• Advancing Drug and Biomaterial Design with Constant pH Molecular Dynamics Simulations

      Tsai, Cheng-chieh; Shen, Jana (2019)
      Molecular dynamics (MD) simulation is a valuable tool for investigating motions of macromolecular systems; however, solution pH, a critical factor for biological and chemical processes, is neglected in conventional MD simulations. To address this weakness, our group developed various continuous constant pH molecular dynamic (CpHMD) tools. In this dissertation, applications and new protocols that utilize CpHMD for drug and biomaterial design are presented. β-secretase 1 (BACE1, implicated in Alzheimers disease) and Src kinase (implicated in cancer) were utilized as model systems for drug design applications. In chapter 2, we applied CpHMD to understand the binding of BACE1 with two small-molecular inhibitors. We discovered that, despite the structural similarity of the two inhibitors, the titration behavior of the protein active site differs and this difference dramatically impacts the protein-ligand interactions and consequently the inhibitor affinity. Further, we tested a new protocol that combines CpHMD titration with free energy simulations to construct the pH-dependent binding free energy profiles. The resulting data showed excellent agreement with experiment and identified one potential allosteric site in BACE1. Next, in chapter 3, we simulated Src kinase to test the capability of CpHMD tostudy kinases. Starting from the crystal structure of the inactive Src, CpHMD can capture the conformational activation along the major inactive states without introducing any biasing potential or mutation. Starting from chapter 4, we studied the chitosan-based hydrogel systems to explore the detailed molecular mechanisms that give rise to macroscopic materials properties. We examined the flexibility of chitosan chains under different environmental conditions such as pH and salt concentration. Simulation data revealed that in addition to electrostatic screening, salt ions enhance the chain flexibility by interrupting the intra-molecular hydrogen bonds and thereby shifting the conformational populations between extended and bent states. In chapter 5, the atomic-level mechanism of pH-responsive chitosan-based hydrogels with switchable mechanical properties was investigated. Our data suggested that the electrostatic crosslinks are formed through the pH-dependent salt-bridge interactions between the chitosan glucosamines and surfactant headgroups. The pKa difference between the chitosan crystallite and the surfactant-bound chitosan is a key for the persistent but erasable gradient in the structural and mechanical properties between the two crosslinked regions. In summary, my work provids insights that will contribute to drug and biomaterial design and highlights the usefulness of CpHMD in these fields.
    • Exploring Protein Dynamics and Folding with Constant pH Molecular Dynamics Simulations

      Yue, Zhi; Shen, Jana; 0000-0002-4231-7474 (2017)
      Solution acidity or pH is a key environmental regulator with profound impact on biological processes such as protein dynamics and functions. Over the past decade, our research group has developed a set of tools to explicitly account for solution pH in molecular dynamics simulations. In this dissertation we demonstrate the new application areas of the constant-pH molecular dynamics tools. First we use constant-pH MD alongside fixed-protonation-state simulations to explore the dynamics of a cytoplasmic heme-binding protein (PhuS) in Pseudomonas aeruginosa, a notorious opportunistic pathogen. Our results provide atomic-level information on how heme binding affects PhuS dynamics which suggests an induced-fit mechanism, in support of the recent hydrogen-deuterium exchange data. Secondly, we apply constant-pH MD to investigate the acid-induced unfolding of BBL, a small component of the ubiquitous pyruvate dehydrogenase multienzyme complex involved in carbohydrate metabolism inside mitochondria. Our data support that BBL is a barrier-limited two-state folder, an issue that has drawn intense debate among experimental groups. More importantly, our simulations reveal that acid-induced unfolding of BBL is triggered by sequential protonation of His166 and Asp162 and thereby offering atomic details unattainable via experimental means. This work is the first time the constant-pH MD in explicit solvent has been applied to protein folding studies. Next, we employ the membrane-enabled constant-pH MD to understand how proton release drives the conformational transition of the transmembrane multi-drug efflux pump AcrB, which is crucial for the intrinsic resistance of E. coli to clinically important antibiotics. Our data address the controversy regarding the proton/drug stoichiometry and reveal the details of how deprotonation of a single residue leads to a global conformational transition in AcrB. This work paves the way for understanding the complete cycle of drug transport in AcrB and validates the membrane-enabled constant-pH MD technique for mechanistic studies of proton-coupled transporters. Lastly, we benchmark the accuracy of the all-atom constant-pH MD with charge-leveling co-ion using titration simulations of five proteins (HP36, BBL, NTL9, HEWL and SNase). The average and maximum absolute errors between the calculated and experimental pKa values based on 10-ns pH-based replica-exchange simulations are 0.7 and 0.9 units, respectively. Detailed analyses indicate that limited sampling is a major source of error. This work demonstrates the all-atom constant-pH MD method a practical tool for accurate prediction of pKa's and atomically detailed studies of proton-dependent conformational dynamics. In summary, my studies offered new mechanistic insights into the various roles of protons in protein dynamics and folding that were previously not well understood. My work further established constant-pH MD as a powerful tool for revealing atomic details of proton-coupled dynamic processes.
    • From Proteases to Antiporters: Advancing Drug Design Efforts with Continuous-Constant pH Molecular Dynamics Simulations

      Henderson, Jack Anthony; Shen, Jana; 0000-0001-6675-7944 (2021)
      Proton-mediated processes play essential roles in biology and human diseases. These processes are challenging to study experimentally and model with traditional simulation techniques because they rely on fixed protonation states. Continuous-constant pH molecular dynamics (CpHMD) is a computer simulation that provides atomic-level details of proton-coupled mechanisms. In this dissertation, several proteases (Beta-secretases 1/2, plasmepsin II, main proteases, and papain-like proteases) and a sodium-proton antiporter (NhaA) are investigated, elucidating protonation states of critical residues and proton-coupled dynamics to aid drug design efforts. In Chapter 2, CpHMD is used to investigate several aspartyl proteases. Human Beta-secretase 1 (BACE1) was considered a lead drug target based on the Beta-amyloid hypothesis for Alzheimer's disease. First, simulations reveal how water plays a vital role in improving the selectivity of an inhibitor for BACE1 over the closely related off-target BACE2. Next, simulations of plasmepsin II, a drug target against malaria, reveal the acid-base role of its catalytic aspartates and how binding of the substrate analog inhibitor pepstatin induces pH-dependent dynamics of its active site. In Chapter 3, simulations of two types of coronavirus cysteine proteases, the papain-like proteases (PLpros) and main proteases (MPros), are performed to aid the broad-spectrum inhibitor design against coronaviruses (CoVs). Here, the protonation states of PLpro from SARS-CoV, SARS-CoV-2, and MERS-CoV reveal the function of the catalytic residues. Moreover, the protonation state of cysteine on the second blocking loop is found to modulate the dynamics of a druggable subpocket. Investigation of the Mpros of SARS-CoV and SARS-CoV-2 uncovers a reactive cysteine residue that covalent inhibitors could target and protonation of a conserved histidine leads to the partial collapse of the S1 pocket. In Chapter 4, simulations are applied to the E. coli sodium-proton antiporter NhaA which facilitates the exchange of two protons for one sodium ion across the lipid bilayer. One proton binding site is generally accepted, while the other is controversial due to a series of mutations study showing retained activity. The simulations show that in the presence of various mutations, an alternative proton binding site can accept the second proton, and long-distance proton coupling occurs in some cases.