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Vasopressin (CYFQNCPRG-NH(2), AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G-protein-coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptors versus the closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three-dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(338-350) prototype as a template. AVP was docked into the receptor-G(alpha) systems. The three lowest-energy pairs of receptor-AVP-G(alpha) (two complexes per each receptor) were selected. The 1-ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER 7.0 force field. Six relaxed receptor-AVP-G(alpha) models were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed.

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HtrA2(Omi), belonging to the high-temperature requirement A (HtrA) family of stress proteins, is involved in the maintenance of mitochondrial homeostasis and in the stimulation of apoptosis, as well as in cancer and neurodegenerative disorders. The protein comprises a serine protease domain and a postsynaptic density of 95 kDa, disk large, and zonula occludens 1 (PDZ) regulatory domain and functions both as a protease and a chaperone. Based on the crystal structure of the HtrA2 inactive trimer, it has been proposed that PDZ domains restrict substrate access to the protease domain and that during protease activation there is a significant conformational change at the PDZ–protease interface, which removes the inhibitory effect of PDZ from the active site. The crystal structure of the HtrA2 active form is not available yet. HtrA2 activity markedly increases with temperature. To understand the molecular basis of this increase in activity, we monitored the temperature-induced structural changes using a set of single-Trp HtrA2 mutants with Trps located at the PDZ–protease interface. The accessibility of each Trp to aqueous medium was assessed by fluorescence quenching, and these results, in combination with mean fluorescence lifetimes and wavelength emission maxima, indicate that upon an increase in temperature the HtrA2 structure relaxes, the PDZ–protease interface becomes more exposed to the solvent, and significant conformational changes involving both domains occur at and above 30 °C. This conclusion correlates well with temperature-dependent changes of HtrA2 proteolytic activity and the effect of amino acid substitutions (V226K and R432L) located at the domain interface, on HtrA2 activity. Our results experimentally support the model of HtrA2 activation and provide an insight into the mechanism of temperature-induced changes in HtrA2 structure.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-012-0355-1) contains supplementary material, which is available to authorized users.

Background: An understanding of the HtrA protease activation mechanism is incomplete with respect to its LA regulatory loop.Results: A theoretical model of the LA structure is provided and experimentally verified. Conclusion: LA intersubunit contacts strongly contribute to the stabilization of the inactive HtrA. Significance: This is the first report that simultaneously offers a theoretical three-dimensional structure of LA and its biophysical and functional properties.

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Proteasomes are responsible for protein turnover in eukaryotic cells, degrading short-lived species but also removing improperly folded or oxidatively damaged ones. Dysfunction of a proteasome results in gradual accumulation of misfolded/damaged proteins, leading to their aggregation. It has been postulated that proteasome activators may facilitate removal of such aggregation-prone proteins and thus prevent development of neurodegenerative disorders. However, the discovery of pharmacologically relevant compounds is hindered by insufficient structural understanding of the activation process. In this study we provide a model peptidic activator of human proteasome and analyze the structure-activity relationship within this novel scaffold. The binding mode of the activator at the relevant pocket within the proteasome has been determined by X-ray crystallography. This crystal structure provides an important basis for rational design of pharmacological compounds. Moreover, by providing a novel insight into the proteasome gating mechanism, our results allow the commonly accepted model of proteasome regulation to be revisited.