Solution structure of a novel T-cell adhesion inhibitor derived from the fragment of ICAM-1 receptor: cyclo(1,8)-Cys-Pro-Arg-Gly-Gly-Ser-Val-Cys

Abstract

This study is aimed at elucidating the structure of a novel T-cell adhesion inhibitor, cyclo(1,8)-CPRGGSVC using one- and two-dimensional (2D) (1)H NMR and molecular dynamics (MD) simulation. The peptide is derived from the sequence of its parent peptide cIBR (cyclo(1,12)-PenPRGGSVLVTGC), which is a fragment of intercellular adhesion molecule-1 (ICAM-1). Our previous results show that the cyclo(1,8)-CPRGGSVC peptide binds to the LFA-1 I-domain and inhibits heterotypic T-cell adhesion, presumably by blocking the LFA-1/ICAM-1 interactions. The structure of the peptide was determined using NMR and MD simulation in aqueous solution. Our results indicate that the peptide adopts type-I beta-turn conformation at the Pro2-Arg3-Gly4-Gly5 (PRGG) sequence. The beta-turn structure at the PRGG motif is well conserved in cIBR peptide and ICAM-1 receptor, which suggests the importance of the PRGG motif for the biological activity of cyclo(1,8)-CPRGGSVC peptide. Meanwhile, the Gly5-Ser6-Val7-Cys8-Cys1 (GSVCC) sequence forms a "turn-like" random coil structure that does not belong to any structured motif. Therefore, cyclo(1,8)-CPRGGSVC peptide has only one structured region at the PRGG sequence, which may play an important role in the binding of the peptide to the LFA-1 I-domain. The conserved beta-turn conformation of the PRGG motif in ICAM-1, cIBR, and cyclo(1,8)-CPRGGSVC peptides can potentially be used to design peptidomimetics. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 633-641, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

Figure 1

(a) Fingerprint region of TOCSY spectrum of cyclo(1,8)-CPRGGSVC peptide. (b) NH-αH region of ROESY spectrum of cyclo(1,8)-CPRGGSVC peptide superimposed with the fingerprint region of TOCSY spectrum. The “backbone walk” of the ROESY/TOCSY spectrum used to determine the sequence of the peptide is shown by the sequential numbers. (c) NH-NH region of ROESY spectrum of cyclo(1,8)-CPRGGSVC peptide showing d_NN(i, i+1) cross-peaks

Figure 2

Chemical shift indices (CSI, above the line) and summary of the inter-residue ROE effects among the backbone NH, αH, and βH (below). The CSI values were equal to 0 or were not calculated for amino acid residues with empty squares. The ROE intensities are reflected by the thickness of the lines.

Figure 3

One-dimensional NMR spectrum taken from F2 row of ROESY spectrum of cyclo(1,8)-CPRGGSVC peptide. The value of ³J_NH-HCα was taken from amide proton peaks on this spectrum. The peak splitting is marked (d = doublet, t = triplet).

Figure 4

Stereo-view of superimposed conformations of 10 structures taken from the last 500 ps molecular dynamics trajectories (a). The trajectory with lowest energy was taken for clarity (b). The hydrogen bonds between the backbone carbonyl group of Pro2 and amide protons of Gly4 and Gly5 are shown by yellow dashed lines. The distance between Cα of Pro2 and Cα of Gly5 is shown by an orange dashed line. The distance between Cα of Cys1 and Cα of Pro2 is shown by a turquoise dashed line.

Figure 5

Chemical shifts of amide protons of cyclo(1,8)-CPRGGSVC peptide at different temperatures. The slope of the curve (Δδ/ΔT) is the temperature coefficient of the amide proton.