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 three-dimensional structure of staphylokinase has been determined at 1.8 A. The puntative site of interaction with plasminogen was identified and epitopes were mapped.

The structure of the Mg 2+ -dependent enzyme human phosphoserine phosphatase (HPSP) was exploited to examine the structural and functional role of the divalent cation in the active site of phosphatases. Most interesting is the biochemical observation that a Ca 2+ ion inhibits the activity of HPSP, even in the presence of added Mg 2+ . Human phosphoserine phosphatase (HPSP) catalyses the last and irreversible step of the de novo biosynthesis of L-serine, i.e. the hydrolysis of phosphoserine leading to the formation of L-serine and inorganic phosphate (Pi). HPSP is a member of the haloacid dehalogenase (HAD) superfamily of which the members are characterized by three short conserved sequence motifs (Fig. 1). The residues of these motifs cluster together to form the active site. All enzymes of the HAD superfamily use the aspartate residue of the first conserved DXXX(T/V) motif as a nucleophilic residue for catalysis [1]. The second motif contains a conserved serine or threonine residue, and the third motif contains a strictly conserved lysine residue followed, at some distance, by less conserved residues and a strictly conserved aspartate. Mutagenesis studies on these conserved residues show that all three motifs play an important role in the catalytic process [2][3][4].Despite the low overall sequence homology among the enzymes of the HAD superfamily, all known structures of enzymes of this superfamily display a conserved fold [5] . Two interesting questions arise from these observations: is there structural evidence for the fact that Mg 2+ in the active site cannot be replaced by another divalent cation without loss of activity, and how does an enzyme manage to select a specific cation from the surrounding fluids that contain a broad variety of cations? A detailed study of the structure of the active site of HPSP with Ca 2+ bound may provide an insight into the biological Correspondence to A. Rabijns,

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.