PDBsum entry 1nub

Extracellular module

PDB id: 1nub

Contents

Protein chain

226 a.a. *

Ligands

NAG-NAG ×2

Metals

_CA ×6

* Residue conservation analysis

PDB id:

1nub

Name:

Extracellular module

Title:

Helix c deletion mutant of bm-40 fs-ec domain pair

Structure:

Basement membrane protein bm-40. Chain: a, b. Fragment: fs-ec domain pair, fs, follistatin-like, ec, extracellular calcium-binding. Synonym: osteonectin, sparc, secreted protein acidic and rich in cysteine. Engineered: yes. Mutation: yes. Other_details: helix c (residues 196-203) deletion mutant

Source:

Homo sapiens. Human. Organism_taxid: 9606. Cell_line: 293-ebna. Organ: kidney. Expressed in: homo sapiens. Expression_system_taxid: 9606. Expression_system_cell_line: 293-ebna

Resolution:

2.80Å R-factor: 0.255 R-free: 0.306

Authors:

E.Hohenester,T.Sasaki,R.Timpl

Key ref:

T.Sasaki et al. (1998). Crystal structure and mapping by site-directed mutagenesis of the collagen-binding epitope of an activated form of BM-40/SPARC/osteonectin. Embo J, 17, 1625-1634. PubMed id: 9501084 DOI: 10.1093/emboj/17.6.1625

Date:

05-Dec-97 Release date: 30-Dec-98

Protein chains

P09486  (SPRC_HUMAN) -  SPARC from Homo sapiens

Seq: 303 a.a.

Struc: 226 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1093/emboj/17.6.1625

Embo J 17:1625-1634 (1998)

PubMed id: 9501084

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

T.Sasaki, E.Hohenester, W.Göhring, R.Timpl.

ABSTRACT

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 alphaA and to be partially masked by helix alphaC. Here we show that deletion of helix alphaC 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 A 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 alphaA, 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 A 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 DeltaI also reduced collagen binding, consistent with the same location of the epitope but in a more cryptic form in intact BM-40.

Selected figure(s)

Figure 2.
Figure 2 Structure of BM-40 I, C. (A) Two orthogonal views (Kraulis, 1991) related by a rotation of 90° about the vertical axis. The FS domain is in green and the EC domain is in red. The calcium ions bound to the EF hand pair in the EC domain (Ca1 and Ca2) are in yellow; the calcium ion bound at the tip of the E - F loop (Ca3) is in blue. Helices in the EC domain are labelled. Note that five residues around the deletion site (marked by a

Figure 3.
Figure 3 Stereo view (Kraulis, 1991) of a C[ ]trace of the BM-40 I, C structure showing the residues that were mutated in this study (see text). Disulfide bridges are shown with thick bonds. Mutated residues are identified by position numbers and their side chains. The three calcium ions are shown as black spheres.

The above figures are reprinted from an Open Access publication published by Macmillan Publishers Ltd: Embo J (1998, 17, 1625-1634) copyright 1998.

Figures were selected by an automated process.