Structure-activity relationships of the RGD loop linker region and C-terminus of Rhodostomin mutants in the recognition of integrins

Abstract

Integrins are ?β heterodimeric receptors that mediate cell-cell and cell-extracellular matrix interactions Because integrins are involved in tumor progression thrombosis and osteoporosis they are important therapeutic targets Disintegrins are a family of potent integrin inhibitors that found in snake venoms Rhodostomin (Rho) is a disintegrin containing a 48PRGDMP motif a 39SRAGKICRI linker region and a 65PRYH C-terminus with six disulfide bonds In this dissertation I used Rho as protein scaffold to study the interactions between integrins and disintegrins Using cell adhesion assay nuclear magnetic resonance (NMR) spectroscopy X-ray crystallography and molecular docking four structure-activity relationships between integrins and Rho mutants were identified: (1) the 48ARGDWN motif and C-terminus of Rho mutants acted synergistically and regulated the recognition of integrin ?IIbβ3 To study the roles of the RGD loop and C-terminal region in disintegrins we expressed Rho 48PRGDMP and 48ARGDWN mutants in Pichia pastoris containing 65P 65PR 65PRYH 65PRNGLYG and 65PRNPWNG C-terminal sequences The effect of C-terminal region on their integrin binding affinities was ?IIbβ3 > ?Vβ3 > ?5β1 The 48ARGDWN-65PRNPWNG protein was the most selective integrin ?IIbβ3 mutant; however the 48PRGDMP-65PRNPWNG mutant did not exhibit any integrin selectivity NMR structural analyses of 48ARGDWN-65PRYH 48ARGDWN-65PRNGLYG and 48ARGDWN-65PRNPWNG mutants demonstrated that their C-terminal regions interacted with the RGD loop In particular the W52 sidechain of 48ARGDWN-65PRYH 48ARGDWN-65PRNGLYG and 48ARGDWN-65PRNPWNG interacted with H68 of 65PRYH L69 of 65PRNGLYG and N70 of 65PRNPWNG respectively The docking of the 48ARGDWN-65PRNPWNG mutant into integrin ?IIbβ3 indicated that the N70 residue formed hydrogen bonds with the ?IIb D159 residue and the W69 residue formed cation-pi interaction with the β3 K125 residue Our results demonstrated that the RGD loop and C-terminus of disintegrins acted in a synergistic manner resulting in their functional and structural differences in integrin binding; (2) Rho 48ARGDDP mutant selectively inhibited integrin ?Vβ3 To study the role of the C-terminal residue adjacent to the ARGD motif we expressed Rho 48ARGD52XP mutants The effect of the 52 residue position on their integrin binding activities was ?5β1 (86-fold) > ?IIbβ3 (41-fold) > ?Vβ3 (14-fold) The 48ARGDDP mutant was integrin ?Vβ3-specific mutant and inhibited integrins ?Vβ3 ?IIbβ3 ?Vβ5 ?Vβ6 and ?5β1 with the IC50 values of 45 3 5117 2 6886 14980 and 5117 2 nM X-ray structural analysis showed that the distance between the ? carbons of Arg49 and Asp52 was 5 9 ? that is consistent with the structural requirement for integrin ?Vβ3-specific antagonist The Met to Asp mutation caused a negative surface charge which might be related to the lower activities toward integrins ?IIbβ3 ?Vβ5 ?Vβ6 and ?5β1 In vitro study showed that Rho 48ARGDDP mutant inhibited HUVEC migration and tube formation in a dose-dependent manner suggesting its potential use as an anti-angiogenic agent; (3) the M to P mutation of the C-terminal residue adjacent to the ARGD motif abolished its binding to integrins The 48ARGDPP mutant was an inactive integrin antagonist which inhibited integrins ?Vβ3 ?5β1 ?IIbβ3 ?Vβ5 and ?Vβ6 with the IC50 values of 41260 62460 64665 35247 and 15055 3 nM X-ray structure analysis showed that Rho 48ARGDPP mutant has two conformations and the P52 residue caused conformational change of the RGD motif Met to Pro mutation in residue 52 caused the cis formation of D51-P52 peptide bond in conformer A and that of P52-P53 in conformer B The distance between the ? carbons of Arg49 and Pro52 was increased up to 8 0 ? indicating the disruption of turn conformation in the RGDX motif caused by the P52 residue According to Ramachandran plot analysis the P52 mutation modulated the D51 residue into an extended conformation and resulted in the loss of function of the RGDX motif Our results demonstrated that the importance of the turn conformation in the RGDX motif of integrin ligands for integrin recognition; and (4) Rho 39KKARTICAR-48GRGDNP-65PRYH (KG) and 39KKARTICAR-48GRGDNP-65PGLYG (KG-P) mutants exhibited lower ?IIbβ3 integrin inhibitory activity The inhibitory activities of platelet aggregation by KG and KG-P mutants were 56 and 384 times lower than that by Rho X-ray structural analyses of KG and KG-P mutants showed that their C-terminal regions interacted with the RGD loop: the R56 residue interacted with the Y67 residue in KG mutant and the D55 residue interacted with the L67 and Y68 residues as well as the R49 and N52 residues interacted with Y68 residue in KG-P mutant They had relatively narrower RGD loop and different electrostatic surface in comparison with those of Rho The docking experiments showed that the positive charge patch formed by the R46 and R66 residues of Rho had salt bridge interactions with the negative charge D159 on the insert-3 region of ?IIb subunit KG and KG-P mutants did not have the positive charge patch due to the lack of the R46 residue in KG mutant and the lack of the R46 and R66 residues in KG-P mutant These results suggested that this positive charge patch may be important for the interaction of integrin ?IIbβ3 with disintegrins Our results demonstrated that the RGD loop the linker region and C-terminus of disintegrins acted in a synergistic manner resulting in their functional and structural differences in integrin binding

Date of Award	2014 Aug 7
Original language	English
Supervisor	Woei-Jer Chuang (Supervisor)