A hinge in the distal end of the PACSIN 2 F-BAR domain may contribute to membrane-curvature sensing.

The protein kinase C and casein kinase 2 substrates in neurons (PACSINs) represent a subfamily of membrane-binding proteins characterized by an amino-terminal Bin-Amphiphysin-Rvs (F-BAR) domain. PACSINs link membrane trafficking with actin dynamics and regulate the localization of distinct cargo molecules. The F-BAR domain forms a dimer essential for lipid binding. We have obtained crystals of authentic murine PACSIN 2 that contain an ordered F-BAR domain, indicating that additional domains are flexibly connected to F-BAR. The structure shares similarity to other BAR domains and exhibits special features unique to PACSINs. These include the uneven distribution of charged residues on the concave molecular surface and a so-called wedge loop that is driven into the membrane upon binding of PACSIN. The murine PACSIN 2 F-BAR domain requires dimerization for sensing of curved membranes, and the present structure also provides a mechanism for higher-order oligomer formation. Importantly, comparison of murine with human and Drosophila PACSIN 2 F-BAR domains reveals stark differences in the orientation of distal helical segments leading to a wider crescent shape of murine PACSIN 2. We define hinge residues for these movements that may help PACSINs sense and concomitantly reinforce membrane curvature.

TIP47 functions in the biogenesis of lipid droplets.

TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

Crystallization and preliminary characterization of the Thermus thermophilus RNA helicase Hera C-terminal domain.

Heat-resistant RNA-dependent ATPase (Hera) from Thermus thermophilus is a DEAD-box RNA helicase. Two constructs encompassing the second RecA-like domain and the C-terminal domain of Hera were overproduced in Escherichia coli and purified to homogeneity. Single crystals of both Hera constructs were obtained in three crystal forms. A tetragonal crystal form belonged to space group P4(1)2(1)2, with unit-cell parameters a = 65.5, c = 153.0 A, and contained one molecule per asymmetric unit. Two orthorhombic forms belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 62.8, b = 70.9, c = 102.3 A (form I) and a = 41.6, b = 67.6, c = 183.5 A (form II). Both orthorhombic forms contained two molecules per asymmetric unit. All crystals diffracted X-rays to beyond 3 A resolution, but the tetragonal data sets displayed high Wilson B values and high mean |E(2) - 1| values, indicating potential disorder and anisotropy. The tetragonal crystal was phased by MAD using a single selenium site.

Structures of the human orotidine-5'-monophosphate decarboxylase support a covalent mechanism and provide a framework for drug design.

UMP synthase (UMPS) catalyzes the last two steps of de novo pyrimidine nucleotide synthesis and is a potential cancer drug target. The C-terminal domain of UMPS is orotidine-5'-monophosphate decarboxylase (OMPD), a cofactor-less yet extremely efficient enzyme. Studies of OMPDs from micro-organisms led to the proposal of several noncovalent decarboxylation mechanisms via high-energy intermediates. We describe nine crystal structures of human OMPD in complex with substrate, product, and nucleotide inhibitors. Unexpectedly, simple compounds can replace the natural nucleotides and induce a closed conformation of OMPD, defining a tripartite catalytic site. The structures outline the requirements drugs must meet to maximize therapeutic effects and minimize cross-species activity. Chemical mimicry by iodide identified a CO(2) product binding site. Plasticity of catalytic residues and a covalent OMPD-UMP complex prompt a reevaluation of the prevailing decarboxylation mechanism in favor of covalent intermediates. This mechanism can also explain the observed catalytic promiscuity of OMPD.

Pseudo-merohedral twinning in monoclinic crystals of human orotidine-5'-monophosphate decarboxylase.

Human UMP synthase is a bifunctional enzyme that catalyzes the penultimate and last steps in the de novo biosynthesis of UMP. In contrast to prokaryotes, UMP synthase from higher eukaryotes combines the orotate phosphoribosyltransferase and the orotidine-5'-monophosphate (OMP) decarboxylase activities on a single polypeptide chain. The decarboxylase activity is unusual in that it represents the fastest rate acceleration of any enzyme studied to date. Although several crystal structures of OMP decarboxylases have been described, the precise decarboxylation mechanism remains elusive. The crystal structure of the OMP decarboxylase domain from human UMP synthase was determined by molecular replacement using data from a highly twinned monoclinic crystal. The space group is P2(1), with unit-cell parameters a = 69.18, b = 61.70, c = 69.17 A, beta = 113.06 degrees. Self-rotation function analysis and various intensity statistics revealed the presence of pseudo-merohedral twinning, but these tests underestimated the true twin fraction of alpha approximately = 0.44. Data analysis, the origin of the twinning and structure determination are discussed.

Crystal structure and nucleotide binding of the Thermus thermophilus RNA helicase Hera N-terminal domain.

DEAD box RNA helicases use the energy of ATP hydrolysis to unwind double-stranded RNA regions or to disrupt RNA/protein complexes. A minimal RNA helicase comprises nine conserved motifs distributed over two RecA-like domains. The N-terminal domain contains all motifs involved in nucleotide binding, namely the Q-motif, the DEAD box, and the P-loop, as well as the SAT motif, which has been implicated in the coordination of ATP hydrolysis and RNA unwinding. We present here the crystal structure of the N-terminal domain of the Thermus thermophilus RNA helicase Hera in complex with adenosine monophosphate (AMP). Upon binding of AMP the P-loop adopts a partially collapsed or half-open conformation that is still connected to the DEAD box motif, and the DEAD box in turn is linked to the SAT motif via hydrogen bonds. This network of interactions communicates changes in the P-loop conformation to distant parts of the helicase. The affinity of AMP is comparable to that of ADP and ATP, substantiating that the binding energy from additional phosphate moieties is directly converted into conformational changes of the entire helicase. Importantly, the N-terminal Hera domain forms a dimer in the crystal similar to that seen in another thermophilic prokaryote. It is possible that this mode of dimerization represents the prototypic architecture in RNA helicases of thermophilic origin.

A general binding mechanism for all human sulfatases by the formylglycine-generating enzyme.

The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme-substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE-substrate complexes with pentamer and heptamer peptides that mimic sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE-peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and sulfatases are explained by this binding mechanism.

Crystal structure of Rab9 complexed to GDP reveals a dimer with an active conformation of switch II.

The small GTPase Rab9 is an essential regulator of vesicular transport from the late endosome to the trans-Golgi network, as monitored by the redirection of the mannose-6-phosphate receptors. The crystal structure of Rab9 complexed to GDP, Mg(2+), and Sr(2+) reveals a unique dimer formed by an intermolecular beta-sheet that buries the switch I regions. Surface area and shape complementarity calculations suggest that Rab9 dimers can form an inactive, membrane-bound pool of Rab9 . GDP that is independent of GDI. Mg(2+)-bound Rab9 represents an inactive state, but Sr(2+)-bound Rab9 . GDP displays activated switch region conformations, mimicking those of the GTP state. A hydrophobic tetrad is formed resembling an effector-discriminating epitope found only in GTP-bound Rab proteins.

Purification, crystallization and preliminary X-ray analysis of the GTP-binding protein Rab9 implicated in endosome-to-TGN vesicle trafficking.

Rab GTP-binding proteins are involved in the regulation of distinct vesicular-transport events involving membrane targeting and fusion. They differ from other small GTPases by the presence of specific loop regions that serve as effector-binding sites in addition to the classical switch I and switch II regions. While the structures of many small GTP-binding proteins of the Ras superfamily are available in both GDP- and GTP-bound forms, Rab proteins are less well characterized than Ras proteins at the structural level. The crystallization of Rab9, a key regulatory component in the recycling of mannose-6-phosphate receptors from endosomes to the trans-Golgi network, is described here.