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Investigating Ligand Binding Kinetics and pH Sensing in Proteins: Insights from Molecular Dynamics Simulations


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14:20 até 14:50 em 26/10/2023

Investigating Ligand Binding Kinetics and pH Sensing in Proteins: Insights from Molecular Dynamics Simulations

Ariane Nunes-Alves
Technical University of Berlin

Proteins are essential molecular entities involved in a wide range of biological processes, and their functional properties can be influenced by ligand binding and by the surrounding environment. Here, we employed molecular dynamics (MD) simulations to obtain mechanistic insights about ligand dissociation and pH sensing in different proteins. Protein-ligand unbinding usually takes milliseconds or more to happen, while MD simulations are limited to the microsecond timescale. Different enhanced sampling methods have been developed to bridge this difference in timescales (1), allowing one to observe unbinding events in simulations. Here, we employed the enhanced sampling method τ-random accelerated molecular dynamics (τRAMD) to investigate the dissociation of benzene and indole from mutants of T4 lysozyme (2) and the dissociation of inhibitors (O2 and CO) from mutants of NiFe hydrogenase (3). The relative residence times obtained from τRAMD reproduced the trends observed in the experiments. In T4 lysozyme, although many ligand dissociation paths were detected, the same preferred path was observed for different mutants. Moreover, the presence of a greater number of metastable states along exit pathways led to slower protein-ligand dissociation. In NiFe hydrogenases, several mutants presented main exit paths which differed from the ones in the wild type protein. These physical insights could be exploited in the rational optimization of drug candidates or in the engineering of O2 - or CO-tolerant NiFe hydrogenases.

Resistance of the human malaria parasite Plasmodium falciparum to chloroquine and related drugs is mediated by the chloroquine resistance transporter (PfCRT), which displays pH-sensitive activity. We employed mutagenesis and conventional MD simulations to elucidate the mechanism of pH sensing in
PfCRT (4). The E207A mutation abolished pH-sensitivity in experiments. MD simulations showed there is relocation of E207 during the transport cycle, forming a salt bridge with residue K80. The importance of the salt bridge between E207 and K80 was further confirmed by mutagenesis. We propose that the
ionized carboxyl group of E207 acts as a hydrogen acceptor, facilitating transport cycle progression, while pH sensing serves as a by-product.

1. F. Sohraby, A. Nunes-Alves. Advances in computational methods for ligand binding kinetics. Trends
Biochem. Sci. 2023, 48: 437-449.
2. A. Nunes-Alves, D. B. Kokh, R. C. Wade. Ligand unbinding mechanisms and kinetics for T4 lysozyme
mutants from τRAMD simulations. Curr. Res. Struct. Biol. 2021, 4: 106-111.
3. F. Sohraby, A. Nunes-Alves. Characterization of ligand unbinding mechanisms and kinetics for NiFe
hydrogenase mutants using τRAMD. Manuscript in preparation.
4. F. Berger, G. M. Gomez, C. P. Sanchez, B. Posch, G. Planelles, F. Sohraby, A. Nunes-Alves, M. Lanzer.
pH-dependence of the Plasmodium falciparum chloroquine resistance transporter is linked to the transport
cycle. Nat. Commun. In press.


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