SummaryProtein tyrosine phosphatases (PTPs) play a critical role in regulating cellular functions by selectively dephosphorylating their substrates. Here we present 22 human PTP crystal structures that, together with prior structural knowledge, enable a comprehensive analysis of the classical PTP family. Despite their largely conserved fold, surface properties of PTPs are strikingly diverse. A potential secondary substrate-binding pocket is frequently found in phosphatases, and this has implications for both substrate recognition and development of selective inhibitors. Structural comparison identified four diverse catalytic loop (WPD) conformations and suggested a mechanism for loop closure. Enzymatic assays revealed vast differences in PTP catalytic activity and identified PTPD1, PTPD2, and HDPTP as catalytically inert protein phosphatases. We propose a “head-to-toe” dimerization model for RPTPγ/ζ that is distinct from the “inhibitory wedge” model and that provides a molecular basis for inhibitory regulation. This phosphatome resource gives an expanded insight into intrafamily PTP diversity, catalytic activity, substrate recognition, and autoregulatory self-association.

Sphingosine 1-phosphate (S1P) is one of several bioactive phospholipids that exert profound mitogenic and morphogenic actions. Originally characterized as a second messenger, S1P is now recognized to achieve many of its effects through cell surface, G protein-coupled receptors. We used a subunit-selective [35 S]GTP␥S binding assay to investigate whether the variety of actions exerted through Edg-1, a recently identified receptor for S1P, might be achieved through multiple G proteins. We found, employing both Sf9 and HEK293 cells, that Edg-1 activates only members of the G i family, and not G s , G q , G 12 , or G 13 . We additionally established that Edg-1 activates G i in response not only to S1P but also sphingosylphosphorylcholine; no effects of lysophosphatidic acid through Edg-1 were evident. Our assays further revealed a receptor(s) for S1P endogenous to HEK293 cells that mediates activation of G 13 as well as G i . Because several of the biological actions of S1P are assumed to proceed through the G 12/13 family, we tested whether Edg-3 and H218/Edg-5, two other receptors for S1P, might have a broader coupling profile than Edg-1. Indeed, Edg-3 and H218/Edg-5 communicate not only with G i but also with G q and G 13 . These studies represent the first characterization of S1P receptor activity through G proteins directly and establish fundamental differences in coupling.Sphingolipid metabolites, including sphingosine 1-phosphate (S1P), 1 regulate many aspects of cell growth and differentiation. S1P is a mitogen (1-4), opposes ceramide-induced apoptosis (5, 6), inhibits cell motility (2, 7), activates platelets (8), and causes retraction of neurites (9, 10). S1P also elicits diverse biochemical responses, including activation of mitogenactivated protein (MAP) kinases (2, 11), phospholipase C (12-14), phospholipase D (15-17), and I k(ACh) (18,19), inhibition of adenylyl cyclase (16,19), and mobilization of Ca 2ϩ (1, 12, 20 -24).Although postulated to function as an intracellular messenger in some cases (21,22,25,26), S1P exerts many of its effects through cell surface receptors (9,11,16,18,19). Recently, the former orphan receptor Edg-1 (endothelial differentiation gene 1) was identified as a high affinity receptor for S1P (27)(28)(29). In cells overexpressing Edg-1, S1P promotes activation of MAP kinase and inhibition of adenylyl cyclase (28,30,31). Additionally, S1P causes a mobilization of calcium in Chinese hamster ovary cells, although not in Sf9, HEK293, or COS-7 cells, overexpressing Edg-1 (29 -31). The sensitivity of these S1P/Edg-1-induced responses to a pertussis toxin (PTX) indicates that members of the G i family of G proteins mediate them (28 -32). In addition to the functional coupling of Edg-1 with G i proteins, physical interaction of Edg-1 with members of this family has been demonstrated. In transfected HEK293 cells, all four PTXsensitive G ␣ subunits, ␣ i1 , ␣ i2 , ␣ i3 , and ␣ o , associate with the third intracellular loop of Edg-1 in a GTP␥S-sensitive manner, and ␣ i1 and ␣ i3 co-immunop...

ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an openinwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.ABC mitochondrial erythroid | X-ray crystallography | human membrane protein structure | nucleotide complex | cardiolipin