Camiel J. De Ranter

A structural basis for the unique binding features of the human vitamin D-binding protein.

The human serum vitamin D-binding protein (DBP) has many physiologically important functions, ranging from transporting vitamin D3 metabolites, binding and sequestering globular actin and binding fatty acids to functioning in the immune system. Here we report the 2.3 Å crystal structure of DBP in complex with 25-hydroxyvitamin D3, a vitamin D3 metabolite, which reveals the vitamin D-binding site in the N-terminal part of domain I. To more explicitly explore this, we also studied the structure of DBP in complex with a vitamin D3 analog. Comparisons with the structure of human serum albumin, another family member, reveal a similar topology but also significant differences in overall, as well as local, folding. These observed structural differences explain the unique vitamin D3-binding property of DBP.

Structural Basis for Inhibition of Aspergillus niger Xylanase by Triticum aestivum Xylanase Inhibitor-I

Plants developed a diverse battery of defense mechanisms in response to continual challenges by a broad spectrum of pathogenic microorganisms. Their defense arsenal includes inhibitors of cell wall-degrading enzymes, which hinder a possible invasion and colonization by antagonists. The structure of Triticum aestivum xylanase inhibitor-I (TAXI-I), a first member of potent TAXI-type inhibitors of fungal and bacterial family 11 xylanases, has been determined to 1.7-Å resolution. Surprisingly, TAXI-I displays structural homology with the pepsin-like family of aspartic proteases but is proteolytically nonfunctional, because one or more residues of the essential catalytical triad are absent. The structure of the TAXI-I·Aspergillus niger xylanase I complex, at a resolution of 1.8 Å, illustrates the ability of tight binding and inhibition with subnanomolar affinity and indicates the importance of the C-terminal end for the differences in xylanase specificity among different TAXI-type inhibitors.

X-Ray and theoretical structural study of novel 5,6,7,8-tetrahydrobenzo-4H-pyrans

Abstract The X-ray crystal structure of three substituted tetrahydrobenzo-4H-pyrans ( 6a–c ) is presented and the experimental findings compared with the theoretical values calculated at the semiempirical (AM1, PM3) and ab initio (HF/6-31G∗) levels. A good agreement between both experimental and theoretical data is found showing a flattened boat conformation for the 4H-pyran ring and a pseudo-axial orientation of the aryl ring on the C4 position. The geometrical features of these systems ( 6a–c ) reveal that they present the structural requirements to act as calcium antagonists.

His374 of wheat endoxylanase inhibitor TAXI-I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases

Wheat endoxylanase inhibitor TAXI‐I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase‐TAXI‐I complex showed His374 of TAXI‐I to be a key residue in endoxylanase inhibition [Sansen S, De Ranter CJ, Gebruers K, Brijs K, Courtin CM, Delcour JA & Rabijns A (2004) J Biol Chem 279, 36022–36028]. Its role in enzyme–inhibitor interaction was further investigated by site‐directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatumendoxylanases and wild‐type TAXI‐I and TAXI‐I His374 mutants were determined by surface plasmon resonance analysis. Enzyme–inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild‐type TAXI‐I towards the endoxylanases were in the range between 1.96 and 36.1 × 104m−1·s−1 and 0.72–3.60 × 10−4·s−1, respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI‐I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three‐ to 80‐fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI‐I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase–TAXI‐I complex rather than in the docking of inhibitor onto enzyme.

Actin-DBP: the perfect structural fit?

The multifunctional vitamin D binding protein (DBP) is an actin-sequestering protein present in blood. The crystal structure of the actin-DBP complex was determined at 2.4 A resolution. DBP binds to actin subdomains 1 and 3 and occludes the cleft at the interface between these subdomains. Most remarkably, DBP demonstrates an unusually large actin-binding interface, far exceeding the binding-interface areas reported for other actin-binding proteins such as profilin, DNase I and gelsolin. The fast-growing side of actin monomers is blocked completely through a perfect structural fit with DBP, demonstrating how DBP effectively interferes with actin-filament formation. It establishes DBP as the hitherto best actin-sequestering protein and highlights its key role in suppressing and preventing extracellular actin polymerization.

Structural analysis of the jacalin-related lectin MornigaM from the black mulberry (Morus nigra) in complex with mannose

The structures of MornigaM and the MornigaM–mannose complex have been determined at 1.8 Å and 2.0 Å resolution, respectively. Both structures adopt the typical β‐prism motif found in other jacalin‐related lectins and their tetrameric assembly closely resembles that of jacalin. The carbohydrate‐binding cavity of MornigaM readily binds mannose. No major structural rearrangements can be observed in MornigaM upon binding of mannose. These results allow corroboration of the structure–function relationships within the small group of Moraceae lectins.

Polarographic evidence for the interaction of reduced nitroimidazole derivatives with DNA bases

Nitroimidazole drugs require reductive activation to exert antimicrobial and mutagenic activity (e.g. antiprotozoal, antitumour). The polarographic behaviour of several nitroimidazoles has been investigated in the presence and in the absence of the potential biological targets (i.e. adenine, guanine, cytosine and thymine). Adenine, guanine and cytosine caused the half-wave potential to shift in the positive direction. Thymine, however, caused negative shifts of the half-wave potentials. Adsorption is not the cause of the positive shifts observed but is clearly the cause of the negative shift induced by thymine. We conclude that primarily adenine and guanine are susceptible for interaction with reduced nitroimidazoles. The results also showed that the stability of the responsible intermediates is related to the value of the half-wave potential of the parent drug.

High-resolution structure of human phosphoserine phosphatase in open conformation.

The crystal structure of human phosphoserine phosphatase (HPSP) in the open conformation has been determined at a resolution of 1.53 A. The crystals are orthorhombic, belonging to space group C222(1), with unit-cell parameters a = 49.03, b = 130.25, c = 157.29 A. The asymmetric unit contains two molecules. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using a selenomethionine-derivative crystal of HPSP. The structure was refined using CNS to a final crystallographic R value of 21.6% (R(free) = 23.4%). HPSP is a dimeric enzyme responsible for the third and final step of the l-serine biosynthesis pathway. It catalyses the Mg2+-dependent hydrolysis of l-phosphoserine. Recently, the structure of HPSP in complex with an inhibitor bound to the active site has been reported to be the open conformation of the enzyme. Here, the structure of HPSP is reported in the absence of substrate in the active site. Evidence is presented that HPSP in an uncomplexed form is in an even more open conformation than in the inhibitor complex. In this state, the enzyme is partially unfolded to allow the substrate to enter the active site. Binding of the substrate causes HPSP to shift to the closed conformation by stabilizing the partially unfolded region. In the present structure a Ca2+ ion is bound to the active site and an explanation is given why HPSP is not active when in the active site Mg2+ is replaced by a Ca2+ ion.

Crystallization and preliminary X-ray diffraction study of a cell-wall invertase from Arabidopsis thaliana.

Cell-wall invertase 1 (AtcwINV1), a plant protein from Arabidopsis thaliana which is involved in the breakdown of sucrose, has been crystallized in two different crystal forms. Crystal form I grows in space group P3(1) or P3(2), whereas crystal form II grows in space group C222(1). Data sets were collected for crystal forms I and II to resolution limits of 2.40 and 2.15 A, respectively.

Crystallization and X-ray investigation of vitamin D-binding protein from human serum. Identification of the crystal content.

Vitamin D-binding protein (DBP), a multifunctional, highly polymorphic glycoprotein responsible for the transport of vitamin D and for sequestering extracellular actin, was isolated from human serum and crystallized using vapour diffusion methods. The crystals were grown from 7.5% v/v polyethylene glycol 400 and 0.1 M acetate buffer at pH 4.6. These crystals show diffraction patterns consistent with the tetragonal space groups P4(1) and P4(3) with unit cell dimensions a = b = 135.5(4) A and c = 75.9(4) A. They diffract to 2.3 A. Using polyacrylamide gel electrophoresis it was shown that according to their electrophoretic mobility the O-glycosylated isoforms, with a terminal sialic acid residue, are absent in the crystals.