Incentivizing Commuter Cycling by Financial and Non-Financial Rewards.

Current mobility patterns over-rely on transport modes that do not benefit sustainable and healthy lifestyles. To explore the potential for active mobility, we conducted a randomized experiment aimed at increasing regular commuter cycling in cities. In designing the experiment, we teamed up with developers of the "Cyclers" smartphone app to improve the effectiveness of the app by evaluating financial and non-financial motivational features. Participants in the experiment were recruited among new users of the app, and were randomly assigned to one of four different motivational treatments (smart gamification, two variants of a financial reward, and a combination of smart gamification and a financial reward) or a control group (no specific motivation). Our analysis suggests that people can be effectively motivated to engage in more frequent commuter cycling with incentives via a smartphone app. Offering small financial rewards seems to be more effective than smart gamification. A combination of both motivational treatments-smart gamification and financial rewards-may work the same or slightly better than financial rewards alone. We demonstrate that small financial rewards embedded in smartphone apps such as "Cyclers" can be effective in nudging people to commute by bike more often.

Nucleoplasmin-like domain of FKBP39 from Drosophila melanogaster forms a tetramer with partly disordered tentacle-like C-terminal segments.

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a ß-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins.

Discovery and Characterization of a Biologically Active Non-ATP-Competitive p38 MAP Kinase Inhibitor.

Mitogen-activated protein kinase (MAPK) p38 is part of a broad and ubiquitously expressed family of MAPKs whose activity is responsible for mediating an intracellular response to extracellular stimuli through a phosphorylation cascade. p38 is central to this signaling node and is activated by upstream kinases while being responsible for activating downstream kinases and transcription factors via phosphorylation. Dysregulated p38 activity is associated with numerous autoimmune disorders and has been implicated in the progression of several types of cancer. A number of p38 inhibitors have been tested in clinical trials, with none receiving regulatory approval. One characteristic shared by all of the compounds that failed clinical trials is that they are all adenosine triphosphate (ATP)-competitive p38 inhibitors. Seeing this lack of mechanistic diversity as an opportunity, we screened ~32,000 substances in search of novel p38 inhibitors. Among the inhibitors discovered is a compound that is both non-ATP competitive and biologically active in cell-based models for p38 activity. This is the first reported discovery of a non-ATP-competitive p38 inhibitor that is active in cells and, as such, may enable new pharmacophore designs for both therapeutic and basic research to better understand and exploit non-ATP-competitive inhibitors of p38 activity.

Structure of a lectin from the sea mussel Crenomytilus grayanus (CGL).

CGL is a 150 amino-acid residue lectin that was originally isolated from the sea mussel Crenomytilus grayanus. It is specific for binding GalNAc/Gal-containing carbohydrate moieties and in general does not share sequence homology with other known galectins or lectins. Since CGL displays antibacterial, antifungal and antiviral activities, and interacts with high affinity with mucin-type receptors, which are abundant on some cancer cells, knowledge of its structure is of significant interest. Conditions have been established for the expression, purification and crystallization of a recombinant variant of CGL. The crystal structure of recombinant CGL was determined and refined at a resolution of 2.12 Å. The amino-acid sequence of CGL contains three homologous regions (73% similarity) and the folded protein has a ß-trefoil topology. Structural comparison of CGL with the closely related lectin MytiLec allowed description of the glycan-binding pockets.

Intrinsically disordered and pliable Starmaker-like protein from medaka (Oryzias latipes) controls the formation of calcium carbonate crystals.

Fish otoliths, biominerals composed of calcium carbonate with a small amount of organic matrix, are involved in the functioning of the inner ear. Starmaker (Stm) from zebrafish (Danio rerio) was the first protein found to be capable of controlling the formation of otoliths. Recently, a gene was identified encoding the Starmaker-like (Stm-l) protein from medaka (Oryzias latipes), a putative homologue of Stm and human dentine sialophosphoprotein. Although there is no sequence similarity between Stm-l and Stm, Stm-l was suggested to be involved in the biomineralization of otoliths, as had been observed for Stm even before. The molecular properties and functioning of Stm-l as a putative regulatory protein in otolith formation have not been characterized yet. A comprehensive biochemical and biophysical analysis of recombinant Stm-l, along with in silico examinations, indicated that Stm-l exhibits properties of a coil-like intrinsically disordered protein. Stm-l possesses an elongated and pliable structure that is able to adopt a more ordered and rigid conformation under the influence of different factors. An in vitro assay of the biomineralization activity of Stm-l indicated that Stm-l affected the size, shape and number of calcium carbonate crystals. The functional significance of intrinsically disordered properties of Stm-l and the possible role of this protein in controlling the formation of calcium carbonate crystals is discussed.

Calponin-like Chd64 is partly disordered.

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function.

Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti.

The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD-NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors.

The molecular basis of conformational instability of the ecdysone receptor DNA binding domain studied by in silico and in vitro experiments.

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule.

Conformational changes in the DNA-binding domains of the ecdysteroid receptor during the formation of a complex with the hsp27 response element.

The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein-DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5'- and/or 3'-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5'-end of the regulatory element and moved close to the 3'-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented.

Starmaker exhibits properties of an intrinsically disordered protein.

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.

Insect juvenile hormone binding protein shows ancestral fold present in human lipid-binding proteins.

Low molecular weight juvenile hormone binding proteins (JHBPs) are specific carriers of juvenile hormone (JH) in the hemolymph of butterflies and moths. As hormonal signal transmitters, these proteins exert a profound effect on insect development. The crystal structure of JHBP from Galleria mellonella shows an unusual fold consisting of a long alpha-helix wrapped in a highly curved antiparallel beta-sheet. JHBP structurally resembles the folding pattern found in tandem repeats in some mammalian lipid-binding proteins, with similar organization of one cavity and a disulfide bond between the long helix and the beta-sheet. JHBP reveals, therefore, an archetypal fold used by nature for hydrophobic ligand binding. The JHBP molecule possesses two hydrophobic cavities. Several lines of experimental evidence conclusively indicate that JHBP binds JH in only one cavity, close to the N- and C-termini, and that this binding induces a structural change. The second cavity, located at the opposite end of the molecule, could bind another ligand.

Novel DNA-binding element within the C-terminal extension of the nuclear receptor DNA-binding domain.

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95 A structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an alpha-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the alpha-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs.

The DNA-binding domain of the ultraspiracle drives deformation of the response element whereas the DNA-binding domain of the ecdysone receptor is responsible for a slight additional change of the preformed structure.

Ecdysteroids control molting and metamorphosis in insects via a heterodimeric complex of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle protein (Usp). We used fluorescence resonance energy transfer (FRET) to study the topology of the natural pseudopalindromic element from the hsp27 gene (hsp27pal) in complex with the DNA-binding domains of Usp and EcR (UspDBD and EcRDBD, respectively). Steady-state data revealed shortening of the end-to-end distance of the hsp27pal-derived probe. For the 70.8 +/- 0.6 A distance obtained for the UspDBD-complexed DNA a bend of about 23.1 +/- 2.9 degrees was measured. Nearly the same value (23.0 +/- 3.4 degrees) was obtained for the DNA complexed with the UspDBD/EcRDBD heterodimer. The respective bend angles estimated using fluorescence decay measurements were 19.0 +/- 2.1 degrees and 20.9 +/- 3.6 degrees . Thus, the FRET data suggest for the first time that the UspDBD defines the architecture of the UspDBD/EcRDBD heterocomplex due to the significant deformation of the hsp27pal. This suggestion has been further reinforced using gel retardation experiments, which, in conjunction with high-resolution DNase I footprinting, indicate that the main contribution to the observed bend is given by the UspDBD itself, while binding of the EcRDBD molecule brings on a slight additional change of the preformed structure.