N-terminal Backbone Pairing Shifts in CCL5-12AAA14 Dimer Interface: Structural Significance of the FAY Sequence.

Jin-Ye Li,Yi-Chen Chen,Shih-Che Sue,Yi-Zong Lee,Chun-Hsiang Huang

doi:10.3390/ijms21051689

Abstract

CC-type chemokine ligand 5 (CCL5) has been known to regulate immune responses by mediating the chemotaxis of leukocytes. Depending on the environment, CCL5 forms different orders of oligomers to interact with targets and create functional diversity. A recent CCL5 trimer structure revealed that the N-terminal conversed F12-A13-Y14 (12FAY14) sequence is involved in CCL5 aggregation. The CCL5-12AAA14 mutant with two mutations had a deficiency in the formation of high-order oligomers. In the study, we clarify the respective roles of F12 and Y14 through NMR analysis and structural determination of the CCL5-12AAA14 mutant where F12 is involved in the dimer assembly and Y14 is involved in aggregation. The CCL5-12AAA14 structure contains a unique dimer packing. The backbone pairing shifts for one-residue in the N-terminal interface, when compared to the native CCL5 dimer. This difference creates a new structural orientation and leads to the conclusion that F12 confines the native CCL5 dimer configuration. Without F12 anchoring in the position, the interfacial backbone pairing is permitted to slide. Structural plasticity occurs in the N-terminal interaction. This is the first case to report this structural rearrangement through mutagenesis. The study provides a new idea for chemokine engineering and complements the understanding of CCL5 oligomerization and the role of the 12FAY14 sequence.

Highlights

CC-type chemokine ligand 5 (CCL5) known as RANTES is expressed and produced by many types of cells including platelets, macrophages, eosinophils, fibroblasts, and epithelial cells [1]
At pH 3.2, CCL5 existed in a monomer-dimer equilibrium and the dimer formation was dominant
We concluded that CCL5 starts to form large and soluble oligomers and only a very low content of protein was still in the dimeric formation

Summary

Introduction

CC-type chemokine ligand 5 (CCL5) known as RANTES (regulated on activation, normal T cell expressed and secreted) is expressed and produced by many types of cells including platelets, macrophages, eosinophils, fibroblasts, and epithelial cells [1]. Many chemokines show similar features to cooperatively execute their functions in a concentration-dependent manner for binding to G protein-coupled receptors (GPCRs) as monomers at low concentrations and interact with glycosaminoglycans (GAGs) as oligomer formations at high concentrations [5,6,7,8,9]. CCL5 has a similar concentration-dependent manner, but a more complicated regulatory mechanism [10,11,12,13,14]. The aggregation property correlates with CCL5 chemotaxis activity as disaggregated mutants lose their inflammatory properties, such as T cell activation [15,16]. There are at least three phases for CCL5 oligomerization and aggregation that depend on pH, concentration, and temperature [14]. At pH 7.0, massive aggregation can be observed if the concentration is higher than μM

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