Conformational changes in the A1 domain of von Willebrand factor modulating the interaction with platelet glycoprotein Ibalpha.
The interaction between von Willebrand factor (vWF) A1 domain and platelet glycoprotein Ib alpha occurs in the presence of high shear stress or when vWF becomes immobilized onto a surface but not appreciably in the normal circulation. To investigate the structural properties regulating A1 domain function, we have used recombinant fragments prepared either in cyclic form with oxidized Cys509-Cys695 disulfide bond or reduced and alkylated. Interaction with glycoprotein Ibalpha was assessed by testing inhibition of monoclonal antibody LJ-Ib1 binding to platelets and inhibition of shear-induced platelet aggregation mediated by native vWF. Fragments exposed to pH between 2.5 and 3.5 adopted the molten globule conformation with loosened tertiary structure intermediate between native and completely unordered state. Maximal receptor binding activity was observed when fragments kept at acidic pH, particularly after reduction of the Cys509-Cys695 disulfide bond, were subjected to quick refolding by rapid pH increase. In contrast, slow refolding by incremental pH change over several hours resulted in at least 20-fold lower activity. A specific single point mutation (I546V) resulted in enhanced receptor binding, whereas another mutation (S561G) caused markedly reduced binding. These results provide experimental evidence that conformational transitions can modulate function of the vWF A1 domain in solution.
Miyata
S
S
Roon Research Center for Arteriosclerosis and Thrombosis, Scripps Research Institute, La Jolla, California 92037.
Goto
S
S
Federici
A B
AB
Ware
J
J
Ruggeri
Z M
ZM
D000478:Alkylation; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D014364:Tryptophan