Cellular metal chelation is a vital plant stress response mechanism that enables plants to survive in metal-polluted soils, which could be valuable in crop engineering for agricultural use and phytoremediation of metal-polluted soils. The study of metal-chelating proteins is crucial in understanding these biological processes. This project aims to uncover the mechanism of copper ion binding in the metal-chelating BURP domain protein, AhyBURP. By employing cutting-edge computational chemistry techniques and experimental structural characterization, we will determine the structure of AhyBURP and the geometric and electronic properties of its Cu-binding sites. The goal is to investigate the role of AhyBURP in the biosynthesis of metal-chelating peptides in BURP-domain peptide cyclases, which play an essential role in a wide range of biological activities.
Previous research on analogous proteins, along with our X-ray diffraction (XRD) and native mass spectrometry results, support the hypothesis that AhyBURP binds Cu through two histidines and one methionine in two predicted binding sites. To examine this hypothesis, a combination of experimental and computational methods will be used. The first step will be to determine the overall structure of AhyBURP using nuclear magnetic resonance (NMR) and Electron Paramagnetic Resonance (EPR) spectroscopy, and computational methods such as AlphaFold. Molecular dynamics simulations will then be used to investigate any structural changes that may occur during the catalytic cycle.
Quantum mechanics/molecular mechanics (QM/MM) methods will provide a comprehensive understanding of the copper-binding sites and the underlying mechanism of the protein. The optimization of the active site's geometry and the calculation of X-ray absorption near edge structure (XANES) spectra for the copper-bound AhyBURP at different stages of the catalytic cycle will be performed using density functional theory (DFT) and time-dependent DFT (TDDFT) calculations.
We propose the NMR experimental and computational part of this project to be performed at the Environmental Molecular Sciences Laboratory (EMSL) using its advanced experimental and computational resources. The XAS experiments will be performed at the Stanford Synchrotron Radiation Lightsource (SSRL) and combined with the computational analysis results to provide a comprehensive understanding of the protein's behavior. The FEFF package will be used to fit and calculate Extended X-ray Absorption Fine Structure (EXAFS) measurements, which will provide detailed electronic and structural information about the protein.
This investigation holds significant potential to enhance our comprehension of Cu-binding mechanisms in metal-chelating proteins, laying the groundwork for future studies on the roles of these proteins in metal homeostasis and other biological processes. The collaboration between SSRL and EMSL will strengthen ties between the institutions and promote interdisciplinary research in the field. The findings will contribute to improving our understanding of metal stress in crops and its potential for crop engineering and phytoremediation.