Differential recognition of snake venom proteins expressing specific Arg-Gly-Asp (RGD) sequence motifs by wild-type and variant integrin alphaIIbbeta3: further evidence for distinct sites of RGD ligand recognition exhibiting negative allostery.
Several studies have demonstrated that the amino acid residues flanking the Arg-Gly-Asp (RGD) sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. Because of the absence of structural data for integrin complexes with bound ligand, the molecular basis for this specificity modulation remains obscure. In a previous paper [Rahman, Lu, Kakkar and Authi (1995) Biochem. J. 312, 223-232] we demonstrated that two genetically distinct venom-derived RGD proteins, kistrin and dendroaspin (both containing the sequence PRGDMP), were simple competitors, indicating the recognition of an identical binding site on the alpha(IIb)beta(3) complex. Furthermore, both kistrin and dendroaspin inhibited the binding of the disintegrin elegantin (containing the sequence ARGDNP) via a non-competitive mechanism, suggesting that the binding of elegantin to the alpha(IIb)beta(3) complex was at a remote site and down-regulated via an allosteric mechanism. Here we present further evidence for distinct RGD ligand recognition sites on the alpha(IIb)beta(3) complex that exhibit a negative allosteric relationship. A panel of well-characterized recombinant dendroaspin and elegantin derivatives were employed for this study. These recombinant molecules were constructed as glutathione S-transferase fusion proteins with either an Ala or Pro residue N-terminal to the RGD sequence in combination with either a Met or an Asn residue immediately C-terminal. Equilibrium competition experiments showed that elegantin binding to ADP-treated platelets was inhibited by derivatives Eleg. AM (ARGDMP) and Eleg. PM (PRGDMP) via an allosteric competitive mechanism, providing direct evidence that modulation of the RGD motif can alter competitive behaviour. In addition, recombinant kistrin and dendroaspin both inhibited elegantin binding via a non-competitive mechanism, confirming our previous observations. Further evidence for distinct binding sites employing an independent approach was obtained by analysing the binding of the panel of venom proteins to the functionally defective heterodimer alpha(IIb)beta(3) Ser(123)-->Ala expressed on Chinese hamster ovary cells. These studies demonstrated that simple competitors kistrin and dendroaspin bound with high affinity to the variant integrin complex. In contrast, the binding of elegantin and most significantly, recombinant Dendro. PN (PRGDNP) and Dendro. AN (ARGDNP) were abolished. These observations, taken together, are consistent with a model depicting the presence of distinct sites of RGD ligand recognition on the alpha(IIb)beta(3) complex that show the preferential recognition of specific RGD motifs. Competition experiments demonstrate a negative allosteric relationship between these RGD recognition sites.
Rahman
S
S
The Coagulation Research Laboratory, GKT Medical School, St. Thomas' Hospital, Lambeth Palace Road, London SE1 7EH, U.K. s.rahman@kcl.ac.uk
Flynn
G
G
Aitken
A
A
Patel
Y
Y
Hussain
F
F
Lu
X
X
Loftus
J C
JC
French
D
D
Wijelath
E
E
Strand
K
K
Savidge
G F
GF
D000409:Alanine; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells / Q000378:metabolism; D006224:Cricetinae; D004546:Elapid Venoms / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine; D012910:Snake Venoms / Q000737:chemistry*
D004546:Elapid Venoms; D009842:Oligopeptides; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011993:Recombinant Fusion Proteins; D012910:Snake Venoms; C064594:elegantin; C074772:mambin; D012694:Serine; C047981:arginyl-glycyl-aspartic acid; C067623:kistrin; D000409:Alanine