Detailed Characterization of the Cooperative Binding of Piperine with Heat Shock Protein 70 by Molecular Biophysical Approaches.

Gabriel Zazeri,Marinônio Lopes Cornélio,Ana Paula Ribeiro Povinelli,Marcelo De Freitas Lima

doi:10.3390/biomedicines8120629

Abstract

In this work, for the first time, details of the complex formed by heat shock protein 70 (HSP70) independent nucleotide binding domain (NBD) and piperine were characterized through experimental and computational molecular biophysical methods. Fluorescence spectroscopy results revealed positive cooperativity between the two binding sites. Circular dichroism identified secondary conformational changes. Molecular dynamics along with molecular mechanics Poisson Boltzmann surface area (MM/PBSA) reinforced the positive cooperativity, showing that the affinity of piperine for NBD increased when piperine occupied both binding sites instead of one. The spontaneity of the complexation was demonstrated through the Gibbs free energy (∆G < 0 kJ/mol) for different temperatures obtained experimentally by van’t Hoff analysis and computationally by umbrella sampling with the potential of mean force profile. Furthermore, the mean forces which drove the complexation were disclosed by van’t Hoff and MM/PBSA as being the non-specific interactions. In conclusion, the work revealed characteristics of NBD and piperine interaction, which may support further drug discover studies.

Highlights

Heat shock proteins (HSPs) constitute the first line of protection for cells exposed to stressful conditions [1]
Steady-state and time-resolved fluorescence showed that nucleotide binding domain (NBD) in the presence of piperine presented a static-quenching process, which means that a complex was formed
Fluorescence spectroscopy revealed with a Scatchard plot and Hill’s method an important feature about the interaction of NBD and piperine, which was the presence of positive cooperativity between the two binding sites

Summary

Introduction

Heat shock proteins (HSPs) constitute the first line of protection for cells exposed to stressful conditions [1]. Asea et al [3] initiated a paradigm in the understanding of the function of one of the heat shock proteins (HSP70), revealing that this protein may be found in the extracellular medium acting as an inflammatory cytokine that stimulates innate immune response through the activation of Nuclear. HSP70 is a 70 kDa protein that consists of an independent conserved N-terminal nucleotide binding domain (NBD ≈ 40 kDa) with ATPase activity, a substrate binding domain (SBD ≈ 25 kDa). The interaction with ATP leads the NBD structure to conformational changes that affect the affinity of HSP70 for its receptors (TLRs), revealing the plasticity of this protein [8]. Considering the NBD structural flexibility, the key to inhibit the cytokine function of HSP70 may be the search for small molecules able to induce conformational changes that reduce the affinity for the receptors

Methods

Results

Conclusion