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

Abstract

The human chemokine interleukin-8 (IL-8; CXCL8) is a key mediator of innate immune and inflammatory responses. This small, soluble protein triggers a host of biological effects upon binding and activating CXCR1, a G proteincoupled receptor, located in the cell membrane of neutrophils. Here, we describe 1 H-detected magic angle spinning solid-state NMR studies of monomeric IL-8 (1-66) bound to full-length and truncated constructs of CXCR1 in phospholipid bilayers under physiological conditions. Cross-polarization experiments demonstrate that most backbone amide sites of IL-8 (1-66) are immobilized and that their chemical shifts are perturbed upon binding to CXCR1, demonstrating that the dynamics and environments of chemokine residues are affected by interactions with the chemokine receptor. Comparisons of spectra of IL-8 (1-66) bound to full-length CXCR1 (1-350) and to N-terminal truncated construct NT-CXCR1 (39-350) identify specific chemokine residues involved in interactions with binding sites associated with N-terminal residues (binding site-I) and extracellular loop and helical residues (binding site-II) of the receptor. Intermolecular paramagnetic relaxation enhancement broadening of IL-8 (1-66) signals results from interactions of the chemokine with CXCR1 (1-350) containing Mn 2þ chelated to an unnatural amino acid assists in the characterization of the receptor-bound form of the chemokine.

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

“…Similarly, in a radiolytic footprinting study, detergent-solubilized ACKR3 protected the same CXCL12 residues from radiolytic oxidation [34]. Perturbations of the CXCL8 β1-strand were also reported upon binding of soluble or membrane-bound CXCR1 N-terminus in solution NMR [26,57], and β1-strand mutations weakened the affinity and potency of CXCL8 towards CXCR1/2 [58,59].…”

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

“…The ELR motif is therefore thought to be important for the interaction with the second binding site associated with extracellular loops of CXCR1 and not involved in binding the N-terminal domain (Clark-Lewis et al 1991; Hebert et al 1991; Park et al 2017; Williams et al 1996). This is supported by the lack of chemical shift perturbations for these residues upon interaction with both ND-CXCR1(1–38) and 1TM-CXCR1(1–72).…”

“…Although the characteristic ELR residues (Glu4, Leu5 and Arg6) have been shown to be essential for CXCR1 activation they are not involved in IL-8 binding to N-terminal peptides (Clark-Lewis et al 1991; Hebert et al 1991; Park et al 2017). The main binding sites on IL-8 have been mapped to the N-loop (residues 12–18) and the 40 s loop and third β-strand (residues 44–51) and include both charged and hydrophobic residues (Fernando et al 2004, 2007; Ravindran et al 2009; Skelton et al 1999; Park et al 2011a, b; Clubb et al 1994; Williams et al 1996; Kendrick et al 2014).…”

GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures

“…This enables the determination of structures of GPCR ligands and measurement of their conformational dynamics while bound to the receptor and, in principle, their drug pharmacokinetics. Some examples from a very large array of literature data on this topic include the determination of the structure of the agonist dynorphin bound to the κ-opioid receptor 132 , the cytokine interleukine-8 and its interactions with the chemokine receptor CXCR1 133,134 , lipids of the free fatty acid receptor 135 , and peptide agonists of the bradykinin receptors 136 . Importantly, NMR can be used to monitor the relatively weak interactions of low-molecular-mass compounds with GPCRs, and therefore can support fragment-based lead discovery approaches.…”