Bidirectional Approach to Interrogate CCL11-CCR3 Interaction: Liquid and Solid State NMR

Chemokines and their receptors are critical messengers in immune signaling, and an unregulated chemokine-receptor axis is associated with inflammation, infection, and cancer. The C-C motif ligand 11 (CCL11) and C-C motif chemokine receptor 3 (CCR3) play vital roles in allergic responses, neurodegenerative diseases, and tumor metastasis. Although structures of CCL11-CCR3's N-terminal have been reported previously, there is no report about orthosteric pocket binding. Using high-field solution Nuclear Magnetic Resonance (NMR) spectroscopy (800 MHz and 1.1 GHz), we determined a high-resolution structure of CCL11. Compared to the earlier model, the new ensemble exhibits more defined secondary structural features as well as restricted N-terminal flexibility. Docking studies using ClusPro 2.0 and RRCS displayed that the N-terminus of CCL11 directly interacts with the residues in the orthosteric pocket of CCR3, consistent with previously reported mutagenesis and chemotaxis data. Ensemble-based root-mean-square fluctuation (RMSF) analysis on NMR structures of CCLs, including CCL11, further suggested that reduced N-terminal dynamics are close to those of other CCLs. These findings may come from the shorter purification artifact or the usage of synthetic protein. We explored CCR3 interactions using Solid-State NMR (SSNMR) in a phosphatidylcholine:cholesterol proteoliposome environment. Optimization of temperature in various experiments revealed that enhanced cross-polarization and spectral resolution were related to the lipid phase transition (~–30 °C). Application of proper temperature improved the detection of protein-lipid coupling and dynamic restraint. Based on the data from CCR3, preliminary 1H-15N SSNMR spectra of the CCL11 (labeled)- CCR3 (non-labeled) complex are being interrogated.