Dick Van der Helm

Crystal structure of the outer membrane active transporter FepA from Escherichia coli.

Integral outer membrane receptors for iron chelates and vitamin B 12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton–motive force of the inner membrane through physical interaction with TonB–ExbB–ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 Å resolution. Two distinct functional domains are revealed: (i) a 22–stranded β–barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N–terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB–mediated transport. The blockage of the pore suggests that the N–terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

Metallorganische verbindungen der lanthanoide: XXXI. Synthese und molekülstruktur einiger cyclopentadienyllutetiumhydride☆

Abstract The reaction of alkyl- and aryl-(dicyclopentadienyl)lutetium complexes ( 1 ) with H 2 or D 2 gives dimeric dicyclopentadienyllutetiumhydride ( 2 ) or -deuteride ( 3 ). Dicyclopentadienyllutetiumchloride ( 4 ) reacts with sodium in tetrahydrofuran (THF) with formation of [Na(THF) 6 ][(Cp 2 LuH) 3 H] ( 5 ). Tricyclopentadienyllutetium ( 6 ) reacts with NaH or NaD to give the complexes [Na(THF) 6 ][(Cp 3 Lu) 2 H]·2(THF) ( 7 ) or [Na(THF) 6 ][(Cp 3 Lu) 2 D]·2(THF) ( 8 ), respectively. The molecular structures of 2 and 7 were determined by single crystal X-ray diffraction.

Molecular mechanism of ferricsiderophore passage through the outer membrane receptor proteins of Escherichia coli

Iron is an essential nutrient for all microorganisms with a few exceptions. Microorganisms use a variety of systems to acquire iron from the surrounding environment. One such system includes production of an organic molecule known as a siderophore by many bacteria and fungi. Siderophores have the capacity to specifically chelate ferric ions. The ferricsiderophore complex is then transported into the cell via a specific receptor protein located in the outer membrane. This is an energy dependent process and is the subject of investigation in many research laboratories. The crystal structures of three outer membrane ferricsiderophore receptor proteins FepA, FhuA and FecA from Escherichia coli and two FpvA and FptA from Pseudomonas aeruginosa have recently been solved. Four of them, FhuA, FecA, FpvA and FptA have been solved in ligand-bound forms, which gave insight into the residues involved in ligand binding. The structures are similar and show the presence of similar domains; for example, all of them consist of a 22 strand-β-barrel formed by approximately 600 C-terminal residues while approximately 150 N-terminal residues fold inside the barrel to form a plug domain. The plug domain obstructs the passage through the barrel; therefore our research focuses on the mechanism through which the ferricsiderophore complex is transported across the receptor into the periplasm. There are two possibilities, one in which the plug domain is expelled into the periplasm making way for the ferricsiderophore complex and the second in which the plug domain undergoes structural rearrangement to form a channel through which the complex slides into the periplasm. Multiple alignment studies involving protein sequences of a large number of outer membrane receptor proteins that transport ferricsiderophores have identified several conserved residues. All of the conserved residues are located within the plug and barrel domain below the ligand binding site. We have substituted a number of these residues in FepA and FhuA with either alanine or glutamine resulting in substantial changes in the chemical properties of the residues. This was done to study the effect of the substitutions on the transport of ferricsiderophores. Another strategy used was to create a disulfide bond between the residues located on two adjacent β-strands of the plug domain or between the residues of the plug domain and the β-barrel in FhuA by substituting appropriate residues with cysteine. We have looked for the variants where the transport is affected without altering the binding. The data suggest a distinct role of these residues in the mechanism of transport. Our data also indicate that these transporters share a common mechanism of transport and that the plug remains within the barrel and possibly undergoes rearrangement to form a channel to transport the ferricsiderophore from the binding site to the periplasm.

Siderophore Mediated Iron(III) Uptake in Gliocladium virens: 1. Properties of cis-Fusarinine, trans-Fusarmine, Dimerum Acid, and Their Ferric Complexes

Gliocladium virens (ATCC 24290) produces two monohydroxamates (cis- and trans-fusarinine) and a dihydroxamate (dimerum acid) as the major siderophores in the culture filtrate. This fungus also produces minor quantities of three trihydroxamates (the deferri forms of ferricrocin, coprogen B, and coprogen). Structural features of the free ligands and the metal complexed forms of cis-fusarinine (cF), trans-fusarinine (tF), and dimerum acid (DA) have been investigated using electronic (visible), circular dichroism (CD), and NMR spectroscopy. In aqueous solution, in the pH range of 6.5-8.0, all of the ferric complexes of cF (and tF) exist as 3:1 chelates. Fe(cF)3 [or Fe(tF)3] forms both lambda and delta coordination isomers, but the former in a slight excess. DA forms a 3:2 ferric complex in the pH range of 5.0-8.0. Iron coordination in Fe2(DA)3 is predominantly delta. DA ligands in Ga2(DA)3 exist as two different conformers at a ratio of 2:1. In mixed solution cF, tF, and DA form a large number of homogeneous and heterogeneous Fe(III) chelates.

Identification and mutational studies of conserved amino acids in the outer membrane receptor protein, FepA, which affect transport but not binding of ferric-enterobactin in Escherichia coli.

Many gram-negative bacteria produce and excrete siderophores, which complex iron with high affinity in the environment. The ferric siderophore complexes are transported across the outer membrane by receptor proteins. This process requires energy and is TonB dependent and must involve conformational changes in the receptor proteins to allow the transport of the ferric siderophores from the extracellular binding site to the periplasm. There is a large variety in the structures, molecular weights and charges among the siderophores. It was therefore realized that when the sequences of the many different receptor proteins were compared, simultaneously, all identities and close similarities, found in this manner, could only be due to residues involved in the conformational changes and transport mechanism, common to all the proteins, and not be due to the specificity of ligand recognition. Once the crystal structures of FepA, FhuA and FecA became available, it was immediately clear that the sequence similarities which were found in the simultaneous alignment, were all localized in a few structural domains, which are identical in the three structures and can therefore be expected to be maintained in all the proteins in this family. One of these domains, tentatively named the lock region, consists of 10 residues with a central quadrupole formed by two arginines and two glutamates, from the plug region and the beta barrel. We mutated several of these residues in FepA. All showed normal binding in quantitative binding studies. Some showed normal transport as well, however, the majority showed moderate to severe defective transport with ferric enterobactin. The results therefore show the validity of the hypothesis that the simultaneous sequence alignment will select the residues involved in the transport function of the receptor proteins. In addition the results allow to relate the severity of the transport deficiency to be correlated with the structure of the lock region while it is also possible to propose a function of this region in the conformational changes of the protein during the transport of the ligand from the binding site to the periplasm.

Amido-3-hydroxypyridin-4-ones as iron(III) ligands.

The synthesis and physicochemical properties of a range of 2- and 6-amido-3-hydroxypyridin-4-ones are described. All the amido-substituted 3-hydroxypyridin-4-ones have lower pK(a) values than 1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone). This is due to the inductive effect of the amido group. Furthermore, the pK(a) values of the 3-hydroxy group in 1-nonsubstituted pyridinones are dramatically lower than those of the corresponding 1-alkyl analogues, indicating that a strong hydrogen bond exists between the 2-amido function and the 3-oxygen anion, which stabilises the anion. As a result of the decreased competition with protons, the pFe(3+) values of this group of molecules are higher than that of deferiprone. The distribution coefficients of these molecules are also increased despite the lack of a hydrophobic 1-alkyl substituent and this is ascribed to the intramolecular hydrogen bond. X-ray diffraction studies confirm the existence of the intramolecular hydrogen bond.

Synthesis of triorganostannate esters of dicarboxylic acids. Crystal structure of bis(dicyclohexylammonium) trisoxalatotetrakis(tri-n-butylstannate) · 2 ethanol

Abstract Triphenyltin hydroxide has been shown to react with dicyclohexylammonium oxalate to give [(cyclo-C6H11)2NH2]+ [(C6H5)3SnOC(O)C(O)O]−, and to give with dicyclohexylammonium malonate [(cyclo-C6H11)2NH2]+ &{;[(C6H5)3SnOC(O)CH2C(O)O]2H&0;−. Bis(tri-n-butyltin) oxide afforded 2[(cyclo-C6H11)2NH2]+ &{;[(n-C4H9)3Sn]4[OC(O)CH2C(O)O]3{2− on treatment with dicyclohexylammonium malonate, but with dicyclohexylammonium oxalate it yielded a stannate which crystallized with two molecules of ethanol. A low-temperature X-ray diffraction study of this ethanol-containing stannate, 2[(cyclo-C6H11)2NH2]+ [C2H5OH · (n-C4H9)3SnOC(CO2)(O)(n-C4H9)3SnOC(O)]22− revealed the presence of centrosymmetric chains, each consisting of four [(n-C4H9)3Sn]+ cations linked by three [OC(O)C(O)]2− anions. The chains are hydrogen bonded by [(cyclo-C6H11)2NH2]+ cations and ethanol molecules into a three-dimensional lattice. The geometry around the ethanol-coordinated (SnO 2.465(4) A) terminal tin atom is essentially a trans-C3SnO2 trigonal bipyramid, but is distorted towards a C3SnO3 skew-trapezoidal bipyramid; the tin atom is part of a five-membered SnOCCO ring in which the OSnO angle is 63.4(1)° and SnO distances are 2.191(3) and 2.890(3) A. The geometry around the inner tin atom is a less distorted trans-C3SnO2 trigonal bipyramid; the four SnOCO (Sn ··· O 3.043(3) A) atoms do not form a ring owing to the small chelate bite.

Separation of ferrichromes and other hydroxamate siderophores of fungal origin by reversed-phase chromatography

Iron(III) chelates of nineteen trihydroxamate siderophores of fungal origin, including ferrichromes, coprogen and triacetylfusarinine C, were separated on a preparative scale with a reversed-phase column using the octadecyl silica gels LRP-1 or LRP-2 as the stationary phase and a water-methanol gradient as the mobile phase. Using this system in combination with silica gel column chromatography, most siderophores can be obtained in pure form. Factors affecting the mobility of these compounds in the reversed-phase system are discussed.

Structure–stability relationships of 3-hydroxypyridin-4-one complexes

The structural features of the ligand 1-ethyl-3-hydroxy-2-methylpyridin-4-one HL1 and its tris complexes [ML13]·3H2O (M = Fe 1, Ga 2 and Al 3) have been characterized by single-crystal X-ray diffraction. The crystal structures of complexes 1–3 are isomorphous. The comparisons among HL1 and 1–3 indicate that several resonance forms play an important role in determining their structures. It is concluded that the absolute stabilities of the complexes of Al3+ and Ga3+ are similar and both greater than that of the iron(III) complex. A structural comparison between ligand HL1 and the closely related 1-butyl-3-hydroxypyridin-2-one provides an explanation for the higher affinity of 3-hydroxypyridin-4-ones for Fe3+ over 3-hydroxypyridin-2-ones. The complexes of Al3+ and Ga3+ with HL1 can be crystallized both as tri- and dodeca-hydrates. It is shown by comparing the same complex in different environments that neither the interplanar angle nor the twist angle is a reliable structural parameter to characterize a metal complex.

Nα-Dimethylcoprogens Three novel trihydroxamate siderophores from pathogenic fungi

SummaryThree novel siderophores have been isolated from a highly pathogenic strain ofAlternaria longipes (ATCC 26293). The compounds are Nα-dimethylated analogs of coprogen, neocoprogen I and isoneocoprogen I. Structures of the compounds have been determined by1H- and13C-NMR, fast-atom-bombardment (FAB) mass spectroscopy and partial hydrolysis. One of the new compounds, Nα -dimethylcoprogen, is also produced, as the major siderophore, in another fungus,Fusarium dimerum.