Protein tyrosine phosphatase SHP2 functions as a key regulator of cell cycle control, and activating mutations cause several cancers. Here, we dissect the energy landscape of wild-type SHP2 and the oncogenic mutation E76K. NMR spectroscopy and X-ray crystallography reveal that wild-type SHP2 exchanges between closed, inactive and open, active conformations. E76K mutation shifts this equilibrium toward the open state. The previously unknown open conformation is characterized, including the active-site WPD loop in the inward and outward conformations. Binding of the allosteric inhibitor SHP099 to E76K mutant, despite much weaker, results in an identical structure as the wild-type complex. A conformational selection to the closed state reduces drug affinity which, combined with E76K’s much higher activity, demands significantly greater SHP099 concentrations to restore wild-type  activity levels. The differences in structural ensembles and drug-binding kinetics of cancer-associated SHP2 forms may stimulate innovative ideas for developing more potent inhibitors for activated SHP2 mutants.

Adaptive thermogenesis has attracted much attention because of its ability to raise systemic energy expenditure and counter obesity and diabetes 1,2,3 . Recent data have indicated that thermogenic fat cells utilize creatine to stimulate futile substrate cycling, dissipating chemical energy as heat 4,5 . This model was based on the super-stoichiometric relationship between creatine added to mitochondria and O 2 consumed. Here we provide direct evidence for the molecular basis of this futile creatine cycling (FCC) activity. Thermogenic fat cells contain robust phosphocreatine phosphatase activity, attributable to tissue-nonspecific alkaline phosphatase (TNAP). TNAP hydrolyzes phosphocreatine to initiate a futile cycle of creatine dephosphorylation and phosphorylation. Remarkably, unlike in other cells, TNAP is localized to mitochondria of thermogenic fat cells, where FCC occurs. TNAP expression is powerfully induced when animals are subjected to cold exposure. Moreover, the essential role of TNAP in the FCC is illustrated by the loss of this cycle when TNAP is inhibited in isolated mitochondria. Finally, genetic ablation of TNAP in adipocytes reduces whole body energy expenditure and causes rapid-onset obesity, with Reprints and permissions information is available at www.nature.com/reprints.

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Fibronectin is a modular extracellular matrix protein that is essential for vertebrate development. The 3rd type III domain (3FN3) in fibronectin interacts with other parts of fibronectin and with anastellin, a protein fragment that causes fibronectin aggregation. 3FN3 opens readily both as an isolated domain in solution and when part of fibronectin in stretched fibrils, and it was proposed that this opening is important for anastellin binding. We determined the structure of 3FN3 using nuclear magnetic resonance spectroscopy and we investigated its stability, folding and unfolding. Similar to most other FN3 domains, 3FN3 contains two antiparallel β-sheets that are composed of three (A, B and E) and four (C, D, F and G) β-strands, respectively, and are held together by a conserved hydrophobic interface. Cis-trans isomerization of P847 at the end of β-strand C leads to observable conformational heterogeneity in 3FN3, with a cis peptide bond present in almost one quarter of the molecules. The chemical stability of 3FN3 is relatively low, but the folding rate constant in the absence of denaturant is in the same range as those of other, more stable FN3 domains. Interestingly, the unfolding rate constant in the absence of denaturant is several orders of magnitude higher than the unfolding rate constants of other FN3 domains investigated to date. This unusually fast rate is comparable to the rate of 3FN3 binding to anastellin at saturating anastellin concentrations, consistent with the model that 3FN3 has to unfold in order to interact with anastellin.

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RIZ (retinoblastoma protein-interacting zinc finger protein), also denoted PRDM2, is a transcriptional regulator and tumor suppressor. It was initially identified because of its ability to interact with another well-established tumor suppressor, the retinoblastoma protein (Rb). A short motif, IRCDE, in the acidic region (AR) of RIZ was reported to play an important role in the interaction with the pocket domain of Rb. The IRCDE motif is similar to a consensus Rb-binding sequence LXCXE (where X denotes any amino acid) that is found in several viral Rb-inactivating oncoproteins. To improve our understanding of the molecular basis of binding of Rb to RIZ, we investigated the interaction between purified recombinant AR and the pocket domain of Rb using nuclear magnetic resonance spectroscopy, isothermal titration calorimetry, and fluorescence anisotropy experiments. We show that AR is intrinsically disordered and that it binds the pocket domain with submicromolar affinity. We also demonstrate that the interaction between AR and the pocket domain is mediated primarily by the short stretch of residues containing the IRCDE motif and that the contribution of other parts of AR to the interaction with the pocket domain is minimal. Overall, our data provide clear evidence that RIZ is one of the few cellular proteins that can interact directly with the LXCXE-binding cleft on Rb.

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