Interaction between S100P and the anti-allergy drug cromolyn

https://doi.org/10.1016/j.bbrc.2014.10.048Get rights and content

Highlights

  • The interaction between S100P–cromolyn was investigated by fluorescence spectroscopy.

  • The interfacial residues on S100P and cromolyn contact surface were mapped by 1H-15N HSQC experiments.

  • S100P–cromolyn complex model was generated from NMR restraints using HADDOCK program.

  • The stability of the S100P–cromolyn complex was studied using molecular dynamics simulations.

Abstract

The S100P protein has been known to mediate cell proliferation by binding the receptor for advanced glycation end products (RAGE) to activate signaling pathways, such as the extracellular regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways. S100P/RAGE signaling is involved in a variety of diseases, such as cancer, metastasis, and diabetes. Cromolyn is an anti-allergy drug that binds S100P to block the interaction between S100P and RAGE. In the present study, we characterized the properties of the binding between cromolyn and calcium-bound S100P using various biophysical techniques. The binding affinity for S100P and cromolyn was measured to be in the millimolar range by fluorescence spectroscopy. NMR-HSQC titration experiments and HADDOCK modeling was employed to determine the spatial structure of the proposed heterotetramer model of the S100P–cromolyn complex. Additional MD simulation results revealed the important properties in the complex stability and conformational flexibility of the S100P–cromolyn complex. This proposed model has provided an understanding of the molecular level interactions of S100P–cromolyn complex.

Introduction

The S100 proteins are calcium-binding EF-hand proteins. The calcium-induced conformational change in S100 proteins results in exposure of the hydrophobic surface between a loop region (between helix-2 and helix-3) and either helix-4 or the subsequent C-terminus to bind protein targets and regulate biological functions [1], [2]. These S100 proteins are known to be expressed in a tissue-specific and cell-specific pattern. Importantly, S100 proteins influence different functions both extracellularly and intracellularly by direct interaction with specific target proteins. Misregulation of these interactions is observed in a wide range of pathologies, thus making S100 proteins potential drug targets [3], [4]. For instance, the S100P protein is over-expressed in pancreatic, breast, lung and colon cancer cells and regulates biological function upon binding to RAGE extracellularly [5]. Thus, it is used as a clinical marker of various cancers. The direct interaction between S100P and RAGE has been confirmed by co-immunoprecipitation and activation of downstream signaling pathways, such as the extracellular regulated kinases (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) pathways [6], [7]. Therefore, elevated levels of S100P can activate the downstream pathways, including the mitogen-activated protein (MAP) kinase, ERK and NF-κB pathways, and are associated with the tumor growth in various types of cancer [7], [8], [9], [10].

Cromolyn is used to treat various diseases, including allergic rhinitis, allergic conjunctivitis, and mastocytosis [11], [12], [13]. Previous studies using affinity chromatography reported that S100P binds to cromolyn and blocks the interaction between S100P and RAGE, leading to inhibition of S100P-stimulated cell growth, invasion, and NF-κB activity in S100P-expressing BxPC-3 and MPanc-96 pancreatic cancer cells, but not in Panc-1 cells without endogenous S100P [14]. Recently, cromolyn analogs were reported to block the interaction between S100P and RAGE [15]. In the current study, we aimed to map the binding interface of S100P for cromolyn and characterized the model structure of S100P–cromolyn. Overall, the results provide significant insights into S100P–RAGE interactions.

Section snippets

Reagents and chemicals

15NH4Cl and D2O were obtained from Cambridge Isotope Laboratories. Luria Broth was obtained from AMRESCO. Cromolyn was purchased from Sigma–Aldrich. All used chemicals were analytical grade.

Expression and purification of S100P

Recombinant wild-type S100P (95 amino acids) was cloned into the pET-20b (+) T7 expression vector, over-expressed in the Escherichia coli BL21 (DE3) strain and purified using a previously reported procedure [16]. The S100P protein was eluted as a dimer from a Superdex-75 column (1.6 × 60 cm; Pharmacia). The

Fluorescence spectroscopy studies of S100P–cromolyn interactions

The cromolyn molecule showed two distinct UV absorption bands at ∼265 nm and ∼326 nm (Fig. S-1). The 326 nm absorption band of cromolyn was selected for this study. Upon excitation at 326 nm, changes in the emission of cromolyn were observed in the range from 400 to 600 nm. As shown in Fig. 1A, the addition of S100P to a 34 μM solution of cromolyn in 20 mM Tris buffer, pH = 7.5, 100 mM KCl, and 4 mM CaCl2 resulted in both a gradual shift in the emission maxima and a blue shift in the lower energy

Acknowledgments

We sincerely thank the 700 MHz Nuclear Magnetic Resonance Facility in the Department of Chemistry, National Tsing Hua University, Taiwan. We acknowledge financial support from the Ministry of Science and Technology, Taiwan (Grant number: MOST 103-2113-M-007-017-MY3 to Chin Yu) and China Medical University, Taiwan (Grant number: CMU101-N2-04 to Ruey-Hwang Chou).

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