Structural recognition of an ICAM‐1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1, 12)‐Pen‐Pro‐Arg‐Gly‐Gly‐Ser‐Val‐Leu‐Val‐Thr‐Gly‐Cys‐OH

Abstract

The purpose of this study is to elucidate the solution conformation of cyclic peptide 1 (cIBR), cyclo (1, 12)-Pen1-Pro2-Arg3-Gly4-Gly5-Ser6-Val7-Leu8-V al9-Thr10-Gly11-Cys12-OH, using NMR, circular dichroism (CD) and molecular dynamics (MD) simulation experiments. cIBR peptide (1), which is derived from the sequence of intercellular adhesion molecule-1 (ICAM-1, CD54), inhibits homotypic T-cell adhesion in vitro. The peptide hinders T-cell adhesion by inhibiting the leukocyte function-associated antigen-1 (LFA-1, CD11a/CD18) interaction with ICAM-1. Furthermore, Molt-3 T cells bind and internalize this peptide via cell surface receptors such as LFA-1. Peptide internalization by the LFA-1 receptor is one possible mechanism of inhibition of T-cell adhesion. The recognition of the peptide by LFA-1 is due to its sequence and conformation; therefore, this study can provide a better understanding for the conformational requirement of peptide-receptor interactions. The solution structure of 1 was determined using NMR, CD and MD simulation in aqueous solution. NMR showed a major and a minor conformer due to the presence of cis/trans isomerization at the X-Pro peptide bond. Because the contribution of the minor conformer is very small, this work is focused only on the major conformer. In solution, the major conformer shows a trans-configuration at the Pen1-Pro2 peptide bond as determined by HMQC NMR. The major conformer shows possible beta-turns at Pro2-Arg3-Gly4-Gly5, Gly5-Ser6-Val7-Leu8, and Val9-Thr10-Gly11-Cys12. The first beta-turn is supported by the ROE connectivities between the NH of Gly4 and the NH of Gly5. The connectivities between the NH of Ser6 and the NH of Val7, followed by the interaction between the amide protons of Val7 and Leu8, support the presence of the second beta-turn. Furthermore, the presence of a beta-turn at Val9-Thr10-Gly11-Cys12 is supported by the NH-NH connectivities between Thr10 and Gly11 and between Gly11 and Cys12. The propensity to form a type I beta-turn structure is also supported by CD spectral analysis. The cIBR peptide (1) shows structural similarity at residues Pro2 to Val7 with the same sequence in the X-ray structure of D1-domain of ICAM-1. The conformation of Pro2 to Val7 in this peptide may be important for its binding selectivity to the LFA-1 receptor.

Adhesion Molecule Interactions Facilitate Human Immunodeficiency Virus Type 1-Induced Virological Synapse Formation between T Cells

“…The reduction of conjugate frequency by the cyclic peptides in the absence of a reduction in VS assembly implies that the peptides may simply be reducing the stability of cell-cell adhesion without inducing or altering adhesion molecule-mediated signaling to the VS. A second, nonexclusive explanation is that LFA-1-ICAM-3 interactions are more important than LFA-1-ICAM-1 interactions in conjugate and VS formation when CXCRX4-tropic viruses infect naïve or resting CD4 ϩ CXCR4 ϩ T cells and that the peptides more effectively inhibit the binding of LFA-1 to ICAM-1 than to ICAM-3. Since the cIBR peptide sequence is derived from the ICAM-1 D1 domain (14), it is unlikely to inhibit LFA-1-ICAM-3 binding as efficiently as LFA-1-ICAM-1 binding, especially given the fact that a much larger region of the ICAM-3 D1 domain contributes to LFA-1 association (2). Furthermore, the cLAB.L peptide is derived from the LFA-1 I domain and has been reported to bind less well to ICAM-3 than to ICAM-1 (35).…”

“…Other MAbs increased the frequency of conjugate formation (BT-1, MHM23) while reducing that of VS formation (MHM23). To further exclude nonspecific inhibition of conjugate and VS formation by MAbs, we tested cyclic peptides designed to inhibit LFA-1-ICAM interactions (14,35) for their effects on these processes. Target CD4 ϩ T cells were preincubated with the cIBR or cLAB.L peptide at 37°C, mixed with effector cells, incubated for 1 h, fixed, and processed for LSCM.…”

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“…[25,26] and the NMR structure of cIBR peptide (magenta) [30]. The cIBL, cIBC, and cIBR peptides are located along the face of the A, A' and B strands on D1 of ICAM-1.…”

“…the cIBR and cIBC peptides have an overlapping Pro-Arg-Gly-Gly (PRGG) sequence with a β-turn structure [30,31]. This epitope has a similar β-turn conformation in the X-ray structure of D1 of ICAM-1 ( Figure 5).…”

NMR Spectroscopy for Studying Integrin Antagonists

“…The structure of cIBR was determined by NMR spectroscopy in aqueous solution (Gursoy et al, 1999). The resonances were assigned using conventional through-bond (J-mediated) 2D-1H NMR spectra and inter-residue correlations from NOESY type experiments which were also used to obtain distance based constraints.…”