Solution structure of γ‐bungarotoxin: The functional significance of amino acid residues flanking the RGD motif in integrin binding

Abstract

Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins.

Effects of the RGD loop and C-terminus of rhodostomin on regulating integrin αIIbβ3 recognition

“…For example, disintegrins with an ARGD W sequence exhibit a higher affinity for binding with integrin αIIbβ3, whereas disintegrins with an ARGD N sequence preferentially bind with integrins αvβ3 and α5β1 [10]. The amino acid sequences of RGD loop of rhodostomin (Rho) was mutated from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in the inhibition of integrin αIIbβ3 [12]. Replacing the N-terminal alanine with the proline of the RGD motif of elagantin (a disintegrin with an A RGDMP sequence) diminishes its ability to bind to integrin α5β1 [13].…”

Effect of P to A Mutation of the N-Terminal Residue Adjacent to the Rgd Motif on Rhodostomin: Importance of Dynamics in Integrin Recognition

“…The RGD sequence is recognized by half of the 24 known integrins, whereas alternative short peptide sequences are recognized by other integrins [4]. In addition to adhesive proteins, the RGD sequence is found in many proteins, including dendroaspin [5], decorsin [6], savignygrin [7], streptopain [8], γ-bungarotoxin [9], human herpesvirus 8 envelope glycoprotein B [10], and disintegrins [11]. Disintegrins are the peptides found in snake venoms of the viper family and mainly inhibit the functions of β1- and β3-associated integrins.…”

Dynamics and functional differences between dendroaspin and rhodostomin: Insights into protein scaffolds in integrin recognition

“…12 The RGD motif is located at the apex of a long loop, between two b strands of the protein, protruding 10-17 Å from the protein core. [6][7][8][9][10][11][12] The flexibility and solvent exposure of the RGD loop are responsible for the recognition and fitting the interface between integrin a and b subunits. Studies 35,37,38 also show that the dynamic properties of the RGD motif may be important in its interaction with integrins.…”

“…The synergy between structure and dynamics is essential to the function of integrin complexes. [6][7][8][9][10][11][12] Many studies have compared the binding affinity of RGD-containing peptide with their 3D conformations and backbone dynamics. 27,36,38,43 As shown in Table IV, the residues of the RGD loop of these proteins exhibited extensive flexibility on fast motion on the picasecond per nanosecond time scale.…”

“…[3][4][5] Many toxins are obtained from a variety of species, such as snakes, ticks, and leeches, and contain an arginylglycyl-aspartic acid (RGD) motif with different protein scaffolds. [6][7][8][9][10][11] They specifically inhibit the integrin-binding function and are potent integrin antagonists. Many of these proteins have potential as therapeutic agents that can directly target integrins.…”