Jochen Wuerges

Vitamin B12: unique metalorganic compounds and the most complex vitamins.

The chemistry and biochemistry of the vitamin B12 compounds (cobalamins, XCbl) are described, with particular emphasis on their structural aspects and their relationships with properties and function. A brief history of B12, reveals how much the effort of chemists, biochemists and crystallographers have contributed in the past to understand the basic properties of this very complex vitamin. The properties of the two cobalamins, the two important B12 cofactors Ado- and MeCbl are described, with particular emphasis on how the Co-C bond cleavage is involved in the enzymatic mechanisms. The main structural features of cobalamins are described, with particular reference to the axial fragment. The structure/property relationships in cobalamins are summarized. The recent studies on base-off/base-on equilibrium are emphasized for their relevance to the mode of binding of the cofactor to the protein scaffold. The absorption, transport and cellular uptake of cobalamins and the structure of the B12 transport proteins, IF and TC, in mammals are reviewed. The B12 transport in bacteria and the structure of the so far determined proteins are briefly described. The currently accepted mechanisms for the catalytic cycles of the AdoCbl and MeCbl enzymes are reported. The structure and function of B12 enzymes, particularly the important mammalian enzymes methyltransferase (MetH) and methyl-malonyl-coenzymeA mutase (MMCM), are described and briefly discussed. Since fast proliferating cells require higher amount of vitamin B12 than that required by normal cells, the study of B12 conjugates as targeting agents has recently gained importance. Bioconjugates have been studied as potential agents for delivering radioisotopes and NMR probes or as various cytotoxic agents towards cancer cells in humans and the most recent studies are described. Specifically, functionalized bioconjugates are used as “Trojan horses” to carry into the cell the appropriate antitumour or diagnostic label. Possible future developments of B12 work are summarized.

Structural study on ligand specificity of human vitamin B12 transporters.

Studies comparing the binding of genuine cobalamin (vitamin B12) to that of its natural or synthetic analogues have long established increasing ligand specificity in the order haptocorrin, transcobalamin and intrinsic factor, the high-affinity binding proteins involved in cobalamin transport in mammals. In the present study, ligand specificity was investigated from a structural point of view, for which comparative models of intrinsic factor and haptocorrin are produced based on the crystal structure of the homologous transcobalamin and validated by results of published binding assays. Many interactions between cobalamin and its binding site in the interface of the two domains are conserved among the transporters. A structural comparison suggests that the determinant of specificity regarding cobalamin ligands with modified nucleotide moiety resides in the beta-hairpin motif beta3-turn-beta4 of the smaller C-terminal domain. In haptocorrin, it provides hydrophobic contacts to the benzimidazole moiety through the apolar regions of Arg357, Trp359 and Tyr362. Together, these large side chains may compensate for the missing nucleotide upon cobinamide binding. Intrinsic factor possesses only the tryptophan residue and transcobalamin only the tyrosine residue, consistent with their low affinity for cobinamide. Relative affinity constants for other analogues are rationalized similarly by analysis of steric and electrostatic interactions with the three transporters. The structures also indicate that the C-terminal domain is the first site of cobalamin-binding since part of the beta-hairpin motif is trapped between the nucleotide moiety and the N-terminal domain in the final holo-proteins.

Interactions between the nucleosome histone core and Arp8 in the INO80 chromatin remodeling complex

Actin-related protein Arp8 is a component of the INO80 chromatin remodeling complex. Yeast Arp8 (yArp8) comprises two domains: a 25-KDa N-terminal domain, found only in yeast, and a 75-KDa C-terminal domain (yArp8CTD) that contains the actin fold and is conserved across other species. The crystal structure shows that yArp8CTD contains three insertions within the actin core. Using a combination of biochemistry and EM, we show that Arp8 forms a complex with nucleosomes, and that the principal interactions are via the H3 and H4 histones, mediated through one of the yArp8 insertions. We show that recombinant yArp8 exists in monomeric and dimeric states, but the dimer is the biologically relevant form required for stable interactions with histones that exploits the twofold symmetry of the nucleosome core. Taken together, these data provide unique insight into the stoichiometry, architecture, and molecular interactions between components of the INO80 remodeling complex and nucleosomes, providing a first step toward building up the structure of the complex.

Axial bonding in alkylcobalamins: DFT analysis of the inverse versus normal trans influence.

Density functional theory has been applied to study the origin of the inverse and normal trans influence in alkylcobalamins. In order to cover the X-ray structural data available for alkylcobalamins with a variety of axial substituents, geometries of 28 related corrin-containing models have been optimized and analyzed. The BP86/6-31G(d) level of theory was applied which showed good reliability in reproducing the axial bond lengths. Comparison of experimental and calculated data allowed to conclude that the inverse trans influence is not a general feature of cobalamins, as it appeared from the experimental data analysis alone. Inverse trans influence is observed for the series of R groups with increasing bulk and electron donating ability. For the series of R groups having similar medium bulk, but differing significantly in the electron donating ability, normal trans influence was found. Finally, it was determined, that the axial bond lengths correlate well but differently in the two series of R groups with the orbital energies of the six molecular orbitals essential in axial interligand bonding.

Vitamin B12 Transport Proteins: Crystallographic Analysis of β‐axial Ligand Substitutions in Cobalamin Bound to Transcobalamin

Cobalamin (Cbl, vitamin B12) is an essential micronutrient that is synthesized only by bacteria. Mammals have developed a complex system for internalization of this vitamin from the diet. Three binding proteins (haptocorrin, intrinsic factor, transcobalamin (TC)) and several specific cell surface receptors are involved in the process of intestinal absorption, plasma transport and cellular uptake. The recent literature on the binding proteins is briefly reviewed. A structural study is presented addressing a unique feature of TC among the three proteins, i.e., the displacement of the weak Co(III)‐ligand H2O at the upper (or β) axial side of H2O‐Cbl by a histidine side chain. We have investigated crystallographically the β‐ligand exchange on Cbl bound to TC by crystallization of bovine holo‐TC in the presence of either cyanide or sulfite. The resulting electron density maps show that the histidine side chain has been displaced by an exogenous ligand CN‐ or to a lower extent than expected based on their higher affinity for Co and excess concentration with respect to histidine. This may reflect either reduced affinities of CN‐ and or the advantageous binding of the protein‐integrated His‐residue when competing for the β‐site of Cbl bound to TC. The loop hosting the histidine residue appears more flexible after disruption of the coordination bond His‐Cbl but no other differences are observed in the overall structure of holo‐TC. These structural results are discussed in relation to a possible physiological role of histidine substitution for H2O and regarding the role of β‐conjugated Cbl‐analogues recently proposed for targeted delivery of imaging agents. IUBMB Life, 59: 722‐729, 2007

X-ray studies on ternary complexes of maltodextrin phosphorylase.

We report crystal structures of ternary complexes of maltodextrin phosphorylase with natural oligosaccharide and phosphate mimicking anions: nitrate, sulphate and vanadate. Electron density maps obtained from crystals grown in presence of Al(NO3)3 show a nitrate ion instead of the expected AlF4- in the catalytic site. The trigonal NO3- is coplanar with the Arg569 guanidinium group and mimics three of the four oxygen atoms of phosphate. The ternary complex with sulphate shows a partial occupancy of the anionic site. The low affinity of the sulphate ion, observed when the alpha-glucosyl substrate is present in the catalytic channel, is ascribed to restricted space for the anion. Even lower occupancy is observed for the larger vanadate anion. The Malp/G5/VO43- structure shows the partial occupancy of the oligosaccharide and the dislocation of the 380s loop. This has been attributed to the formation of oligosaccharide vanadate derivatives (confirmed by capillary electrophoresis) that reduces their effective concentration. The difficulty to trap a ternary complex mimicking the ground state has been correlated to the apparent lower affinity that natural substrates show regarding the intermediates of the enzymatic reaction.

A potent HIV protease inhibitor identified in an epimeric mixture by high-resolution protein crystallography.

Structure-based drug design uses structural data obtained by protein crystallography, NMR spectroscopy, and computational chemistry to guide the synthesis of potential drugs. The structural information can be used to explain the basis of their activity (SAR) and to improve the potency and specificity of new lead compounds. Indeed, there are currently several drugs on the market that originated from this structure-based approach to drug design. The most commonly cited are antiHIV drugs such as amprenavir (Agenerase) and nelfinavir (Viracept), which were developed by using the structural information obtained from the X-ray crystallographic structure of HIV-1 protease, an essential hydrolase in the retroviral life cycle. The use of X-ray and NMR techniques has been extended beyond pure structural characterization to new approaches for structure-based lead discovery. 7] Both NMRand X-raybased screening of mixtures have been introduced in pharmaceutical research. In general, crystallography has the advantage over NMR techniques for the definition of ligand-binding sites. However, the detection of ligands in protein sites is highly dependent on the resolution limit of diffraction data. For a typical protein structure with resolution worse than 2 =, it may be very difficult to unambiguously recognize the ligand from inspection of the electron-density map of the catalytic site. To overcome this difficulty, a method has been developed recently that uses the soaking of a protein crystal in a cocktail of molecules with different shapes that can be easily distinguished in the electron density map. In recent years, various technical improvements, ranging from better crystallization techniques to the use of synchrotron sources and cryogenic techniques for the measurement of diffraction data, have made it possible to improve the resolution limit considerably, and biocrystallographic studies with resolution values between 1.5 and 0.9 = are becoming more frequent. At this level of resolution, the individual atoms can be clearly distinguished. This leads to the precise identification of the stereochemistry of a bound inhibitor and often provides important clues for the rationalization of its affinity and selectivity. In particular, the possibility to distinguish the stereoisomer recognized by an enzyme is crucial for the development of chiral drugs. Currently, racemates and diastereomeric mixtures are almost absent in the list of FDA-approved drugs. All potent and selective inhibitors of HIV protease are single enantiomers and were designed as mimics of the viral substrates with non-cleavable structures replacing the scissile bond. The HIV-1 protease specifically processes the gag and gag–pol polyproteins into mature viral replication enzymes (reverse transcriptase, integrase, and protease) and structural proteins (p6, p7, p17, and p24). The HIV-1 protease, unlike other proteases, is able to cleave Tyr–Pro and Phe–Pro sequences in the viral polyprotein. As the amide bonds of Pro residues are not susceptible to easy cleavage by mammalian endopeptidases, peptidomimetic molecules that incorporate non-hydrolysable Phe–Pro isosteres are expected to have the potential advantage of higher selectivity. Recently, we described a new synthetic approach to dihydroxyethylene Xaa–Pro isosteres (in which Xaa=variable residue) and applied it to the synthesis of a new generation of protease inhibitors based on a novel Phe–Pro isostere (Figure 1). The presence of two amino termini in this non-hydrolysable moiety permits the development of pseudosymmetric peptidomimetic inhibitors that can match the twofold symmetry of the homodimeric structure of the HIV-1 protease (Figure 2). The pseudo-hexapeptide TS-126, which has an acetylated Trp–Val dipeptide at both termini of the central unit (Figure 1), has IC50 values in the nanomolar range, similar to those of commercial drugs. However, during the synthesis of this new inhibitor, an epimerization of the two C (Val) stereocenters occurred and a mixture of four stereoisomers was obtained. To determine which stereoisomer is associated with the strong enzyme inhibition, biocrystallographic techniques were used to screen the epimeric mixture. The