No abstract

Nop56p and Nop58p are two core proteins of the box C/D snoRNPs that interact concurrently with fibrillarin and snoRNAs to function in enzyme assembly and catalysis. Here we report the 2.9 A resolution co-crystal structure of an archaeal homolog of Nop56p/Nop58p, Nop5p, in complex with fibrillarin from Archaeoglobus fulgidus (AF) and the methyl donor S-adenosyl-L-methionine. The N-terminal domain of Nop5p forms a complementary surface to fibrillarin that serves to anchor the catalytic subunit and to stabilize cofactor binding. A coiled coil in Nop5p mediates dimerization of two fibrillarin-Nop5p heterodimers for optimal interactions with bipartite box C/D RNAs. Structural analysis and complementary biochemical data demonstrate that the conserved C-terminal domain of Nop5p harbors RNA-binding sites. A model of box C/D snoRNP assembly is proposed based on the presented structural and biochemical data.

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Activation of certain classes of G protein-coupled receptors (GPCRs) can lead to alterations in the actin cytoskeleton, gene transcription, cell transformation, and other processes that are known to be regulated by Rho family small-molecular-weight GTPases. Although these responses can occur indirectly via cross-talk from canonical heterotrimeric G protein cascades, it has recently been demonstrated that Dbl family Rho guanine nucleotide exchange factors (RhoGEFs) can serve as the direct downstream effectors of heterotrimeric G proteins. Heterotrimeric G␣ 12/13 , G␣ q , and G␤␥ subunits are each now known to directly bind and regulate RhoGEFs. Atomic structures have recently been determined for several of these RhoGEFs and their G protein complexes, providing fresh insight into the molecular mechanisms of signal transduction between GPCRs and small molecular weight G proteins. This review covers what is currently known about the structure, function, and regulation of these recently recognized effectors of heterotrimeric G proteins.Heterotrimeric G proteins are master regulators of cell homeostasis. By coordinating signaling between the ϳ800 G protein-coupled receptors (GPCRs) in the human genome and a relatively small handful of effector enzymes and channels in the cell, they control processes such as muscle contractility, glycogen metabolism, neurotransmission, and the concentration of intracellular ions. Their profound impact on nearly all cellular processes and their therapeutic potential have rendered them one of the most intensely studied signal transduction paradigms at the biochemical and molecular level (Sprang et al., 2007).When heterotrimeric G proteins are in their inactive, GDPbound state, they exist as an inert complex composed of ␣, ␤, and ␥ subunits (G␣␤␥). In this state, they are substrates for activated GPCRs, which catalyze nucleotide exchange on the ␣ subunit (G␣). When bound to GTP, G␣ releases the effector binding surface of the ␤ and ␥ heterodimer (G␤␥) so that both G␣ and G␤␥ can interact with and modulate the activity of specific downstream enzymes and channels. The G␣ subunit has weak guanine nucleotide triphosphatase (GTPase) activity that slowly returns the G protein to its GDP-bound state. G␣⅐GDP then becomes resequestered by G␤␥. Beyond serving as conduits for extracellular signals, heterotrimeric G proteins contribute to the fidelity, duration, and amplitude of GPCR signaling. A given class of heterotrimeric G protein can typically recognize only a subset of GPCRs, and can only interact with one or a few downstream effector targets, ensuring the specificity of signaling from receptor to effector. The rate of GTP hydrolysis on G␣ dictates the length of time that its signal is in play, and this rate -bound conformation. The Ras-like domain is colored cyan, and the ␣-helical domain is gray. The three nucleotide-dependent switch regions (switch I-III) are red. The canonical effector docking site, a shallow canyon formed between switch II and the ␣3 helix, is indicated by the tr...

Psychrophilic organisms such as micro‐organisms and other ectothermic species living in polar, deep‐ sea or any constantly low temperature environments, produce enzymes adapted to function at low temperature. These enzymes are characterized by a high catalytic efficiency at low and moderate temperatures but are rather thermolabile. Due to their high specific activity and their rapid inactivation at temperatures as low as 30°C, they offer, along with the producing micro‐organisms, a great potential in biotechnology. The molecular basis of the adaptation of cold α‐amylase, subtilisin, triose phosphate isomerase from Antarctic bacteria and of trypsin from fish living in North Atlantic and in Antarctic sea waters have been studied. The comparison of the 3D structures obtained either by protein modelling or by X‐ray crystallography (North Atlantic trypsin) with those of their mesophilic counterparts indicates that the molecular changes tend to increase the flexibility of the structure by a weakening of the intramolecular interactions and by an increase of the interactions with the solvent. For each enzyme, the most appropriate strategy enabling it to accommodate the substrate at a low energy cost is selected. There is a price to pay in terms of thermosensibility because the selective pressure is essentially oriented towards the harmonization of the specific activity with ambient thermal conditions. However, as demonstrated by site‐directed mutagenesis experiments carried out on the Antarctic subtilisin, the possibility remains to stabilize the structure of these enzymes without affecting their high catalytic efficiency.

The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25°C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heatlabile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.

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Gα q directly activates p63RhoGEF and closely related catalytic domains found in Trio and Kalirin, thereby linking G q -coupled receptors to the activation of RhoA. Although the crystal structure of Gα q in complex with the catalytic domains of p63RhoGEF is available, the molecular mechanism of activation has not yet been defined. In this study, we show that membrane translocation does not appear to play a role in Gα q -mediated activation of p63RhoGEF, as it does in some other RhoGEFs. Gα q instead must act allosterically. We next identify specific structural elements in the PH domain that inhibit basal nucleotide exchange activity, and provide evidence that Gα q overcomes this inhibition by altering the conformation of the α6-αN linker that joins the DH and PH domains, a region that forms direct contacts with RhoA. We also identify residues in Gα q that are important for the activation of p63RhoGEF and that contribute to Gα subfamily selectivity, including a critical residue in the Gα q C-terminal helix, and demonstrate the importance of these residues for RhoA activation in living cells.