High phosphatidylinositol 4-phosphate (PI4P)-dependent ATPase activity for the Drs2p-Cdc50p flippase after removal of its N- and C-terminal extensions.

P4-ATPases, also known as phospholipid flippases, are responsible for creating and maintaining transbilayer lipid asymmetry in eukaryotic cell membranes. Here, we use limited proteolysis to investigate the role of the N and C termini in ATP hydrolysis and auto-inhibition of the yeast flippase Drs2p-Cdc50p. We show that limited proteolysis of the detergent-solubilized and purified yeast flippase may result in more than 1 order of magnitude increase of its ATPase activity, which remains dependent on phosphatidylinositol 4-phosphate (PI4P), a regulator of this lipid flippase, and specific to a phosphatidylserine substrate.

A GTPase chimera illustrates an uncoupled nucleotide affinity and release rate, providing insight into the activation mechanism.

The release of GDP from GTPases signals the initiation of a GTPase cycle, where the association of GTP triggers conformational changes promoting binding of downstream effector molecules. Studies have implicated the nucleotide-binding G5 loop to be involved in the GDP release mechanism. For example, biophysical studies on both the eukaryotic Gα proteins and the GTPase domain (NFeoB) of prokaryotic FeoB proteins have revealed conformational changes in the G5 loop that accompany nucleotide binding and release.

A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate.

P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required.

The cation-dependent G-proteins: in a class of their own.

G-proteins are some of the most important and abundant enzymes, yet their intrinsic nucleotide hydrolysis reaction is notoriously slow and must be accelerated in vivo. Recent experiments on dynamin and GTPases involved in ribosome assembly have demonstrated that their hydrolysis activities are stimulated by potassium ions. This article presents the hypothesis that cation-mediated activation of G-proteins is more common than currently realised, and that such GTPases represent a structurally and functionally unique class of G-proteins.

The structure of an N11A mutant of the G-protein domain of FeoB.

The uptake of ferrous iron in prokaryotes is mediated by the G-protein-coupled membrane protein FeoB. The protein contains two N-terminal soluble domains that are together called `NFeoB'. One of these is a G-protein domain, and GTP hydrolysis by this domain is essential for iron transport.

A suite of Switch I and Switch II mutant structures from the G-protein domain of FeoB.

The acquisition of ferrous iron in prokaryotes is achieved by the G-protein-coupled membrane protein FeoB. This protein possesses a large C-terminal membrane-spanning domain preceded by two soluble cytoplasmic domains that are together termed 'NFeoB'. The first of these soluble domains is a GTPase domain (G-domain), which is then followed by an entirely α-helical domain.

Molecular basis of the cooperative binding of Cu(I) and Cu(II) to the CopK protein from Cupriavidus metallidurans CH34.

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions. Upon copper challenge, it upregulates the periplasmic protein CopK (8.3 kDa).

The initiation of GTP hydrolysis by the G-domain of FeoB: insights from a transition-state complex structure.

The polytopic membrane protein FeoB is a ferrous iron transporter in prokaryotes. The protein contains a potassium-activated GTPase domain that is essential in regulating the import of iron and conferring virulence to many disease-causing bacteria. However, the mechanism by which the G-domain of FeoB hydrolyzes GTP is not well understood.

Conserved beta-hairpin recognition by the GYF domains of Smy2 and GIGYF2 in mRNA surveillance and vesicular transport complexes.

The yeast suppressor of myosin 2 protein (Smy2) interacts with mRNA-processing proteins through recognition of proline-rich sequences (PRS). Here, we describe the crystal structure of the GYF domain of Smy2 in association with a PRS from the yeast branch point binding protein (BBP/ScSF1). Complex formation requires that the beta-hairpin of the central PPGL motif of the ligand is accommodated by an extended hydrophobic cleft in the domain-a specificity feature that is maintained in the human protein GIGYF2.

Potassium-activated GTPase reaction in the G Protein-coupled ferrous iron transporter B.

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe(2+)). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe(2+) membrane transport mechanism.

Unprecedented binding cooperativity between Cu(I) and Cu(II) in the copper resistance protein CopK from Cupriavidus metallidurans CH34: implications from structural studies by NMR spectroscopy and X-ray crystallography.

The bacterium Cupriavidus metallidurans CH34 is resistant to high environmental concentrations of many metal ions, including copper. This ability arises primarily from the presence of a large plasmid pMOL30 which includes a cluster of 19 cop genes that respond to copper. One of the protein products CopK is induced at high levels and is expressed to the periplasm as a small soluble protein (8.