Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.
We previously reported that the fibronectin (FN) type III domains of FN may unfold to interact with anastellin and form FN aggregates. In the present study, we have focused on the interaction between anastellin and the third FN type III domain (III3), which is a key anastellin binding site on FN. Anastellin binding to III3 was monitored by 8-anilino-1-naphthalene sulfonate (ANS) fluorescence. ANS binding to anastellin dramatically increased its emission intensity, but this was reduced to half by the addition of III3, suggesting that ANS and III3 share a common hydrophobic binding site on anastellin. An engineered mutant of III3 that was stabilized by an intrachain disulfide bond did not interact with anastellin, as seen by its failure to interfere with ANS binding to anastellin. We also mutated hydrophobic core residues to destabilize III3 and found that these mutants were still capable of interacting with anastellin. Anastellin binding to III3 was also monitored using an intramolecular green fluorescent protein (GFP)-based fluorescence resonance energy transfer (FRET) construct, in which III3 was flanked by two GFP variants (III3-FRET). Anastellin bound to III3-FRET and caused an increase in the FRET signal. The dissociation constant was estimated to be approximately 210 nM. The binding kinetics of anastellin to III3-FRET fit a first-order reaction with a half-time of approximately 30 s; the kinetics with destabilized III3 mutants were even faster. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry suggested that the middle part of III3 became destabilized and protease sensitive upon anastellin binding. Thus, the stability of III3 seems to be a key factor in anastellin binding.
Ohashi
Tomoo
T
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA. t.ohashi@cellbio.duke.edu
Augustus
Anne Marie
AM
Erickson
Harold P
HP