Crystal structure of a membrane-embedded H+-translocating pyrophosphatase.

H(+)-translocating pyrophosphatases (H(+)-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PP(i)) hydrolysis. H(+)-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H(+)-PPases are unclear. Here we report the crystal structure of a Vigna radiata H(+)-PPase (VrH(+)-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35 Å resolution. Each VrH(+)-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg(2+) ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PP(i) hydrolysis, and H(+) is then transported into the vacuolar lumen through a pathway consisting of Arg 242, Asp 294, Lys 742 and Glu 301. We propose a working model of the mechanism for the coupling between proton pumping and PP(i) hydrolysis by H(+)-PPases.

Structural characterizations of the chloroplast translocon protein Tic110.

Tic110 is a major component of the chloroplast protein import translocon. Two functions with mutually exclusive structures have been proposed for Tic110: a protein-conducting channel with six transmembrane domains and a scaffold with two N-terminal transmembrane domains followed by a large soluble domain for binding transit peptides and other stromal translocon components. To investigate the structure of Tic110, Tic110 from Cyanidioschyzon merolae (CmTic110) was characterized. We constructed three fragments, CmTic110A , CmTic110B and CmTic110C , with increasing N-terminal truncations, to perform small-angle X-ray scattering (SAXS) and X-ray crystallography analyses and Dali structural comparison. Here we report the molecular envelope of CmTic110B and CmTic110C determined by SAXS, and the crystal structure of CmTic110C at 4.2 Å. Our data indicate that the C-terminal half of CmTic110 possesses a rod-shaped helix-repeat structure that is too flattened and elongated to be a channel. The structure is most similar to the HEAT-repeat motif that functions as scaffolds for protein-protein interactions.

Structure of the Trichomonas vaginalis Myb3 DNA-binding domain bound to a promoter sequence reveals a unique C-terminal ß-hairpin conformation.

Trichomonas vaginalis Myb3 transcription factor (tvMyb3) recognizes the MRE-1 promoter sequence and regulates ap65-1 gene, which encodes a hydrogenosomal malic enzyme that may play a role in the cytoadherence of the parasite. Here, we identified tvMyb3(53-180) as the essential fragment for DNA recognition and report the crystal structure of tvMyb3(53-180) bound to MRE-1 DNA. The N-terminal fragment adopts the classical conformation of an Myb DNA-binding domain, with the third helices of R2 and R3 motifs intercalating in the major groove of DNA. The C-terminal extension forms a ß-hairpin followed by a flexible tail, which is stabilized by several interactions with the R3 motif and is not observed in other Myb proteins. Interestingly, this unique C-terminal fragment does not stably connect with DNA in the complex structure but is involved in DNA binding, as demonstrated by NMR chemical shift perturbation, (1)H-(15)N heteronuclear-nuclear Overhauser effect and intermolecular paramagnetic relaxation enhancement. Site-directed mutagenesis also revealed that this C-terminal fragment is crucial for DNA binding, especially the residue Arg(153) and the fragment K(170)KRK(173). We provide a structural basis for MRE-1 DNA recognition and suggest a possible post-translational regulation of tvMyb3 protein.

Crystal structure of the Klebsiella pneumoniae NFeoB/FeoC complex and roles of FeoC in regulation of Fe2+ transport by the bacterial Feo system.

Feo is a transport system commonly used by bacteria to acquire environmental Fe(2+). It consists of three proteins: FeoA, FeoB, and FeoC. FeoB is a large protein with a cytosolic N-terminal domain (NFeoB) that contains a regulatory G protein domain and a helical S domain. The C-terminal region of FeoB is a transmembrane domain that likely acts as the Fe(2+) permease. NFeoB has been shown to form a trimer pore that may function as an Fe(2+) gate. FeoC is a small winged-helix protein that possesses four conserved cysteine residues with a consensus sequence that likely provides binding sites for the [Fe-S] cluster. Therefore, FeoC is presumed to be an [Fe-S] cluster-dependent regulator that directly controls transcription of the feo operon. Despite the apparent significance of the Feo system, however, the function of FeoC has not been experimentally demonstrated. Here, we show that Klebsiella pneumoniae FeoC (KpFeoC) forms a tight complex with the intracellular N-terminal domain of FeoB (KpNFeoB). The crystal structure of the complex reveals that KpFeoC binds to KpNFeoB between the switch II region of the G protein domain and the effector S domain and that the long KpFeoC W1 loop lies above the KpNFeoB nucleotide-binding site. These interactions suggest that KpFeoC modulates the guanine nucleotide-mediated signal transduction process. Moreover, we showed that binding of KpFeoC disrupts pore formation by interfering with KpNFeoB trimerization. These results provide strong evidence suggesting that KpFeoC plays a crucial role in regulating Fe(2+) transport in Klebsiella pneumonia in addition to the presumed gene regulator role.

Structure of the catalytic domain of the Clostridium thermocellum cellulase CelT.

Cellulases hydrolyze cellulose, a major component of plant cell walls, to oligosaccharides and monosaccharides. Several Clostridium species secrete multi-enzyme complexes (cellulosomes) containing cellulases. C. thermocellum CelT, a family 9 cellulase, lacks the accessory module(s) necessary for activity, unlike most other family 9 cellulases. Therefore, characterization of the CelT structure is essential in order to understand its catalytic mechanism. Here, the crystal structure of free CelTΔdoc, the catalytic domain of CelT, is reported at 2.1 Šresolution. Its structure differs in several aspects from those of other family 9 cellulases. CelTΔdoc contains an additional α-helix, α-helices of increased length and two additional surface-exposed ß-strands. It also contains three calcium ions instead of one as found in C. cellulolyticum Cel9M. CelTΔdoc also has two flexible loops at the open end of its active-site cleft. Movement of these loops probably allows the substrate to access the active site. CelT is stable over a wide range of pH and temperature conditions, suggesting that CelT could be used to convert cellulose biomass into biofuel.

Structure of the sodium-dependent phosphate transporter reveals insights into human solute carrier SLC20.

Inorganic phosphate (Pi) is a fundamental and essential element for nucleotide biosynthesis, energy supply, and cellular signaling in living organisms. Human phosphate transporter (hPiT) dysfunction causes numerous diseases, but the molecular mechanism underlying transporters remains elusive. We report the structure of the sodium-dependent phosphate transporter from Thermotoga maritima (TmPiT) in complex with sodium and phosphate (TmPiT-Na/Pi) at 2.3-angstrom resolution. We reveal that one phosphate and two sodium ions (Pi-2Na) are located at the core of TmPiT and that the third sodium ion (Nafore) is located near the inner membrane boundary. We propose an elevator-like mechanism for sodium and phosphate transport by TmPiT, with the TmPiT-Na/Pi complex adopting an inward occluded conformation. We found that disease-related hPiT variants carry mutations in the corresponding sodium- and phosphate-binding residues identified in TmPiT. Our three-dimensional structure of TmPiT provides a framework for understanding PiT dysfunction and for future structure-based drug design.

Roles of the Hydrophobic Gate and Exit Channel in Vigna radiata Pyrophosphatase Ion Translocation.

Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H+ and/or Na+) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH+-PPase), the VrH+-PPase-2Pi complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three VrH+-PPases complexes (Pi-, 2Pi- and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2Pi-bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among VrH+-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225-R562 is at the way out of the exit channel of VrH+-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225-R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer.

Crystal structure of Helicobacter pylori spermidine synthase: a Rossmann-like fold with a distinct active site.

Spermidine synthase (putrescine aminopropyltransferase, PAPT) catalyzes the transfer of the aminopropyl group from decarboxylated S-adenosylmethionine to putrescine during spermidine biosynthesis. Helicobacter pylori PAPT (HpPAPT) has a low sequence identity with other PAPTs and lacks the signature sequence found in other PAPTs. The crystal structure of HpPAPT, determined by multiwavelength anomalous dispersion, revealed an N-terminal beta-stranded domain and a C-terminal Rossmann-like domain. Structural comparison with other PAPTs showed that HpPAPT has a unique binding pocket between two domains, numerous non-conserved residues, a less acidic electrostatic surface potential, and a large buried space within the structure. HpPAPT lacks the gatekeeping loop that facilitates substrate binding in other PAPTs. PAPTs are essential for bacterial cell viability; thus, HpPAPT may be a potential antimicrobial drug target for H. pylori owing to its characteristic PAPT sequence and distinct conformation.

Kinetic and structural properties of inorganic pyrophosphatase from the pathogenic bacterium Helicobacter pylori.

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate (PPi) to orthophosphate (Pi) and controls the level of PPi in cells. PPase plays an essential role in energy conservation and provides the energy for many biosynthetic pathways. The Helicobacter pylori pyrophosphatase (HpPPase) gene was cloned, expressed, purified, and found to have a molecular weight of 20 kDa. The K(m) and V (max) of HpPPase were determined as 214.4 microM and 594 micromol Pi min(-1) mg(-1), respectively. PPi binds Mg(2+) to form a true substrate that activates the enzyme. However, free PPi could be a potent inhibitor for HpPPase. The effects of the inhibitors NaF, ATP, iminodiphosphate, and N-ethylmaleimide on HpPPase activity were evaluated. NaF showed the highest inhibition of the enzyme. Crystal structures of HpPPase and the PPi-HpPPase complex were determined. HpPPase comprises three alpha-helices and nine beta-strands and folds as a barrel structure. HpPPase forms a hexamer in both the solution and crystal states, and each monomer has its own PPi-binding site. The PPi binding does not cause a significant conformational change in the PPi-HpPPase complex, which might represent an inhibition state for HpPPase in the absence of a divalent metal ion.

Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism.

Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5-6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a 'molecular mousetrap', repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.

Proton/sodium pumping pyrophosphatases: the last of the primary ion pumps.

Membrane-bound pyrophosphatases (M-PPases) are homodimeric enzymes that couple the generation and utilization of membrane potentials to pyrophosphate (PPi) hydrolysis and synthesis. Since the discovery of the link between PPi use and proton transport in purple, non-sulphur bacteria in the 1960s, M-PPases have been found in all three domains of life and have been shown to have a crucial role in stress tolerance and in plant maturation. The discovery of sodium-pumping and sodium/proton-pumping M-PPases showed that the pumping specificity of these enzymes is not limited to protons, further suggesting that M-PPases are evolutionarily very ancient. The recent structures of two M-PPases, the Vigna radiata H(+)-pumping M-PPase and Thermotoga maritima Na(+)-pumping M-PPase, provide the basis for understanding the functional data. They show that M-PPases have a novel fold and pumping mechanism, different to the other primary pumps. This review discusses the current structural understanding of M-PPases and of ion selection among various M-PPases.