Zinc-dependent dimers observed in crystals of human endostatin

Abstract

The crystal structure of human endostatin reveals a zinc-binding site. Atomic absorption spectroscopy indicates that zinc is a constituent of both human and murine endostatin in solution. The human endostatin zinc site is formed by three histidines at the N terminus, residues 1, 3, and, 11, and an aspartic acid at residue 76. The N-terminal loop ordered around the zinc makes a dimeric contact in human endostatin crystals. The location of the zinc site at the amino terminus, immediately adjacent to the precursor cleavage site, suggests the possibility that the zinc may be involved in activation of the antiangiogenic activity following cleavage from the inactive collagen XVIII precursor or in the cleavage process itself.

Figure 1

The structure of human endostatin. β-strands (cyan) are labeled in sequential order A–P, α helices are violet, and connecting loops are pink. Residues 1–6 are blue; zinc is a black circle. Human and murine endostatin are very similar (rms deviation = 0.46 Å for 196 Cα pairs; cf. Fig. 3B in ref. 3).

Figure 2

The N-terminal loop and zinc binding site of human endostatin. The zinc (black circle) ligands histidine-1, -3, and -11 and aspartic acid-76, as well as the second shell of interactions that position the zinc ligands, glutamic acid-175, residue 11 carbonyl oxygen, and arginine-4 from the N-terminal loop of an adjacent molecule in a dimer are shown as ball-and-stick models. The zinc is coordinated approximately tetrahedrally between the three histidines and one aspartic acid. Both oxygens of the aspartic acid may bond to the zinc, as shown, but the electron density at this resolution is not unambiguous.

Figure 3

A zinc dependent dimer in human endostatin crystals. (A) The zinc (black circle) site N-terminal loops of two monomers contact across a central dyad axis (colors as in Fig. 1). Glutamine-7, phenylalanine-6, and arginine-4 of the loop project from one monomer to the next. Also shown are two phenylalanine rings, residues 31 and 34, that project from an endostatin α helix and form another dimeric contact in the crystal (see text). (B) Contacts at the interface of the dimer seen in crystals of human endostatin. Zinc (black sphere) ligands have open bonds, interface residues have solid colored bonds. The path of the polypeptide chains of the two monomers are shown as pink and green tubes. The solvent accessible surface buried in this dimer interface is 403 Ų per monomer (probe size = 1.4 Å).

Figure 4

A positively charged surface formed by arginines on the human endostatin dimer. Stereo diagram, surface glutamines (yellow), asparagines (cyan), lysines (green), and arginines (blue) are shown.

Figure 5

Electron density of the zinc site. This electron density map was calculated with the terminal residues 1–9 and 178–183 omitted, using the murine molecular replacement model. The map was iteratively averaged (fourfold) and solvent flattened with a molecular envelope determined from the final structure.