Erhard Hohenester

Crystal structure of the angiogenesis inhibitor endostatin at 1.5 Å resolution

A number of extracellular proteins contain cryptic inhibitors of angiogenesis. Endostatin is a 20 kDa C‐terminal proteolytic fragment of collagen XVIII that potently inhibits endothelial cell proliferation and angiogenesis. Therapy of experimental cancer with endostatin leads to tumour dormancy and does not induce resistance. We have expressed recombinant mouse endostatin and determined its crystal structure at 1.5 Å resolution. The structure reveals a compact fold distantly related to the C‐type lectin carbohydrate recognition domain and the hyaluronan‐binding Link module. The high affinity of endostatin for heparin is explained by the presence of an extensive basic patch formed by 11 arginine residues. Endostatin may inhibit angiogenesis by binding to the heparan sulphate proteoglycans involved in growth factor signalling.

Crystal structure of a pair of follistatin-like and EF-hand calcium-binding domains in BM-40

BM‐40 (also known as SPARC or osteonectin) is an anti‐adhesive secreted glycoprotein involved in tissue remodelling. Apart from an acidic N‐terminal segment, BM‐40 consists of a follistatin‐like (FS) domain and an EF‐hand calcium‐binding (EC) domain. Here we report the crystal structure at 3.1 Å resolution of the FS–EC domain pair of human BM‐40. The two distinct domains interact through a small interface that involves the EF‐hand pair of the EC domain. Residues implicated in cell binding, inhibition of cell spreading and disassembly of focal adhesions cluster on one face of BM‐40, opposite the binding epitope for collagens and the N‐linked carbohydrate. The elongated FS domain is structurally related to serine protease inhibitors of the Kazal family. Notable differences are an insertion into the inhibitory loop in BM‐40 and a protruding N‐terminal β‐hairpin with striking similarities to epidermal growth factor. This hairpin is likely to act as a rigid spacer in proteins containing tandemly repeated FS domains, such as follistatin and agrin, and forms the heparin‐binding site in follistatin.

Crystal structure of a scavenger receptor cysteine-rich domain sheds light on an ancient superfamily.

Scavenger receptor cysteine-rich (SRCR) domains are found widely in cell surface molecules and in some secreted proteins, where they are thought to mediate ligand binding. We have determined the crystal structure at 2.0 Å resolution of the SRCR domain of Mac-2 binding protein (M2BP), a tumor-associated antigen and matrix protein. The structure reveals a curved six-stranded β-sheet cradling an α-helix. Structure-based sequence alignment demonstrates that the M2BP SRCR domain is a valid template for the entire SRCR protein superfamily. This allows an interpretation of previous mutagenesis data on ligand binding to the lymphocyte receptor CD6.

Crystal structure and mapping by site‐directed mutagenesis of the collagen‐binding epitope of an activated form of BM‐40/SPARC/osteonectin

The extracellular calcium‐binding domain (positions 138–286) of the matrix protein BM‐40 possesses a binding epitope of moderate affinity for several collagen types. This epitope was predicted to reside in helix αA and to be partially masked by helix αC. Here we show that deletion of helix αC produces a 10‐fold increase in collagen affinity similar to that seen after proteolytic cleavage of this helix. The predicted removal of the steric constraint was clearly demonstrated by the crystal structure of the mutant at 2.8 Å resolution. This constitutively activated mutant was used to map the collagen‐binding site following alanine mutagenesis at 13 positions. Five residues were crucial for binding, R149 and N156 in helix αA, and L242, M245 and E246 in a loop region connecting the two EF hands of BM‐40. These residues are spatially close and form a flat ring of 15 Å diameter which matches the diameter of a triple‐helical collagen domain. The mutations showed similar effects on binding to collagens I and IV, indicating nearly identical binding sites on both collagens. Selected mutations in the non‐activated mutant ΔI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM‐40.

Extracellular calcium-binding proteins.

Point mutations in Ca2+-binding sites of extracellular matrix proteins have been identified as the cause of human disorders such as Marfansyndrome and pseudoachondroplasia. Although the modes of Ca2+ binding and the effects of point mutations are not yet understood in these two cases, new insight was recently gained by X-ray and NMR structure determinations of several other extracellular proteins; these studies revealed a diversity of functions of Ca2+ ions. Ca2+ may induce a profound conformational change within a single domain, may bridge adjacent domains and thus direct the relative domain orientation and supramolecular structure, or may be involved in carbohydrate and membrane binding.

Human serum amyloid P component is a single uncomplexed pentamer in whole serum.

BackgroundSerum amyloid P component (SAP) is a universal constituent of amyloid deposits and contributes to their pathogenesis. SAP also has important normal functions in the handling of chromatin in vivo and resistance to bacterial infection. The atomic resolution crystal structure of SAP is known, but its physiological oligomeric assembly remains controversial. In the absence of calcium, isolated human SAP forms stable decamers composed of two cyclic disk-like pentamers interacting face to face. However, in the presence of its specific low molecular weight ligands and calcium, SAP forms stable pentamers. In the presence of calcium, but without any ligand, isolated human SAP aggressively autoaggregates and precipitates, imposing severe constraints on methods for molecular mass determination.Materials and MethodsGel filtration chromatography and density gradient ultracentrifugation were used to compare SAP with the closely related molecule, C-reactive protein (CRP; which is known to be a single pentamer) and the effect of human serum albumin on SAP autoaggregation was investigated.ResultsIn most physiological buffers and with the necessary absence of calcium, SAP, whether isolated or from whole serum samples, eluted from gel filtration columns clearly ahead of CRP. This is consistent with the existence of a monodisperse population of SAP decamers, as previously reported. However, in Tris/phosphate buffer, SAP was pentameric, suggesting that decamerization involved ionic interactions. On density gradients formed in undiluted normal human serum, SAP sedimented as single pentamers not complexed with any macromolecular ligand, regardless of the presence or absence of calcium. The calcium-dependent autoaggregation of isolated SAP was completely inhibited by physiological concentrations of albumin and the SAP remained pentameric.ConclusionsHuman SAP exists within serum as single uncomplexed pentamers in the presence or absence of calcium. This oligomeric assembly, thus, does not require a calcium-dependent small molecule interaction. The usual >2000-fold molar excess of albumin over SAP in plasma is apparently sufficient to keep SAP in its physiological conformation.

Structural and functional aspects of calcium binding in extracellular matrix proteins.

Ca2+ ions play crucial roles in many matrix-matrix, cell-matrix and cell-cell contacts. Recent X-ray and NMR structure determinations have revealed an intriguing diversity of Ca(2+)-binding sites in extracellular proteins, ranging from the stabilization of isolated domains to intimate involvement in the superstructure of macromolecular assemblies. The central role of Ca2+ in extracellular proteins is illustrated by the molecular characterization of hereditary connective tissue disorders in humans. Point mutations of Ca(2+)-binding residues in fibrillin and cartilage oligomeric matrix protein are responsible for Marfan syndrome and pseudoachondroplasia, respectively. We also discuss the possibility that structure and function of extracellular proteins may be regulated by physiologically relevant Ca2+ gradients.