membrane Growth factor stimulation activates the mitogen-activated protein kinase module, in which Raf activates MEK, which in turn activates ERK. Kinase suppressor of Ras-1 (KSR-1) requires a phospholipid-binding atypical C1 domain to associate with the plasma membrane to promote assembly of this module. Koveal et al. identified a sterile α motif (SAM) adjacent to a coiled coil (CC) domain in KSR-1. Structural and biochemical analysis of just the CC and SAM regions showed that CC-SAM formed a single, structural module. CC-SAM bound directly to micelles and bicelles in vitro and was targeted to the plasma membrane in growth factor–treated cells. Structure-based point mutations in helix α3 of the CC motif prevented the binding of CC-SAM to micelles and bicelles. Full-length KSR-1 with these mutations did not localize to the plasma membrane or interact with B-Raf when expressed in growth factor–treated cells. Thus, KSR-1 requires the atypical C1 and CC-SAM domains to associate with the plasma membrane. Kinase suppressor of Ras-1 (KSR-1) is an essential scaffolding protein that coordinates the assembly of the mitogen-activated protein kinase (MAPK) module, consisting of the MAPK kinase kinase Raf, the MAPK kinase MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase), and the MAPK ERK (extracellular signal–regulated kinase) to facilitate activation of MEK and thus ERK. Although KSR-1 is targeted to the cell membrane in part by its atypical C1 domain, which binds to phospholipids, other domains may be involved. We identified another domain in KSR-1 that we termed CC-SAM, which is composed of a coiled coil (CC) and a sterile α motif (SAM). The CC-SAM domain targeted KSR-1 to specific signaling sites at the plasma membrane in growth factor–treated cells, and it bound directly to various micelles and bicelles in vitro, indicating that the CC-SAM functioned as a membrane-binding module. By combining nuclear magnetic resonance spectroscopy and experiments in cultured cells, we found that membrane binding was mediated by helix α3 of the CC motif and that mutating residues in α3 abolished targeting of KSR-1 to the plasma membrane. Thus, in addition to the atypical C1 domain, the CC-SAM domain is required to target KSR-1 to the plasma membrane.

Ligand Binding Reduces Conformational Flexibility in the Active Site of Tyrosine Phosphatase Related to Biofilm Formation A (TpbA) from Pseudomonasaeruginosa

Tyrosine phosphatase related to biofilm formation A (TpbA) is a periplasmic dual-specificity phosphatase (DUSP) that controls biofilm formation in the pathogenic bacterium Pseudomonas aeruginosa. While DUSPs are known to regulate important cellular functions in both prokaryotes and eukaryotes, very few structures of bacterial DUSPs are available. Here, we present the solution structure of TpbA in the ligand-free open conformation, along with an analysis of the structural and dynamic changes that accompany ligand/phosphate binding. While TpbA adopts a typical DUSP fold, it also possesses distinct structural features that distinguish it from eukaryotic DUSPs. These include additional secondary structural elements, β0 and α6, and unique conformations of the variable insert, the α4-α5 loop and helix α5 that impart TpbA with a flat active-site surface. In the absence of ligand, the protein tyrosine phosphatase loop is disordered and the general acid loop adopts an open conformation, placing the catalytic aspartate, Asp105, more than 11Å away from the active site. Furthermore, the loops surrounding the active site experience motions on multiple timescales, consistent with a combination of conformational heterogeneity and fast (picosecond to nanosecond) timescale dynamics, which are significantly reduced upon ligand binding. Taken together, these data structurally distinguish TpbA and possibly other bacterial DUSPs from eukaryotic DUSPs and provide a rich picture of active-site dynamics in the ligand-free state that are lost upon ligand binding.

The Differential Regulation of p38α by the Neuronal Kinase Interaction Motif Protein Tyrosine Phosphatases, a Detailed Molecular Study

The MAP kinase p38α is essential for neuronal signaling. To better understand the molecular regulation of p38α we used atomistic and molecular techniques to determine the structural basis of p38α regulation by the two neuronal tyrosine phosphatases, PTPSL/PTPBR7 (PTPRR) and STEP (PTPN5). We show that, despite the fact that PTPSL and STEP belong to the same family of regulatory proteins, they interact with p38α differently and their distinct molecular interactions explain their different catalytic activities. Although the interaction of PTPSL with p38α is similar to that of the previously described p38α:HePTP (PTPN7) complex, STEP binds and regulates p38α in an unexpected manner. Using NMR and small-angle X-ray scattering data, we generated a model of the p38α:STEP complex and define molecular differences between its resting and active states. Together, these results provide insights into molecular regulation of p38α by key regulatory proteins.

Docking interactions of hematopoietic tyrosine phosphatase with MAP kinases ERK2 and p38α.

Hematopoietic tyrosine phosphatase (HePTP) regulates orthogonal MAP kinase signaling cascades by dephosphorylating both extracellular signal-regulated kinase (ERK) and p38. HePTP recognizes a docking site (D-recruitment site, DRS) on its targets using a conserved N-terminal sequence motif (D-motif). Using solution nuclear magnetic resonance spectroscopy and isothermal titration calorimetry, we compare, for the first time, the docking interactions of HePTP with ERK2 and p38α. Our results demonstrate that ERK2-HePTP interactions primarily involve the D-motif, while a contiguous region called the kinase specificity motif also plays a key role in p38α-HePTP interactions. D-Motif-DRS interactions for the two kinases, while similar overall, do show some specific differences.

Interaction of kinase-interaction-motif protein tyrosine phosphatases with the mitogen-activated protein kinase ERK2.

The mitogen-activation protein kinase ERK2 is tightly regulated by multiple phosphatases, including those of the kinase interaction motif (KIM) PTP family (STEP, PTPSL and HePTP). Here, we use small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to show that the ERK2:STEP complex is compact and that residues outside the canonical KIM motif of STEP contribute to ERK2 binding. Furthermore, we analyzed the interaction of PTPSL with ERK2 showing that residues outside of the canonical KIM motif also contribute to ERK2 binding. The integration of this work with previous studies provides a quantitative and structural map of how the members of a single family of regulators, the KIM-PTPs, differentially interact with their corresponding MAPKs, ERK2 and p38α.

Backbone and sidechain 1H, 15N and 13C assignments of Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa

The backbone and side chain resonance assignments of the Tyrosine Phosphatase related to Biofilm formation A (TpbA) of Pseudomonas aeruginosa have been determined based on triple-resonance experiments using uniformly [13C,15N]-labeled protein. This assignment is the first step towards the determination of the 3-dimensional structure of TpbA.

Backbone and side chain 1H, 15N and 13C assignments of the KSR1 CA1 domain.

The backbone and side chain resonance assignments of the murine KSR1 CA1 domain have been determined based on triple-resonance experiments using uniformly [13C, 15N]-labeled protein. This assignment is the first step towards the determination of the three-dimensional structure of the unique KSR1 CA1 domain.