Dimer model for the microfibrillar protein fibulin-2 and identification of the connecting disulfide bridge.
Fibulin-2 is a novel extracellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrillin. The entire protein and its predicted domains were obtained as recombinant products and examined by ultracentrifugation and electron microscopy. This demonstrated a disulfide-linked homodimer of 175 kDa subunits. Partial reduction to monomers identified specifically an odd Cys574 residue responsible for dimer formation in one of three anaphylatoxin-like modules that constitute the central globular domain I (13 kDa) of fibulin-2. Furthermore, a Cys574-Ser mutation abolished disulfide connection but not non-covalent dimerization of fibulin-2. The C-terminal region (85 kDa) was shown to represent a 35-nm-long rod consisting of 11 calcium-binding EGF-like modules (domain II) and a small terminal globe (domain III). The unique N-terminal domain N (55 kDa) was also rod-shaped (approximately 38 nm) and rich in galactosamine indicating extensive O-glycosylation. A dimer model is proposed indicating mainly a rod-like shape of 80 nm length based on an anti-parallel association of two subunits through their domains I. This model also implies alignment of domains II and N between different subunits. This was demonstrated by surface plasmon resonance assay which showed a distinct interaction between domains N and II with a Kd of approximately 0.7 microM.
Sasaki
T
T
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Mann
K
K
Wiedemann
H
H
Göhring
W
W
Lustig
A
A
Engel
J
J
Chu
M L
ML
Timpl
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D003553:Cystine / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D014461:Ultracentrifugation
D002135:Calcium-Binding Proteins; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C084612:fibulin 2; D003553:Cystine