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 A 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.

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.

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.

The structure of the bark lectin RPbAI (isoform A4) from Robinia pseudoacacia has been determined by protein crystallography both in the free form and complexed with N-acetylgalactosamine. The free form is refined at 1.80 A resolution to an R-factor of 18.9% whereas the complexed structure has an R-factor of 19.7% at 2.05 A resolution. Both structures are compared to each other and to other available legume lectin structures. The polypeptide chains of the two structures exhibit the characteristic legume lectin tertiary fold. The quaternary structure resembles that of the Phaseolus vulgaris lectin, the soybean agglutinin, and the Dolichos biflorus lectin, but displays some unique features leading to the extreme stability of this lectin.

1,25(OH) 2 D 3 has a central rigid CD-bicyclic ring portion to which two flexible entities, the side chain and the seco-B,Aring, are connected. The characteristic seco structure of vitamin D 3 originates from the photolytic cleavage of the provitamin 7-dehydrocholesterol. The resulting previtamin triene (previtamin D 3 ) then reversibly thermo-isomerizes to the vitamin form in its less stable 6-s-cis conformation. Rapid rotation about the C6-C7 single bond eventually yields vitamin D 3 in its more stable extended 6-s-trans conformation (2, 3). Vitamin D 3 but also its hydroxylated metabolites are in constant equilibrium with their previtamin D 3 isomers. Previous studies with analogs of 1,25(OH) 2 D 3 that are either structurally blocked in the previtamin form (4, 5) or that only slowly isomerize to the vitamin form (6) indicated that the previtamin form of 1,25(OH) 2 D 3 is a poor activator of the genomic signaling pathway with minimal effects on cell proliferation and differentiation (7). However, the previtamin D 3 form is a potent activator of the non-genomic signal transduction pathway (5-7).The aim of this study is to investigate the biological profile and the genomic signaling pathway of four different locked 19-nor-1,25(OH) 2 -(pre)-vitamin D 3 analogs, with or without a modified CD-ring. Indeed, the sigmatropic shift required for the previtamin-vitamin D conversion cannot occur in these analogs because of the deletion of C19 (4). First, the affinity to VDR was determined as well as the stability of VDR conformation after interaction with the ligand. Second, the potency of the analogs to induce interaction of the VDR-RXR heterodimer with DNA (vitamin D-responsive elements) and TIF2 (as a representative of the p160 family of coactivators; Ref. 8) was investigated. Third, the ability of 19-nor-1,25(OH) 2 -(pre)-vitamin D 3 analogs to induce VDR-dependent transcriptional activation was studied in transient transfection experiments and by determining the induction of vitamin D 24-hydroxylase (CYP 24) gene expression. Finally, docking of the analogs in the ligand binding domain (LBD) of VDR was studied by evaluation