Structural characterization of S100A15 reveals a novel zinc coordination site among S100 proteins and altered surface chemistry with functional implications for receptor binding

Jill I Murray,Benjamin Farnell,Fraser Hof,Michelle L Tonkin,Jay T Cullen,Amanda L Whiting,Fangni Peng,Martin J Boulanger

doi:10.1186/1472-6807-12-16

Abstract

BackgroundS100 proteins are a family of small, EF-hand containing calcium-binding signaling proteins that are implicated in many cancers. While the majority of human S100 proteins share 25-65% sequence similarity, S100A7 and its recently identified paralog, S100A15, display 93% sequence identity. Intriguingly, however, S100A7 and S100A15 serve distinct roles in inflammatory skin disease; S100A7 signals through the receptor for advanced glycation products (RAGE) in a zinc-dependent manner, while S100A15 signals through a yet unidentified G-protein coupled receptor in a zinc-independent manner. Of the seven divergent residues that differentiate S100A7 and S100A15, four cluster in a zinc-binding region and the remaining three localize to a predicted receptor-binding surface.ResultsTo investigate the structural and functional consequences of these divergent clusters, we report the X-ray crystal structures of S100A15 and S100A7D24G, a hybrid variant where the zinc ligand Asp24 of S100A7 has been substituted with the glycine of S100A15, to 1.7 Å and 1.6 Å resolution, respectively. Remarkably, despite replacement of the Asp ligand, zinc binding is retained at the S100A15 dimer interface with distorted tetrahedral geometry and a chloride ion serving as an exogenous fourth ligand. Zinc binding was confirmed using anomalous difference maps and solution binding studies that revealed similar affinities of zinc for S100A15 and S100A7. Additionally, the predicted receptor-binding surface on S100A7 is substantially more basic in S100A15 without incurring structural rearrangement.ConclusionsHere we demonstrate that S100A15 retains the ability to coordinate zinc through incorporation of an exogenous ligand resulting in a unique zinc-binding site among S100 proteins. The altered surface chemistry between S100A7 and S100A15 that localizes to the predicted receptor binding site is likely responsible for the differential recognition of distinct protein targets. Collectively, these data provide novel insight into the structural and functional consequences of the divergent surfaces between S100A7 and S100A15 that may be exploited for targeted therapies.

Highlights

S100 proteins are a family of small, EF-hand containing calcium-binding signaling proteins that are implicated in many cancers
S100A15 retains the ability to coordinate zinc To investigate the differences in zinc binding between S100A7 and S100A15, we recombinantly produced human S100A15 in E. coli and purified the protein to homogeneity using nickel affinity chromatography
Similar to S100A7, S100A15 eluted as a stable dimer from a size exclusion column (SEC) indicating that the seven divergent residues (R21G, D24G, D27E, T83A, T51I, N52H and D56T – S100A7 residues are shown in italics and S100A15 residues are underlined) (Figure 1A) did not disrupt the multimeric structure

Summary

Introduction

S100 proteins are a family of small, EF-hand containing calcium-binding signaling proteins that are implicated in many cancers. S100A7 and S100A15 serve distinct roles in inflammatory skin disease; S100A7 signals through the receptor for advanced glycation products (RAGE) in a zinc-dependent manner, while S100A15 signals through a yet unidentified G-protein coupled receptor in a zinc-independent manner. The S100 calcium-binding protein family of vertebrate, metal-regulated proteins plays pivotal roles in a wide variety of intracellular and extracellular functions including cell growth, inflammation, membrane remodeling and chemotaxis [1,2] and are implicated in many diseases including cancer [3,4,5,6]. While most S100 proteins share moderate sequence identity, S100A7 and S100A15 are 93% identical in sequence yet intriguingly display divergent functions. S100A7 interacts with RAGE (receptor for advanced glycated end products) to mediate chemotaxis of leukocytes [20,22] while S100A15 appears to mediate chemotaxis through an unidentified G protein coupled receptor [20]

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Conclusion