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Variant rs351855-G/A is a commonly occurring single-nucleotide polymorphism of coding regions in exon 9 of the fibroblast growth factor receptor FGFR4 (CD334) gene (c.1162G>A). It results in an amino-acid change at codon 388 from glycine to arginine (p.Gly388Arg) in the transmembrane domain of the receptor. Despite compelling genetic evidence for the association of this common variant with cancers of the bone (1), breast (2), colon (3), prostate (4,5), skin (6), lung (7,8), head and neck (9), as well as soft-tissue sarcomas and non-Hodgkin lymphoma, the underlying biological mechanism has remained elusive. Here we show that substitution of the conserved glycine 388 residue to a charged arginine residue alters the transmembrane spanning segment and exposes a membrane-proximal cytoplasmic signal transducer and activator of transcription 3 (STAT3) binding site [Y.sup.390]-(P)XXQ (393). We demonstrate that such membrane-proximal STAT3 binding motifs in the germline of type I membrane receptors enhance STAT3 tyrosine phosphorylation by recruiting STAT3 proteins to the inner cell membrane. Remarkably, such germline variants frequently co-localize with somatic mutations in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Using Fgfr4 single nucleotide polymorphism knock-in mice and transgenic mouse models for breast and lung cancers, we validate the enhanced STAT3 signalling induced by the FGFR4 Arg388-variant in vivo. Thus, our findings elucidate the molecular mechanism behind the genetic association of rs351855 with accelerated cancer progression and suggest that germline variants of cell-surface molecules that recruit STAT3 to the inner cell membrane are a significant risk for cancer prognosis and disease progression., We previously reported that the FGFR4 Arg388 allele (rs351855-A) located in exon 9 of the FGFR4 gene (Extended Data Fig. 1a) is associated with cancer progression and poor prognosis (6,7,9,10). [...]

Bile acids are synthesized from cholesterol in hepatocytes and secreted through the biliary tract into the small intestine, where they aid in absorption of lipids and fat-soluble vitamins. Through a process known as enterohepatic recirculation, more than 90% of secreted bile acids are then retrieved from the intestine and returned to the liver for resecretion (1). In humans, there are two [Na.sup.+]-dependent bile acid transporters involved in enterohepatic recirculation, the [Na.sup.+]-taurocholate co-transporting polypeptide (NTCP; also known as SLC10A1) expressed in hepatocytes, and the apical sodium-dependent bile acid transporter (ASBT; also known as SLC10A2) expressed on enterocytes in the terminal ileum (2). In recent years, ASBT has attracted much interest as a potential drug target for treatment of hypercholesterolaemia, because inhibition of ASBT reduces reabsorption of bile acids, thus increasing bile acid synthesis and consequently cholesterol consumption (3,4). However, a lack of three-dimensional structures of bile acid transporters hampers our ability to understand the molecular mechanisms of substrate selectivity and transport, and to interpret the wealth of existing functional data (2,5-8). The crystal structure of an ASBT homologue from Neisseria meningitidis ([ASBT.sub.NM]) in detergent was reported recently (9), showing the protein in an inward-open conformation bound to two [Na.sup.+] and a taurocholic acid. However, the structural changes that bring bile acid and [Na.sup.+] across the membrane are difficult to infer from a single structure. To understand the structural changes associated with the coupled transport of [Na.sup.+] and bile acids, here we solved two structures of an ASBT homologue from Yersinia frederiksenii ([ASBT.sub.Yf]) in a lipid environment, which reveal that a large rigid-body rotation of a substrate-binding domain gives the conserved 'crossover' region, where two discontinuous helices cross each other, alternating accessibility from either side of the cell membrane. This result has implications for the location and orientation of the bile acid during transport, as well as for the translocation pathway for [Na.sup.+]., Purified [ASBT.sub.Yf], when reconstituted into liposomes, mediates [Na.sup.+]-dependent transport of the conjugated bile acid taurocholic acid (TCA) with an apparent Michaelis constant ([K.sub.m]) of 46.8 6 7.4 µM (Fig. 1a, [...]

Voltage-gated calcium ([Ca.sub.V]) channels catalyse rapid, highly selective influx of [Ca.sup.2+] into cells despite a 70-fold higher extracellular concentration of [Na.sup.+] .How [Ca.sub.V] channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial [Na.sub.V] channel [Na.sub.V]Ab. Our results reveal interactions of hydrated [Ca.sup.2+] with two high-affinity [Ca.sup.2+] -binding sites followed by a third lower-affinity site that would coordinate [Ca.sup.2+] as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining [Ca.sup.2+] selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations [Cd.sup.2+] and [Mn.sup.2+], with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated [Ca.sup.2+] ions bound in the selectivity filter and supports a 'knock-off' mechanism of ion permeation through a stepwisebinding process. The multi-ion selectivity filter of our [Ca.sub.V]Ab model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate [Ca.sub.V] channels., [Ca.sup.2+] ions flow through [Ca.sub.V] channels at a rate of ~[10.sup.6] ions [s.sup.-1], yet [Na.sup.+] conductance is >500-fold lower (1). Such high-fidelity, high-throughput [Ca.sub.V] channel performance is important in regulating [...]

The contraction and relaxation of muscle cells is controlled by the successive rise and fall of cytosolic [Ca.sup.2+], initiated by the release of [Ca.sup.2+] from the sarcoplasmic reticulum and terminated by re-sequestration of [Ca.sup.2+] into the sarcoplasmic reticulum as the main mechanism of [Ca.sup.2+] removal. Re-sequestration requires active transport and is catalysed by the sarcoplasmic reticulum [Ca.sup.2+]-ATPase (SERCA), which has a key role in defining the contractile properties of skeletal and heart muscle tissue. The activity of SERCA is regulated by two small, homologous membrane proteins called phospholamban (PLB, also known as PLN) and sarcolipin (SLN) (1, 2). Detailed structural information explaining this regulatory mechanism has been lacking, and the structural features defining the pathway through which cytoplasmic [Ca.sup.2+] enters the intramembranous binding sites of SERCA have remained unknown. Here we report the crystal structure of rabbit SERCA1a (also known as ATP2A1) in complex with SLN at 3.1 Å resolution. The regulatory SLN traps the [Ca.sup.2+]-ATPase in a previously undescribed E1 state, with exposure of the [Ca.sup.2+] sites through an open cytoplasmic pathway stabilized by [Mg.sup.2+]. The structure suggests a mechanism for selective [Ca.sup.2+] loading and activation of SERCA, and provides new insight into how SLN and PLB inhibition arises from stabilization of this E1 intermediate state without bound [Ca.sup.2+]. These findings may prove useful in studying how autoinhibitory domains of other ion pumps modulate transport across biological membranes., The regulation of SERCA by SLN and PLB is a key determinant of the contractile properties of both skeletal and heart muscle tissue. In humans, SLN is primarily expressed in [...]

P-type ATPases are ATP-powered ion pumps that establish ion concentration gradients across biological membranes, and are distinct from other ATPases in that the reaction cycle includes an autophosphorylation step. The best studied is [Ca.sup.2+]-ATPase from muscle sarcoplasmic reticulum (SERCA1a), a [Ca.sup.2+] pump that relaxes muscle cells after contraction, and crystal structures have been determined for most of the reaction intermediates (1,2). An important outstanding structure is that of the E1 intermediate, which has empty high-affinity [Ca.sup.2+]-binding sites ready to accept new cytosolic [Ca.sup.2+]. In the absence of [Ca.sup.2+] and at pH 7 or higher, the ATPase is predominantly in E1, not in E2 (low affinity for [Ca.sup.2+]) (3), and if millimolar [Mg.sup.2+] is present, one [Mg.sup.2+] is expected to occupy one of the [Ca.sup.2+]-binding sites with a millimolar dissociation constant (4,5). This [Mg.sup.2+] accelerates the reaction cycle (4), not permitting phosphorylation without [Ca.sup.2+] binding. Here we describe the crystal structure of native SERCA1a (from rabbit) in this E1 x [Mg.sup.2+] state at 3.0 [Anstrom] resolution in addition to crystal structures of SERCA1a in E2 free from exogenous inhibitors, and address the structural basis of the activation signal for phosphoryl transfer. Unexpectedly, sarcolipin (6), a small regulatory membrane protein of [Ca.sup.2+]-ATPase (7), is bound, stabilizing the E1 x [Mg.sup.2+] state. Sarcolipin is a close homologue of phospholamban, which is a critical mediator of β-adrenergic signal in [Ca.sup.2+] regulation in heart (for reviews, see, for example, refs 8-10), and seems to play an important role in muscle-based thermogenesis (11). We also determined the crystal structure of recombinant SERCA1a devoid of sarcolipin, and describe the structural basis of inhibition by sarcolipin/phospholamban. Thus, the crystal structures reported here fill a gap in the structural elucidation of the reaction cycle and provide a solid basis for understanding the physiological regulation of the calcium pump., New E2 (Supplementary Fig. 1) crystals of SERCAla free from exogenous inhibitors were grown in either 40 mM MgS[O.sub.4] (E2 x S[O.sub.4.sup.2-] crystals) or Mg[Cl.sub.2] (E2 crystals) at pH 6.5 [...]

Invertases are highly regulated enzymes with essential functions in carbohydrate partitioning, sugar signaling, and plant development. Here we present the 2.6 A crystal structure of Arabidopsis cell-wall invertase 1 (INV1) in complex with a protein inhibitor (CIF, or cell-wall inhibitor of [beta]-fructosidase) from tobacco. The structure identifies a small amino acid motif in CIF that directly targets the invertase active site. The activity of INV1 and its interaction with CIF are strictly pH-dependent with a maximum at about pH 4.5. At this pH, isothermal titration calorimetry reveals that CIF tightly binds its target with nanomolar affinity. CIF competes with sucrose (Suc) for the same binding site, suggesting that both the extracellular Suc concentration and the pH changes regulate association of the complex. A conserved glutamate residue in the complex interface was previously identified as an important quantitative trait locus affecting fruit quality, which implicates the invertase-inhibitor complex as a main regulator of carbon partitioning in plants. Comparison of the CIF/INV1 structure with the complex between the structurally CIF-related pectin methylesterase inhibitor (PMEI) and pectin methylesterase indicates a common targeting mechanism in PMEI and CIF. However, CIF and PMEI use distinct surface areas to selectively inhibit very different enzymatic scaffolds. active site | crystallography | post-translational enzyme regulation | protein complex | sugar metabolism doi/ 10.1073/pnas.1004481107

With the advent of Systems Biology, the prediction of whether two proteins form a complex has become a problem of increased importance. A variety of experimental techniques have been applied to the problem, but three-dimensional structural information has not been widely exploited. Here we explore the range of applicability of such information by analyzing the extent to which the location of binding sites on protein surfaces is conserved among structural neighbors. We find, as expected, that interface conservation is most significant among proteins that have a clear evolutionary relationship, but that there is a significant level of conservation even among remote structural neighbors. This finding is consistent with recent evidence that information available from structural neighbors, independent of classification, should be exploited in the search for functional insights. The value of such structural information is highlighted through the development of a new protein interface prediction method, PredUs, that identifies what residues on protein surfaces are likely to participate in complexes with other proteins. The performance of PredUs, as measured through comparisons with other methods, suggests that relationships across protein structure space can be successfully exploited in the prediction of protein-protein interactions. doi/ 10.1073/pnas.1005894107

Inositol phosphates (InsPs) are signaling molecules with multiple roles in cells. In particular Ins(1,2,3,4,5,6)[P.sub.6] ([InsP.sub.6]) is involved in mRNA export and editing or chromatin remodeling among other events. [InsP.sub.6] accumulates as mixed salts (phytate) in storage tissues of plants and plays a key role in their physiology. Human diets that are exclusively grain-based provide an excess of [InsP.sub.6] that, through chelation of metal ions, may have a detrimental effect on human health. Ins (1,3,4,5,6)[P.sub.5] 2-kinase ([InsP.sub.5] 2-kinase or Ipk1) catalyses the synthesis of [InsP.sub.6] from [InsP.sub.5] and ATP, and is the only enzyme that transfers a phosphate group to the axial 2-OH of the myo-inositide. We present the first structure for an [InsP.sub.5] 2-kinase in complex with both substrates and products. This enzyme presents a singular structural region for inositide binding that encompasses almost half of the protein. The key residues in substrate binding are identified, with Asp368 being responsible for recognition of the axial 2-OH. This study sheds light on the unique molecular mechanism for the synthesis of the precursor of inositol pyrophosphates. [InsP.sub.5] 2-kinase | IP5 | IP6 | ipkl | crystal structure www.pnas.org/cgi/doi/10.1073/pnas.0912979107

Recent studies suggest that there are many nonfunctional transcription factor binding sites along a genome. Although these 'decoy' sites compete with the promoter region for binding of transcription factors, they may also protect these proteins from degradation. We show that in the limit of perfect protection, where bound transcription factors are never degraded, the competitive effect of nonfunctional binding sites is completely canceled out by the stability gained from reduced degradation. We examine the response of an autoregulated gene to the total number of transcription factors to quantify the consequences of competition for transcription factors. We show that intuition about this system can be gained by mathematically constructing a single gene with effective parameters that reproduce the behavior of a gene with added decoy sites. In analogy to dressed particles in many-body systems we term this description a 'quasi gene.' We find that protective decoys buffer against noise by reducing correlations between transcription factors, specifically in the case of production of transcription factors in bursts. We show that the addition of protective decoy sites causes the level of gene expression to approach that predicted from deterministic mass action models. Finally, we show that protective decoy sites decrease the size of the region of parameter space that exhibits bistability. autoregulation | noise | stochastic gene expression | nonfunctional binding site doi/10.1073/pnas.0915138107

Crystallographic analysis revealed that the 17-member polyketide antibiotic lankacidin produced by Streptomyces rochei binds at the peptidyl transferase center of the eubacterial large ribosomal subunit. Biochemical and functional studies verified this finding and showed interference with peptide bond formation. Chemical probing indicated that the macrolide lankamycin, a second antibiotic produced by the same species, binds at a neighboring site, at the ribosome exit tunnel. These two antibiotics can bind to the ribosome simultaneously and display synergy in inhibiting bacterial growth. The binding site of lankacidin and lankamycin partially overlap with the binding site of another pair of synergistic antibiotics, the streptogramins. Thus, at least two pairs of structurally dissimilar compounds have been selected in the course of evolution to act synergistically by targeting neighboring sites in the ribosome. These results underscore the importance of the corresponding ribosomal sites for development of clinically relevant synergistic antibiotics and demonstrate the utility of structural analysis for providing new directions for drug discovery. doi/10.1073/pnas.0914100107

The catalytic importance of enzyme active-site interactions is frequently assessed by mutating specific residues and measuring the resulting rate reductions. This approach has been used in bacterial ketosteroid isomerase to probe the energetic importance of active-site hydrogen bonds donated to the dienolate reaction intermediate. The conservative Tyr16Phe mutation impairs catalysis by [10.sup.5]-fold, far larger than the effects of hydrogen bond mutations in other enzymes. However, the less-conservative Tyr16Ser mutation, which also perturbs the Tyr16 hydrogen bond, results in a lesssevere 102-fold rate reduction. To understand the paradoxical effects of these mutations and clarify the energetic importance of the Tyr16 hydrogen bond, we have determined the 1.6-A resolution x-ray structure of the intermediate analogue, equilenin, bound to the Tyr16Ser mutant and measured the rate effects of mutating Tyr16 to Ser, Thr, Ala, and Gly. The nearly identical 200-fold rate reductions of these mutations, together with the 6.4-A distance observed between the Ser16 hydroxyl and equilenin oxygens in the x-ray structure, strongly suggest that the more moderate rate effect of this mutant is not due to maintenance of a hydrogen bond from Ser at position 16. These results, additional spectroscopic observations, and prior structural studies suggest that the Tyr16Phe mutation results in unfavorable interactions with the dienolate intermediate beyond loss of a hydrogen bond, thereby exaggerating the apparent energetic benefit of the Tyr16 hydrogen bond relative to the solution reaction. These results underscore the complex energetics of hydrogen bonding interactions and site-directed mutagenesis experiments. doi/10.10731pnas.0911168107

p53 is a master regulatory, sequence-specific transcription factor that directly controls expression of over 100 genes in response to various stress signals. Transactivation is generally considered to occur through p53 binding to a consensus response element (RE) composed of two 5'-RRRCWWGYYY-3' decamers. Recently, studying the human angiogenesis-related gene FLT1 we discovered that p53 can mediate limited transactivation at a noncanonical 1/2 site and could synergize with the estrogen receptor (ER) acting in cis at a nearby ER 1/2 site. To address the generality of concerted transactivation by p53 and ER, the 1/2 site in the FLT1 promoter was replaced with a variety of 1/2 sites, as well as canonical weak and strong p53 REs of human target genes. The p53 transactivation of all tested sequences was greatly enhanced by ligand-activated ER acting in cis. Furthermore, enhanced transactivation extends to several cancer-associated p53 mutants with altered function, suggesting ER-dependent mutant p53 activity for at least some REs. The enhanced transactivation was also found with p63 and p73. We propose a general synergistic relationship between p53 family and ER master regulators in transactivation of p53 target canonical and noncanonical REs, which might be poorly responsive to p53 on their own. This relationship greatly expands the transcriptional master network regulated by p53 in terms of genes affected and levels of expression and has implications for the appearance and possible treatments of cancer. FLT1 | noncanonical response element | half-site | transcriptional synergy doi/ 10.1073/pnas.0909129107

The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping. EPR | nitroxides | T4 lysozyme doi/10.1073/pnas.0912009106

The outer membrane protein OprP from Pseudomonas aeruginosa forms a phosphate selective pore. To understand the mechanism of phosphate permeation and selectivity, we used three simulation techniques [equilibrium molecular dynamics simulations, steered molecular dynamics, and calculation of a potential of mean force (PMF)]. The PMF for phosphate reveals a deep free energy well midway along the OprP channel. Two adjacent phosphate-binding sites (W1 and W2), each with a well depth of [approximately equal to] 8 kT, are identified close to the L3 loop in the most constricted region of the pore. A dissociation constant for phosphate of 6 [micro]M is computed from the PMF, within the range of reported experimental values. The transfer of phosphate between sites W1 and W2 is correlated with changes in conformation of the sidechain of K121, which serves as a 'charged brush' to facilitate phosphate passage between the two subsites. OprP also binds chloride, but less strongly than phosphate, as calculated from a [Cl.sup.-] PMF. The difference in affinity and hence selectivity is due to the 'Lys-cluster' motif, the positive charges of which interact strongly with a partially dehydrated phosphate ion but are shielded from a [Cl.sup.-] by the hydration shell of the smaller ion. Our simulations suggest that OprP does not conform to the conventional picture of a channel with relatively flat energy landscape for permeant ions, but rather resembles a membrane-inserted binding protein with a high specificity that allows access to a centrally located binding site from both the extracellular and the periplasmic spaces. free energy profile | potential of mean force | anion selectivity | hydration | molecular dynamics simulation doi/10.1073/pnas.0907315106

The site on HIV-1 gp120 that binds to the CD4 receptor is vulnerable to antibodies. However, most antibodies that interact with this site cannot neutralize HIV-1. To understand the basis of this resistance, we determined co-crystal structures for two poorly neutralizing, CD4-binding site (CD4BS) antibodies, F105 and b13, in complexes with gp120. Both antibodies exhibited approach angles to gp120 similar to those of CD4 and a rare, broadly neutralizing CD4BS antibody, b12. Slight differences in recognition, however, resulted in substantial differences in F105- and b13-bound conformations relative to b12-bound gp120. Modeling and binding experiments revealed these conformations to be poorly compatible with the viral spike. This incompatibility, the consequence of slight differences in CD4BS recognition, renders HIV-1 resistant to all but the most accurately targeted antibodies. 5 May 2009; accepted 10 September 2009 10.1126/science.1175868

Rather than maximizing toughness, as needed for silk and muscle titin fibers to withstand external impact, the much softer extracellular matrix fibers made from fibronectin (Fn) can be stretched by cell generated forces and display extraordinary extensibility. We show that Fn fibers can be extended more than 8-fold (>700% strain) before 50% of the fibers break. The Young's modulus of single fibers, given by the highly nonlinear slope of the stress-strain curve, changes orders of magnitude, up to MPa. Although many other materials plastically deform before they rupture, evidence is provided that the reversible breakage of force-bearing backbone hydrogen bonds enables the large strain. When tension is released, the nano-sized Fn domains first contract in the crowded environment of fibers within seconds into random coil conformations (molten globule states), before the force-bearing hydrogen bond networks that stabilize the domain's secondary structures are reestablished within minutes (double exponential). The exposure of cryptic binding sites on Fn type III modules increases steeply upon stretching. Thus fiber extension steadily up-regulates fiber rigidity and cryptic epitope exposure, both of which are known to differentially alter cell behavior. Finally, since stress-strain relationships cannot directly be measured in native extracellular matrix (ECM), the stress-strain curves were correlated with stretch-induced alterations of intramolecular fluorescence resonance energy transfer (FRET) obtained from trace amounts of Fn probes (mechanical strain sensors) that can be incorporated into native ECM. Physiological implications of the extraordinary extensibility of Fn fibers and contraction kinetics are discussed. fibrillogenesis | matrix biology | mechanotransduction | multimodular proteins | supramolecular assembly doi/10.1073/pnas.0907518106

Crystallography and computer modeling have been used to exploit a previously unexplored channel in the glucocorticoid receptor (GR). Highly potent, nonsteroidal indazole amides showing excellent complementarity to the channel were designed with the assistance of the computational technique AlleGrow. The accuracy of the design process was demonstrated through crystallographic structural determination of the GR ligand-binding domain--agonist complex of the D-prolinamide derivative 11. The utility of the channel was further exemplified through the design of a potent phenylindazole in which structural motifs, seen to interact with the traditional GR ligand pocket, were abandoned and replaced by interactions within the new channel. Occupation of the channel was confirmed with a second GR crystal structure of this truncated D-alaninamide derivative 13. Compound 11 displays properties compatible with development as an intranasal solution formulation, whereas oral bioavailability has been demonstrated with a related truncated exemplar 14. Data with the pyrrolidinone amide 12 demonstrate the potential for further elaboration within the 'meta' channel to deliver compounds with selectivity for the desired transrepressive activity of glucocorticoids. The discovery of these interactions with this important receptor offers significant opportunities for the design of novel GR modulators. drug design | AlleGrow | glucocorticoid modulation | intranasal glucocorticoid doi/10.1073/pnas.0909125106

To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Sacchoromyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, dusters of sites shift position in rapidly evolving disordered regions. We propose that the regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase-signaling circuits.

Protein O-GIcNAcylation occurs in all animals and plants and is implicated in modulation of a wide range of cytosolic and nuclear protein functions, including gene silencing, nutrient and stress sensing, phosphorylation signaling, and diseases such as diabetes and Alzheimer's. The limiting factor impeding rapid progress in deciphering the biological functions of protein O-GIcNAcylation has been the inability to easily identify exact residues of modification. We describe a robust, high-sensitivity strategy able to assign O-GIcNAcylation sites of native modified peptides using electron transfer dissociation mass spectrometry. We have studied the murine postsynaptic density pseudoorganelle and report the assignment of 58 modification sites from a single experiment--significantly increasing the number of sites known in the literature. Components of several repressor complexes, such as NCoR1, polyhomeotic-like protein3, and EMSY, are modified. In addition, 28 O-GIcNAc sites were found on the protein Bassoon, effectively matching the number of phosphorylation sites reported previously on this protein. This finding suggests that on certain proteins, O-GIcNAcylation may be as extensive and important as phosphorylation in regulating protein function. Three of the newly discovered O-GIcNAc sites on Bassoon have previously been reported as phosphorylation sites, highlighting the interplay of the modifications. Surprisingly, several peptides with GIcNAc modifications on asparagines within the N-X-S/T consensus sequence were also observed from membrane protein extracellular domains. This powerful strategy fulfills a long-standing need in the biological community by facilitating modification site identifications that will accelerate understanding of the biological significance of this elusive regulatory posttranslational modification. modification site assignment | posttranslational modification | proteomics | Bassoon

The interaction of the antitumor drug cisplatin with insulin was studied using a top-down mass spectrometric approach. In vitro incubations were prepared under acidic and physiological conditions at different insulin/cisplatin molar ratios for different incubation times. Size exclusion chromatography--inductively coupled plasma mass spectrometry (SEC--ICPMS) analysis enabled the specific detection of platinum containing species attributed to the binding of the drug to the protein. Further analysis through matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanoelectrospray ionization mass spectrometry using a linear ion trap (nESI-LIT-MS) allowed the identification of platinated mono-, di-, and even triadducts in the incubations. Platinum binding sites were identified by [CID-MS.sup.n] as B chain N-terminus, His5, and probably His10 residues, which turned out to be the same, regardless of the incubation conditions. Evidence on the binding of Pt to B chain Cys7 was also observed. Working with the LIT zoom scan mode provides enough resolution to discern the isotopic pattern for both precursor and fragment ions, allowing the differentiation of platinum-containing ions. The elucidation of platinum binding sites in a native protein through a top-down approach has been performed for the first time with this type of instrument.

We present a fractional sensitivity analysis of a competitive microfluidic heterogeneous immunoassay for a small molecule analyte. A simple two-dimensional finite element model is used to determine the fractional sensitivity of the assay signal with respect to analyte concentration, flow rate, initial surface density of binding sites, and antibody concentration. The fractional sensitivity analysis can be used to identify (1) the system parameters for which it is most crucial to control or quantify the variability between assays and (2) operating ranges for these parameters that improve assay sensitivity (within the constraints of the experimental system). Experimental assay results for the drug phenytoin, obtained using surface plasmon resonance imaging, are shown to be consistent with the predictions of the model.

Genes are not simply turned on or off, but instead their expression is fine-tuned to meet the needs of a cell. How genes are modulated so precisely is not well understood. The glucocorticoid receptor (GR) regulates target genes by associating with specific DNA binding sites, the sequences of which differ between genes. Traditionally, these binding sites have been viewed only as docking sites. Using structural, biochemical, and cell-based assays, we show that GR binding sequences, differing by as little as a single base pair, differentially affect GR conformation and regulatory activity. We therefore propose that DNA is a sequence-specific allosteric ligand of GR that tailors the activity of the receptor toward specific target genes.

Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78-Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains. crystallography | endoplasmic reticulum-associated protein degradation | gp78 | polyubiquitin chain

Two new crystal structures of Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford evidence that a closed interdomain hinge angle requires a covalent bond between AMP and an occupant of either pyrophosphate or tryptophan subsite. They also are within experimental error of a cluster of structures observed in a nonequilibrium molecular dynamics simulation showing partial active-site assembly. Further, the highest energy structure in a minimum action pathway computed by using elastic network models for Open and Pretransition state (PreTS) conformations for the fully liganded TrpRS monomer is intermediate between that simulated structure and a partially disassembled structure from a nonequilibrium molecular dynamics trajectory for the unliganded PreTS. These mutual consistencies provide unexpected validation of inferences drawn from molecular simulations. conformational transition state | domain movement | induced fit | minimum action pathway | nonequilibrium molecular dynamics

Results of Nowak and collaborators concerning the onset of cancer due to the inactivation of tumor suppressor genes give the distribution of the time until some individual in a population has experienced two prespecified mutations and the time until this mutant phenotype becomes fixed in the population. In this article we apply these results to obtain insights into regulatory sequence evolution in Drosophila and humans. In particular, we examine the waiting time for a pair of mutations, the first of which inactivates an existing transcription factor binding site and the second of which creates a new one. Consistent with recent experimental observations for Drosophila, we find that a few million years is sufficient, but for humans with a much smaller effective population size, this type of change would take >100 million years. In addition, we use these results to expose flaws in some of Michael Behe's arguments concerning mathematical limits to Darwinian evolution.

The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDI[alpha] showed two potential protein kinase A (PKA) phosphorylation motifs, [Ser.sup.174] and [Thr.sup.182]. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDI[alpha]-GST fusion protein and PKA motif-containing GDI[alpha] peptide at [Ser.sup.174], but not [Thr.sup.182]. Transfection of COS-7 cells with mutated full-length GDI[alpha] at [Ser.sup.174] to [Ala.sup.174] (GDI[alpha]Ser.sup.174A]) abrogated the ability of cAMP to phosphorylate GDIct. However, mutation of [Thr.sup.182] to [Ala.sup.182] (GDI[alpha]-[Thr.sup.182]) did not abrogate, and cAMP increased phosphorylation of GDI[alpha] to a similar extent as wild-type GDI[alpha] transfectants. The mutant GDI[alpha]-[Ser.sup.174A], but not GDI[alpha][Thr.sup. 182] was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDI[alpha]-[Ser.sup.174A] was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIct binding with RhoA, suggesting that phosphorylation of [Ser.sup.174] regulated preformed GDI[alpha]-RhoA complexes. The findings provide strong support that the selective phosphorylation at [Ser.sup.174] by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury. adenosine 3',5'-cyclic monophosphate-dependent protein kinase; protein kinase A consensus phosphorylation sites; single-site mutated GDI[alpha]

JNK is a stress-activated protein kinase that modulates pathways implicated in a variety of disease states. JNK-interacting protein-1 (JIP1) is a scaffolding protein that enhances JNK signaling by creating a proximity effect between JNK and upstream kinases. A minimal peptide region derived from JIP1 is able to inhibit JNK activity both in vitro and in cell. We report here a series of small molecules JIP1 mimics that function as substrate competitive inhibitors of JNK. One such compound, BI-78D3, dose-dependently inhibits the phosphorylation of JNK substrates both in vitro and in cell. In animal studies, BI-78D3 not only blocks JNK dependent Con A-induced liver damage but also restores insulin sensitivity in mouse models of type 2 diabetes. Our findings open the way for the development of protein kinase inhibitors targeting substrate specific docking sites, rather than the highly conserved ATP binding sites. In view of its favorable inhibition profile, selectivity, and ability to function in the cellular milieu and in vivo, BI-78D3 represents not only a JNK inhibitor, but also a promising stepping stone toward the development of an innovative class of therapeutics. diabetes | drug discovery | JIP1 | kinase | NMR

In many taxa, males and females differ with respect to their sex chromosomes, and dosage compensation mechanisms have evolved to equalize X-linked gene transcription. In Drosophila, the male-specific lethal (MSL) complex binds to hundreds of sites along the male X chromosome and mediates twofold hyper-transcription of the single male X. Two recent studies found evidence for lineage-specific adaptive evolution in all five core protein-coding genes of the MSL complex in Drosophila melanogaster. In particular, dramatic positive selection was detected in domains shown to be responsible for their specific targeting to the X chromosome. Here I use population genetics to show that three previously characterized MSL-binding DNA segments on the X themselves underwent adaptive evolution in D. melanogaster, but not in its close relatives D. simulans and D. yakuba. MSL components have been shown to not correctly target the D. melanogaster X chromosome in hybrids between D. melanogaster and D. simulans. My finding supports the idea of selection-driven coevolution among DNA-protein interactions of the dosage compensation machinery and suggests that misregulated dosage compensation could contribute to male hybrid inviability in Drosophila.

Single-nucleotide polymorphisms (SNPs) in the human ether-a-go-go-related gene 1, hERG1, are associated with cardiac arrhythmias. The Kv11.1 channels encoded by hERG1 are also essential for rhythmic excitability of the pituitary, where they are regulated by thyroid hormone through a signal transduction cascade involving the phosphatidylinositol 3-kinase (PI3K) and the Ser/Thr-directed protein phosphatase, PP5. Here, we show that the hERG1 polymorphism at codon 897, which is read as a Thr instead of a Lys, creates a phosphorylation site for the Akt protein kinase on the Kv11.1 channel protein. Consequently, hormonal signaling through the PI3K signaling cascade, which normally stimulates K897 channels through PP5-mediated dephosphorylation, inhibits T897 channels through Akt-mediated phosphorylation. Thus, hormonal regulation of Kv11.1 in humans with the T897 polymorphism is predicted to prolong the QT interval of cardiac myocytes. A systematic bioinformatics search for SNPs in human ion channel genes identified 15 additional candidates for such 'phosphorylopathies,' which are predicted to create or destroy putative phosphorylation sites. Changes in protein phosphorylation might represent a general mechanism for the interaction of genetic variation and environment on human health. Kv11.1 | LQT | Akt protein kinase | phosphatidylinositol 3-kinase | thyroid hormone

Positive allosteric modulators of [alpha]7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of [alpha]7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of [alpha]7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of [alpha]7 nAChRs. The amino acids we have identified are located within the [alpha]-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of [EC.sub.20] concentrations of acetylcholine to a tenth of the level seen with wild-type [alpha]7. Reference to homology models of the [alpha]7 nAChR, based on the 4[Angstrom] structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four [alpha]-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with [GABA.sub.A] and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels. allosteric modulators | neurotransmitter receptor

The parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R) belongs to family B of seven-transmembrane-spanning receptors and is activated by PTH and PTHrP. Upon PTH stimulation, the rat PTH1R becomes phosphorylated at seven serine residues. Elimination of all PTH1R phosphorylation sites results in prolonged cAMP accumulation and impaired internalization in stably transfected LLC-PK1 cells. The present study explores the role of individual PTH1R phosphorylation sites in PTH1R signaling through phospholipase C, agonist-dependent receptor internalization, and regulation by G protein-coupled receptor kinases. By means of transiently transfected COS-7 cells, we demonstrate that the phosphorylation-deficient (pd) PTH1R confers dramatically enhanced coupling to Gq/11 proteins upon PTH stimulation predominantly caused by elimination of [Ser.sup.491/492/493], [Ser.sup.501], or [Ser.sup.504]. Reportedly, impaired internalization of the pd PTH1R, however, is not dependent on a specific phosphorylation site. In addition, we show that G protein-coupled receptor kinase 2 interferes with pd PTH1R signaling to [G.sub.q/11] proteins at least partially by direct binding to [G.sub.q/11] proteins. G protein-coupled receptor kinases; phosphorylation

Under hypoxic conditions or upon exposure to low concentrations of nitric oxide, DevR transcriptional regulator mediates the activation of ~50 genes that are believed to assist in dormancy development in Mycobacterium tuberculosis. Most of the strongly induced genes are characterized by the presence of one to four copies of a Dev box-like sequence at an upstream location. Among these are several gene pairs that are transcribed in opposite directions. This arrangement could provide for coordinated control of the adjacent genes under inducing conditions. In this work, we report the first detailed analysis of DevR-mediated hypoxic regulation of narK2-Rv1738 genes that are oppositely oriented in M. tuberculosis. Phosphorylated DevR interacts with intergenic sequences and protects ~80 bp of DNA that contains three binding sites, designated Dev boxes D1, D2, and D3. The hypoxia-specific transcription start points of narK2 and Rv1738 were mapped, and it was noted that the-35 elements of both promoters overlapped with the proximally placed Dev box. DevR bound cooperatively to adjacently placed D2 and D3 boxes while binding to D1 was independent of DevR interaction with the D2 and D3 boxes. Mutational analysis and green fluorescent protein reporter assays established that the three Dev boxes function synergistically to mediate maximal transcriptional induction of both narK2 and Rv1738 in hypoxic cultures of M. tuberculosis. Analysis of narK2 promoter activity indicates that it is under negative regulation in addition to DevR-mediated positive regulation and also reveals differences between M. tuberculosis and Mycobacterium bovis BCG.

podosomes (also termed invadopodia in cancer cells) are actin-rich adhesion structures with matrix degradation activity that develop in various cell types. Despite their significant physiological importance, the molecular mechanism of podosome formation is largely unknown. In this study, we investigated the molecular mechanisms of podosome formation. The expression of various phosphoinositide-binding domains revealed that the podosomes in Src-transformed NIH3T3 (NIH-src) cells are enriched with Ptdlns(3,4)P2, suggesting an important role of this phosphoinositide in podosome formation. Live-cell imaging analysis revealed that Src-expression stimulated podosome formation at focal adhesions of NIH3T3 cells after Ptdlns(3,4)P2 accumulation. The adaptor protein Tks5/FISH, which is essential for podosome formation, was found to form a complex with Grb2 at adhesion sites in an Src-dependent manner. Further, it was found that N-WASP bound all SH3 domains of Tks5/FISH, which facilitated circular podosome formation. These results indicate that augmentation of the N-WASP-Arp2/3 signal was accomplished on the platform of Tks5/FISH-Grb2 complex at focal adhesions, which is stabilized by Ptdlns(3,4)P2.

MicroRNAs (miRNAs) are noncoding RNAs with important roles in regulating gene expression. In studying the earliest nuclear steps of miRNA biogenesis, we observe that primary miRNA (pri-miRNA) transcripts retained at transcription sites due to the deletion of 3'-end processing signals are converted more efficiently into precursor miRNAs (pre-miRNAs) than pri-miRNAs that are cleaved, polyadenylated, and released. Flanking exons, which also increase retention at transcription sites, likewise contribute to increased levels of intronic primiRNAs. Consistently, efficiently processed endogenous pri-miRNAs are enriched in chromatin-associated nuclear fractions. In contrast, pri-miRNAs that accumulate to high nuclear levels after cleavage and polyadenylation because of the presence of a viral RNA element (the ENE of the Kaposi's sarcoma--associated herpes virus polyadenylated nuclear RNA) are not efficiently processed to precursor or mature miRNAs. Exogenous pri-miRNAs unexpectedly localize to nuclear foci containing splicing factor SC35; yet these foci are unlikely to represent sites of miRNA transcription or processing. Together, our results suggest that primiRNA processing is enhanced by coupling to transcription.

Increased Src activity, often associated with tumorigenesis, leads to the formation of invasive adhesions termed podosomes. Podosome formation requires the function of Rho family guanosine triphosphatases and reorganization of the actin cytoskeleton. In addition, Src induces changes in gene expression required for transformation, in part by activating mitogen-activated protein kinase (MAPK) signaling pathways. We sought to determine whether MAPK signaling regulates podosome formation. Unlike extracellular signal--regulated kinase 1/2 (ERK1/2), ERK5 is constitutively activated in Src-transformed fibroblasts. ERK5-deficient cells expressing v-Src exhibited increased RhoA activation and signaling, which lead to cellular retraction and an inability to form podosomes or induce invasion. Addition of the Rho-kinase inhibitor Y27632 to ERK5-deficient cells expressing v-Src led to cellular extension and restored podosome formation. In Src-transformed cells, ERK5 induced the expression of a Rho GTPase-activating protein (RhoGAP), RhoGAP7/DLC-1, via activation of the transcription factor myocyte enhancing factor 2C, and RhoGAP7 expression restored podosome formation in ERK5-deficient cells. We conclude that ERK5 promotes Src-induced podosome formation by inducing RhoGAP7 and thereby limiting Rho activation.

Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II-dependent and mediated through the modulation of Synapsin1 a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.

Osteopontin (Opn) contributes to diverse biological processes that include immune responses, vascularization, and bone formation. Until recently, studies describing the activities of Opn have focused on the cytokine-like properties of the secreted protein. Here, we show that alternative translation of a single Opn mRNA species generates a secreted and intracellular isoform. Utilization of a 5' canonical translation start site generates a protein that includes an N-terminal signal sequence allowing targeting to secretory vesicles and cytokine secretion, whereas usage of a downstream start site generates a shortened protein that lacks the N-terminal signal sequence and localizes mainly to cytoplasm. The coordinated action of these Opn gene products regulates the functional phenotype of subsets of dendritic cells. signal sequence | podosome

Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete Mete and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer. cobalamin-dependent methionine synthase | cobalamin-independent methionine synthase | methyl transferases | zinc enzymes | zinc inversion

Results from a random mutagenesis procedure on the PcaU binding site from Acinetobacter baylyi followed by in vivo and in vitro screening are presented. PcaU is an IclR-type transcriptional regulator from the soil bacterium A. baylyi and is required for the regulated expression of enzymes for protocatechuate and quinate degradation encoded by the pca-qui operon. It binds to a 45 bp area located in the pcaU-pcal intergenic region which consists of three perfect 10 bp sequence repeats forming one palindrome (R1, R2) and an additional direct sequence repeat (R3). In vivo selection for pca-qui gene expression revealed that mutations within the three sequence motifs are tolerated to different extents. The functional requirement for conserved nucleotides was greatest in the external half of the palindrome (R1). Four positions within and directly adjacent to this 10 bp sequence never acquired a mutation, and are therefore considered to be the most important for transcriptional regulation by PcaU. Transcriptional output is affected in different ways; for some of these changes there is a correlation with a reduction in the affinity of PcaU for these sites. Two of these positions were also preserved when in vitro screening was performed for PcaU binding alone. Additional conserved residues are detected by the in vitro approach, indicating that the regions of the PcaU binding site involved in binding differ, at least in part, from those required for functional gene expression.

LysR-type transcriptional regulators represent one of the largest groups of prokaryotic regulators described to date. In the gram-negative legume endosymbiont Sinorhizobium meliloti, enzymes involved in the protocatechuate branch of the [beta]-ketoadipate pathway are encoded within the pcaDCHGB operon, which is subject to regulation by the LysR-type protein PcaQ. In this work, purified PcaQ was shown to bind strongly (equilibrium dissociation constant, 0.54 nM) to a region at positions -78 to -45 upstream of the pcaD transcriptional start site. Within this region, we defined a PcaQ binding site with dyad symmetry that is required for regulation of pcaD expression in vivo and for binding of PcaQ in vitro. We also demonstrated that PcaQ participates in negative autoregulation by monitoring expression of pcaQ via a transcriptional fusion to lacZ. Although pcaQ homologues are present in many [alpha]-proteobacteria, this work describes the first reported purification of this regulator, as well as characterization of its binding site, which is conserved in Agrobacterium tumefaciens, Rhizobium leguminosarum, Rhizobium etli, and Mesorhizobium loti.

The general transcription factor IIH is recruited to the transcription preinitiation complex through an interaction between its p62/Tfb1 subunit and the [alpha]-subunit of the general transcription factor IIE (TFIIE[alpha]). We have determined that the acidic carboxyl terminus of TFIIE[alpha] ([TFIIE[alpha].sub.336-439]) directly binds the amino-terminal PH domain of p62/Tfb1 with nanomolar affinity. NMR mapping and mutagenesis studies demonstrate that the TFIIE[alpha] binding site on p62/Tfb1 is identical to the binding site for the second transactivation domain of p53 (p53 TAD2). In addition, we demonstrate that [TFIIE[alpha].sub.336-439] is capable of competing with p53 for a common binding site on p62/ Tfbl and that [TFIIE[alpha].sub.336-439] and the diphosphorylated form (pS46/ pT55) of p53 TAD2 have similar binding constants. NMR structural studies reveal that [TFIIE[alpha].sub.336-439] contains a small domain (residues 395-433) folded in a novel/[beta][beta][alpha][alpha][alpha] topology. NMR mapping studies demonstrate that two unstructured regions (residues 377-393 and residues 433-439) located on either side of the folded domain appear to be required for [TFIIE[alpha].sub.336-439] binding to p62/Tfb1 and that these two unstructured regions are held close to each other in three-dimensional space by the novel structured domain. We also demonstrate that, like p53, [TFIIE[alpha].sub.336-439] can activate transcription in vivo. These results point to an important interplay between the general transcription factor TFIIE[alpha] and the tumor suppressor protein p53 in regulating transcriptional activation that may be modulated by the phosphorylation status of p53. NMR | phosphatidylinositol 5-phosphate | transcription regulation | activation domains | isothermal titration calorimetry

The ability of Neisseria gonorrhoeae to reduce nitric oxide (NO) may have important immunomodulatory effects on the host during infection. Therefore, a comprehensive understanding of the regulatory mechanism of the nitric oxide reductase gene (norB) needs to be elucidated. To accomplish this, we analysed the functional regions of the norB upstream region. The promoter contains an extended--10 motif (TGNTACAAT) that is required for high-level expression. Deletion and substitution analysis of the norB upstream region revealed that no sequence upstream of the--10 motif is involved in norB regulation under anaerobic conditions or in the presence of NO. However, replacement of a 29 bp inverted repeat sequence immediately downstream of the extended--10 motif gave high levels of aerobic expression of a norB::lacZ fusion. Insertional inactivation of gonococcal nsrR, predicted to bind to this inverted repeat sequence, resulted in the loss of norB repression and eliminated NO induction capacity. Single-copy complementation of nsrR in trans restored regulation of both norB transcription and NorB activity by NO. In Escherichia coil, expression of a gonococcal nsrR gene repressed gonococcal norB; induction of norB occurred in the presence of exogenously added NO. NsrR also regulates aniA and dnrN, as well as its own expression. We also determined that Fur regulates norB by a novel indirect activation method, by preventing the binding of a gonococcal ArsR homologue, a second repressor whose putative binding site overlaps the Fur binding site.

The aim of this study was to explore why in rabbits activation of farnesoid X receptor (FXR) is dominant over activated liver X receptor-[alpha] (LXR[alpha]) in the regulation of CYP7A1. We cloned the rabbit CYP7A1 promoter and found a fetoprotein transcription factor (FTF) binding element embedded within the LXR[alpha] binding site (LXRE). Gel shift assays demonstrated that FTF competes with LXR[alpha] for binding to LXRE. Short heterodimer partner (SHP) enhances the competitive ability of FTF. Studies in HepG2 cells showed that SHP combined with FTF had more powerful effect to offset the stimulation of CYP7A1 by LXR[alpha]. Gel shift and chromatin immunoprecipitation assays demonstrated that SHP with FTF diminished LXR[alpha] binding to the CYP7A1 promoter. In vivo studies in rabbits fed cholesterol for 10 days showed that hepatic expression of SHP but not FTF rose and LXR[alpha]-bound LXRE decreased. We propose that the SHP/FTF heterodimer occupies LXRE via the embedded FTF binding element and blocks LXR[alpha] from recruiting to LXRE. Therefore, activation of FXR, which upregulates SHP expression, will eliminate the stimulatory effect of LXR[alpha] on the CYP7A1 promoter because increased levels of SHP combined with FTF diminish the recruitment of LXR[alpha] to CYP7A1 promoter. cholesterol 7[alpha]-hydroxylase; regulation; LXR binding site; short heterodimer partner; fetoprotein transcription factor; farnesoid X receptor; liver X receptor-[alpha]

Proteins that track growing microtubule (MT) ends are important for many aspects of intracellular MT function, but the mechanism by which these +TIPs accumulate at MT ends has been the subject of a long-standing controversy. In this issue, Bieling et al. (Bieling, P., S. Kandels-Lewis, I.A. Telley, J. van Dijk, C. Janke, and T. Surrey. 2008. J. Cell Biol. 183:1223-1233) reconstitute plus end tracking of EB1 and CLIP-170 in vitro, which demonstrates that CLIP-170 plus end tracking is EB1-dependent and that both +TIPs rapidly exchange between a soluble and a plus end-associated pool. This strongly supports the hypothesis that plus end tracking depends on a biochemical property of growing MT ends, and that the characteristic +TIP comets result from the generation of new +TIP binding sites through MT polymerization in combination with the exponential decay of these binding sites.

On binding to its target hormone, the oestrogen-receptor protein modulates the expression of many genes. Changes in the receptor's interaction with DNA have now been linked to clinical outcome in patients with breast cancer. See Letter p.389, In about two-thirds of breast cancers, tumour growth depends on oestrogen. This hormone binds to and activates its nuclear receptor, which then binds to specific sites on the genome and [...]