KO-42, a polypeptide with 42 amino acid residues has been designed to fold into a hairpin helix−loop−helix motif that dimerizes and forms a four-helix bundle. The solution structure of the folded KO-42 dimer has been determined by NMR and CD spectroscopy and ultracentrifugation. On the surface of the folded polypeptide a reactive site has been engineered that is capable of catalyzing acyl-transfer reactions of reactive esters. The reactive site of KO-42 contains six histidine residues with perturbed pK a values. The pK as of His-15, His-30, and His-34 are close to 5, whereas those of His-11, His-19, and His-26 are close to 7, with nonideal titration curves. The second-order rate constant for the KO-42 catalyzed hydrolysis of mono-p-nitrophenyl fumarate at pH 4.1 and 290 K is 0.1 M-1 s-1, which is 1140 times larger than that of the 4-methylimidazole (4-MeIm) catalyzed reaction, 8.8 × 10-5 M-1 s-1. The second-order rate constant for the KO-42 catalyzed transesterification of mono-p-nitrophenyl fumarate to form the corresponding trifluoroethyl ester in 10 vol % trifluoroethanol at pH 4.1 and 290 K is 0.052 M-1 s-1 which is 620 times larger than that of the 4-MeIm catalyzed reaction, 8.4 × 10-5 M-1 s-1. KO-42 catalyzes the corresponding reactions of other p-nitrophenyl esters with similar rate enhancements. At pH 4.1 in aqueous solution where the rate constant ratio k 2(KO-42)/k 2(4-MeIm) is larger than 103 the predominant reactive species of KO-42 have unprotonated histidines flanked by protonated histidines. The kinetic solvent isotope effect at pH 4.7 is 2.0 which shows that isotopic fractionation occurs in the transition state. The kinetic solvent isotope effect at pH 6.1 is 1.1 which shows that there is neither general acid−general base catalysis nor strong hydrogen bonding in the transition state of the rate-limiting reaction step at that pH. The results suggest that at low pH the dominant catalytic species functions through a mechanism where unprotonated nucleophilic histidines are flanked by protonated histidines that bind to one or both of the ester oxygens in the transition state.

Recently, genome sequences from different fungi have become available. This information reveals that yeasts and filamentous fungi possess up to five aquaporins. Functional analyses have mainly been performed in budding yeast, Saccharomyces cerevisiae, which has two orthodox aquaporins and two aquaglyceroporins. Whereas Aqy1 is a spore-specific water channel, Aqy2 is only expressed in proliferating cells and controlled by osmotic signals. Fungal aquaglyceroporins often have long, poorly conserved terminal extensions and differ in the otherwise highly conserved NPA motifs, being NPX and NXA respectively. Three subgroups can be distinguished. Fps1-like proteins seem to be restricted to yeasts. Fps1, the osmogated glycerol export channel in S. cerevisiae, plays a central role in osmoregulation and determination of intracellular glycerol levels. Sequences important for gating have been identified within its termini. Another type of aquaglyceroporin, resembling S. cerevisiae Yfl054, has a long N-terminal extension and its physiological role is currently unknown. The third group of aquaglyceroporins, only found in filamentous fungi, have extensions of variable size. Taken together, yeasts and filamentous fungi are a fruitful resource to study the function, evolution, role and regulation of aquaporins, and the possibility to compare orthologous sequences from a large number of different organisms facilitates functional and structural studies.

Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.

BAG-family proteins share a conserved protein interaction region, called the 'BAG domain', which binds and regulates Hsp70/Hsc70 molecular chaperones. This family of cochaperones functionally regulates signal transducing proteins and transcription factors important for cell stress responses, apoptosis, proliferation, cell migration and hormone action. Aberrant overexpression of the founding member of this family, BAG1, occurs in human cancers. In this study, a structure-based approach was used to identify interacting residues in a BAG1--Hsc70 complex. An Hsc70-binding fragment of BAG1 was shown by multidimensional NMR methods to consist of an antiparallel three-helix bundle. NMR chemical shift experiments marked surface residues on the second (alpha 2) and third (alpha 3) helices in the BAG domain that are involved in chaperone binding. Structural predictions were confirmed by site-directed mutagenesis of these residues, resulting in loss of binding of BAG1 to Hsc70 in vitro and in cells. Molecular docking of BAG1 to Hsc70 and mutagenesis of Hsc70 marked the molecular surface of the ATPase domain necessary for interaction with BAG1. The results provide a structural basis for understanding the mechanism by which BAG proteins link molecular chaperones and cell signaling pathways.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The ␣ 2 -adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOct␣) belongs to the ␣-adrenoceptor-like subfamily, and the other four (BiOct␤-R1, BiOct␤-R2, BiOct␤-R3, and BiOct␤-R4) belong to the ␤-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of ␤-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.