Ca2+-dependent interface formation in fibrillin-1.
The calcium-binding epidermal growth factor-like (cbEGF) domain is a common structural motif in extracellular and transmembrane proteins. K(d) values for Ca2+ vary from the millimolar to nanomolar range; however the molecular basis for this variation is poorly understood. We have measured K(d) values for six fibrillin-1 cbEGF domains, each preceded by a transforming growth factor beta-binding protein-like (TB) domain. Using NMR and titration with chromophoric chelators, we found that K(d) values varied by five orders of magnitude. Interdomain hydrophobic contacts between TB-cbEGF domains were studied by site-directed mutagenesis and could be correlated directly with Ca2+ affinity. Furthermore, in TB-cbEGF pairs that displayed high-affinity binding, NMR studies showed that TB-cbEGF interface formation was strongly Ca2+-dependent. We suggest that Ca2+ affinity is a measure of interface formation in both homologous and heterologous cbEGF domain pairs, thus providing a measure of flexibility in proteins with multiple cbEGF domains. These data highlight the versatile role of the cbEGF domain in fine tuning the regional flexibility of proteins and provide new constraints for the organization of fibrillin-1 within 10-12-nm microfibrils of the extracellular matrix.
Jensen
Sacha A
SA
Division of Molecular and Cellular Biochemistry, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom.
Corbett
Adam R
AR
Knott
Vroni
V
Redfield
Christina
C
Handford
Penny A
PA
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D006801:Humans; D008840:Microfilament Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D016212:Transforming Growth Factor beta / Q000378:metabolism; D054392:Adipokines
D002118:Calcium; D004815:Epidermal Growth Factor; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; C000605722:FBN1 protein, human; D054392:Adipokines