Influence of Local pH Environment and Zn(II) on the Structure of the Antimicrobial Peptide Clavanin A and its Dynamics with Different Membrane Models in MD Simulations

In the latest report from the Centers for Disease Control and Prevention (CDC) on antimicrobial resistance, this problem remains to be an urgent global threat, infecting about three million people and killing about 36,000 people in the United States annually. Antimicrobial peptides (AMPs) offer a number of advantages over small molecule antibiotics, such as their structural flexibility in response to different local environments, allowing for having environment-responsive modes of action. In this dissertation, we will focus on Clavanin A (ClavA), an AMP that was identified and can be extracted from the tunicate Styela clava. Experimentally, it was shown that ClavA acts via different mechanisms at different pHs, pointing to a more membrane-active mechanism at pH 7 than at pH 5. Its antimicrobial activity is also enhanced by 16-fold in the presence of Zn2+ ions. In Chapter 2, we used unbiased molecular dynamics (MD) simulations to explore the structure of ClavA and its interaction with different membrane models of Escherichia coli in different environments. It was shown that the gain in positive charge increases electrostatic attraction between ClavA and the negatively charged model membrane. We proposed the domino-effect model showing how Zn2+ enhances the initial attraction of ClavA to the model membrane. In Chapter 3, we explored different enhanced sampling techniques to generate pathways of ClavA translocation across a more complex model of an E. coli outer membrane. The advantages and disadvantages of using these techniques in novel AMP-membrane systems were described. The free energy profiles constructed from Hamiltonian replica exchange simulations showed that monomeric translocation of ClavA across the model membrane is unlikely. In Chapter 4, we used principal component analyses with bacterial cytological profiling to identify the mechanisms enforced by ClavA under different environmental conditions. In Chapter 5, the applicability of metadynamics in a different protein-metal ion system was showcased. This dissertation is a significant contribution to complement the existing body of empirical knowledge on Clavanin A. Through this work, we have exposed the strengths and limitations of using molecular dynamics simulations in metal ion-binding AMPs, which is an exciting set of AMPs that can be further studied.