Mutation of a critical tryptophan to lysine in avidin or streptavidin may explain why sea urchin fibropellin adopts an avidin-like domain.
Sea urchin fibropellins are epidermal growth factor homologues that harbor a C-terminal domain, similar in sequence to hen egg-white avidin and bacterial streptavidin. The fibropellin sequence was used as a conceptual template for mutation of designated conserved tryptophan residues in the biotin-binding sites of the tetrameric proteins, avidin and streptavidin. Three different mutations of avidin, Trp-110-Lys, Trp-70-Arg and the double mutant, were expressed in a baculovirus-infected insect cell system. A mutant of streptavidin, Trp-120-Lys, was similarly expressed. The homologous tryptophan to lysine (W-->K) mutations of avidin and streptavidin were both capable of binding biotin and biotinylated material. Their affinity for the vitamin was, however, significantly reduced: from K(d) approximately 10(-15) M of the wild-type tetramer down to K(d) approximately 10(-8) M for both W-->K mutants. In fact, their binding to immobilized biotin matrices could be reversed by the presence of free biotin. The Trp-70-Arg mutant of avidin bound biotin very poorly and the double mutant (which emulates the fibropellin domain) failed to bind biotin at all. Using a gel filtration fast-protein liquid chromatography assay, both W-->K mutants were found to form stable dimers in solution. These findings may indicate that mimicry in the nature of the avidin sequence and fold by the fibropellins is not designed to generate biotin-binding, but may serve to secure an appropriate structure for facilitating dimerization.
Laitinen
O H
OH
Department of Biological and Environmental Science, University of Jyväskylä, FIN-40351, Jyväskylä, Finland.
Airenne
K J
KJ
Marttila
A T
AT
Kulik
T
T
Porkka
E
E
Bayer
E A
EA
Wilchek
M
M
Kulomaa
M S
MS
D000818:Animals; D001360:Avidin / Q000235:genetics*; D001665:Binding Sites; D001710:Biotin / Q000235:genetics; D002853:Chromatography, Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000235:genetics*; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D007700:Kinetics; D008239:Lysine / Q000235:genetics*; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics; D012617:Sea Urchins; D019809:Streptavidin / Q000235:genetics*; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000235:genetics*
D016326:Extracellular Matrix Proteins; D011994:Recombinant Proteins; C062354:fibropellin protein, sea urchin; D001360:Avidin; D004815:Epidermal Growth Factor; D001710:Biotin; D014364:Tryptophan; D019809:Streptavidin; D008239:Lysine