Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM + HDL contained S1P, whereas ApoM − HDL did not. Moreover, HDL in Apom −/− mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM + HDL induced S1P 1 receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM − HDL did not. Importantly, lack of S1P in the HDL fraction of Apom −/− mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P 1 receptor on endothelial cells, is a vasculoprotective constituent of HDL.endothelial function | crystal structure | sphingolipids | vascular permeability | atherosclerosis S phingosine-1-phosphate (S1P), the phosphorylated metabolite of D-sphingosine, binds to five G protein-coupled receptors (S1P 1 -S1P 5 ) and regulates a plethora of biological actions (1-6). In particular, the prototypical S1P 1 receptor is essential for vascular maturation during development and promotes endothelial cell migration, angiogenesis, and barrier functions (7-9). Thus, S1P is required for maintenance of the barrier property of the lung endothelium (10). Plasma S1P, which is derived from several cellular sources (11,12), is associated with HDL (∼65%) and albumin (∼35%) (3, 5). HDLinduced vasorelaxation as well as barrier-promoting and prosurvival actions on the endothelium have been attributed to S1P signaling (2, 4, 13). Hence, much of the endothelium-protective actions of HDL may result from the actions of S1P on the endothelial S1P receptors. However, the molecular nature of the S1P binding to HDL and interaction with S1P receptors has not been characterized.Apolipoprotein M (apoM) is a lipocalin that resides mainly in the plasma HDL fraction (14). The retained hydrophobic NH 2 -terminal signal peptide anchors apoM in the phospholipid layer of the lipoprotein and prevents filtration of the ∼22-kDa protein in the kidney (15). The biological functions of apoM are understood only partly. Studies in apoM gene-modified mice suggest that apoM has antiatherogenic effects, possibly related in part to apoM's ability to increase cholesterol efflux from macrophage foam cells, to increased preβ-HDL formation, and to antioxidative effects (16)(17)(18). The recent elucidation of the crystal structure of human recombinant apoM (r-apoM) demonstrated a typical lipocalin fold characterized by an eightstranded antiparallel β-barrel enclosing an internal binding pocket that probably facilitates binding of small lipophilic ligands (19).Indeed, r-apoM expressed in Escherichia coli was found to co...

Vascular endothelial growth factor (VEGF) is a homodimeric member of the cystine knot family of growth factors, with limited sequence homology to platelet-derived growth factor (PDGF) and transforming growth factor ␤2 (TGF-␤). We have determined its crystal structure at a resolution of 2.5 Å, and identified its kinase domain receptor (KDR) binding site using mutational analysis. Overall, the VEGF monomer resembles that of PDGF, but its N-terminal segment is helical rather than extended. The dimerization mode of VEGF is similar to that of PDGF and very different from that of TGF-␤. Mutational analysis of VEGF reveals that symmetrical binding sites for KDR are located at each pole of the VEGF homodimer. Each site contains two functional ''hot spots'' composed of binding determinants presented across the subunit interface. The two most important determinants are located within the largest hot spot on a short, three-stranded sheet that is conserved in PDGF and TGF-␤. Functional analysis of the binding epitopes for two receptor-blocking antibodies reveal different binding determinants near each of the KDR binding hot spots.

Our findings suggest that the character of antigen-antibody interfaces is similar to that of other protein-protein interfaces, such as ligand-receptor interactions; in the case of VEGF, the principal difference is that the residues essential for binding to the Fab fragment are concentrated in one continuous segment of polypeptide chain, whereas those essential for binding to the receptor are distributed over four different segments and span across the dimer interface.

A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

Pyruvate oxidase from Lactobacillus plantarum is a tetrameric enzyme that decarboxylates pyruvate, producing hydrogen peroxide and the energy-storage metabolite acetylphosphate. Structure determination at 2.1 angstroms showed that the cofactors thiamine pyrophosphate (TPP) and flavin adenine dinucleotide (FAD) are bound at the carboxyl termini of six-stranded parallel beta sheets. The pyrophosphate moiety of TPP is bound to a metal ion and to a beta alpha alpha beta unit corresponding to an established sequence fingerprint. The spatial arrangement of TPP and FAD suggests that the oxidation of the oxyethyl intermediate does not occur by hydride displacement but rather by a two-step transfer of two electrons.