Structure of monomeric Interleukin-8 and its interactions with the N-terminal Binding Site-I of CXCR1 by solution NMR spectroscopy

Abstract

The structure of monomeric human chemokine IL-8 (residues 1–66) was determined in aqueous solution by NMR spectroscopy. The structure of the monomer is similar to that of each subunit in the dimeric full-length protein (residues 1–72), with the main differences being the location of the N-loop (residues 10–22) relative to the C-terminal α-helix and the position of the side chain of phenylalanine 65 near the truncated dimerization interface (residues 67–72). NMR was used to analyze the interactions of monomeric IL-8 (1–66) with ND-CXCR1 (residues 1–38), a soluble polypeptide corresponding to the N-terminal portion of the ligand binding site (Binding Site-I) of the chemokine receptor CXCR1 in aqueous solution, and with 1TM-CXCR1 (residues 1–72), a membrane-associated polypeptide that includes the same N-terminal portion of the binding site, the first trans-membrane helix, and the first intracellular loop of the receptor in nanodiscs. The presence of neither the first transmembrane helix of the receptor nor the lipid bilayer significantly affected the interactions of IL-8 with Binding Site-I of CXCR1.

Interaction of Monomeric Interleukin-8 with CXCR1 Mapped by Proton-Detected Fast MAS Solid-State NMR

“…2 A, (Fig. 2 B), which is similar to that obtained from the wild-type dimeric IL-8 (1-72) (17,33). The binding of IL-8 (1-66) to CXCR1 in phospholipid bilayers at a molar ratio of 1:1 ( Fig.…”

“…Presaturation for water suppression in the solid-state MAS spectrum results in missing or very weak intensity signals from solvent-accessible residues (1-5, 11-15, 18, 20, 33, 35, 37, 44, 45, and 57) compared to the solution NMR spectrum obtained with the WATERGATE pulse sequence (61) for water suppression. This result was verified by amide hydrogendeuterium exchange measurements on IL-8 (1-66) in solution (17).…”

“…This spectrum was obtained with the sample in a standard 5 mm OD solution NMR sample tube that was not spun. All the amide resonances have been assigned to specific residues, with the exception of the mobile C-terminal residue, 66, whose amide hydrogen exchanges rapidly with water at pH 7.3 (17). A spectrum of the same solution sample obtained in a 1.3 mm rotor spinning at 60 kHz using the same pulse sequence, with minor modifications of the t delay for INEPT transfers and the use of continuous wave irradiation (presaturation) for water signal suppression, is shown with black contours in Fig.…”

“…We prepared uniformly 2 H and 15 N labeled monomeric IL-8 (1-66) (17), and examined its interactions with unlabeled full-length CXCR1 (1-350), which contains both binding site-I and binding site-II, and an N-terminal truncated construct of CXCR1, NT-CXCR1 (39-350), which lacks binding site-I associated with the N-terminal region but retains binding site-II associated with the extracellular loops. Changes observed in spectra of IL-8 (1-66) upon binding CXCR1 (1-350) and NT-CXCR1 (39-350) identify chemokine residues involved in the interactions with CXCR1.…”

Crosslinking-guided geometry of a complete CXC receptor-chemokine complex and the basis of chemokine subfamily selectivity

“…These studies established the functional importance of receptor N-termini; however, their interaction geometry with the chemokines remains elusive (S1 Table), which is a critical barrier for our understanding of how chemokines control diverse signaling events. In complementary studies, the interaction geometries have been explored by nuclear magnetic resonance (NMR) with isolated Nterminal peptides of the receptors [24][25][26][27][28][29][30][31] (S1 Table). Although these efforts have captured some important residue pairings, the absence of spatial constraints from the TM domain of intact receptors has led to geometric inconsistencies across the NMR structures and in relation to the crystallographic structures of the TM domains [32,33].…”