A Hybrid Computational/Experimental Approach to Tuning the Specificity of Chemokine Inhibitors
Abstract
Virtually all biochemical communicationÑresponsible for everything from embryogenesis to the immune responseÑis mediated by protein-protein interactions, yet we have little atomic-level understanding of these interactions, and this lack of knowledge limits our ability to design therapeutics based on modifying interprotein communication. Structural biology techniques like NMR and X-ray crystallography can provide a snapshot of proteinprotein complexes, but it remains difficult to determine which observed contacts are important and how the flexibility and motion of the proteins affect the interaction. In this work we propose to establish a new approach to understanding protein-protein complexes using a combination of experimental biochemistry and molecular dynamics (MD) simulations, in a collaborative, iterative process to unravel individual interactions that lead to overall high affinity. Our project will focus on the poxvirus-produced protein vCCI (viral CC chemokine inhibitor), which has potent anti-inflammatory activity based on its ability to bind dozens of proteins in the chemokine (chemotactic cytokine) family. We have investigated the complex of vCCI with several chemokines and chemokine analogs by NMR, fluorescence, isothermal titration calorimetry (ITC), and biolayer interferometry (BLI), and performed matched 1µs molecular dynamics (MD) simulations of vCCI in complex with different chemokines having different binding affinities. These preliminary studies validate the use of a complementary experimental/computational approach to this problem. The long term goal of the research is to decode the Òdesign principlesÓ of how vCCI is able to bind such a large family of proteins. This knowledge will allow us to optimize or manipulate vCCIÕs binding in a controlled manner. In the First Aim we will experimentally validate several predictions made by MD simulations regarding the role of the vCCI loop and Nterminal region in its binding ability. These insights were largely unexpected from the known static structures, and will be tested by mutating and expressing vCCI and examining its complex formation using NMR, ITC and BLI. In the Second Aim, it is proposed to undertake a design challenge to determine the robustness of an iterative simulation/biochemical approach. The goal will be to mutate vCCI and a chemokine binding partner in tandem to allow them to retain affinity for each other, but lose affinity for the wild type versions of each. In this Aim, past biochemical and NMR results will inform the simulations, which will in turn pinpoint areas of interaction between vCCI and a chemokine for mutation, purification, and testing for binding affinity. The Third Aim will further validate this approach by designing changes in vCCI that will allow it to tightly and specifically bind the chemokine TARC/CCL17. Wild type vCCI does not bind TARC with high affinity, and the function of TARC in inflammation makes it a useful target from a practical standpoint as well as a proof of concept. Another important outcome of this project is that it will provide an innovative training experience for students from UC MercedÕs highly diverse student body. Through bi-weekly meetings and student-led training modules, students working on both the experimental and computational portions of this project will gain an understanding of the principles and practice of each otherÕs research tools.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Aug 31, 2020
- Source ID
- W911NF2010268
Entities
People
- Patricia J LiWang
Organizations
- Army Contracting Command
- Office of the Secretary of Defense
- University of California