Sarkar DK, Surpeta B, Brezovsky J, 2024: Incorporating prior knowledge in the seeds of adaptive sampling molecular dynamics simulations of ligand transport in enzymes with buried active sites. Journal of Chemical Theory and Computation, DOI: 10.1021/acs.jctc.4c00452. full text dataset

Because most proteins have buried active sites, protein tunnels or channels play a crucial role in the transport of small molecules into buried cavities for enzymatic catalysis. Tunnels can critically modulate the biological process of protein–ligand recognition. Various molecular dynamics methods have been developed for exploring and exploiting the protein–ligand conformational space to extract high-resolution details of the binding processes, a recent example being energetically unbiased high-throughput adaptive sampling simulations. The current study systematically contrasted the role of integrating prior knowledge while generating useful initial protein–ligand configurations, called seeds, for these simulations. Using a nontrivial system of a haloalkane dehalogenase mutant with multiple transport tunnels leading to a deeply buried active site, simulations were employed to derive kinetic models describing the process of association and dissociation of the substrate molecule. The most knowledge-based seed generation enabled high-throughput simulations that could more consistently capture the entire transport process, explore the complex network of transport tunnels, and predict equilibrium dissociation constants, k_off/k_on, on the same order of magnitude as experimental measurements. Overall, the infusion of more knowledge into the initial seeds of adaptive sampling simulations could render analyses of transport mechanisms in enzymes more consistent even for very complex biomolecular systems, thereby promoting drug development efforts and the rational design of enzymes with buried active sites.

Sequeiros-Borja C, Bartlomiej Surpeta, Thirunavukarasu AS, Dongmo Foumthuim CJ, Igor Marchlewski, Brezovsky J, 2024: Water will find its way: transport through narrow tunnels in hydrolases. Journal of Chemical Information and Modeling, DOI: 10.1021/acs.jcim.4c00094. full text dataset-Hal dataset-Epx dataset-Lip dataset-hEpx dataset-E470G dataset-interactions dataset-Hal with different MD settings

An aqueous environment is vital for life as we know it, and water is essential for nearly all biochemical processes at the molecular level. Proteins utilize water molecules in various ways. Consequently, proteins must transport water molecules across their internal network of tunnels to reach the desired action sites, either within them or by functioning as molecular pipes to control cellular osmotic pressure. Despite water playing a crucial role in enzymatic activity and stability, its transport has been largely overlooked, with studies primarily focusing on water transport across membrane proteins. The transport of molecules through a protein’s tunnel network is challenging to study experimentally, making molecular dynamics simulations the most popular approach for investigating such events. In this study, we focused on the transport of water molecules across three different α/β-hydrolases: haloalkane dehalogenase, epoxide hydrolase, and lipase. Using a 5 μs adaptive simulation per system, we observed that only a few tunnels were responsible for the majority of water transport in dehalogenase, in contrast to a higher diversity of tunnels in other enzymes. Interestingly, water molecules could traverse narrow tunnels with subangstrom bottlenecks, which is surprising given the commonly accepted water molecule radius of 1.4 Å. Our analysis of the transport events in such narrow tunnels revealed a markedly increased number of hydrogen bonds formed between the water molecules and protein, likely compensating for the steric penalty of the process. Overall, these commonly disregarded narrow tunnels accounted for ∼20% of the total water transport observed, emphasizing the need to surpass the standard geometrical limits on the functional tunnels to properly account for the relevant transport processes. Finally, we demonstrated how the obtained insights could be applied to explain the differences in a mutant of the human soluble epoxide hydrolase associated with a higher incidence of ischemic stroke.

Bharadwaj P, Shet SM, Bisht M, Sarkar DK, Franklin G, Nataraj SK, Mondal D, 2023: Suitability of adenosine derivatives in improving the activity and stability of cytochrome c under stress: Insights into the effect of phosphate groups. The Journal of Physical Chemistry B doi: 10.1021/acs.jpcb.3c05996. full text

It is well known that adenosine and its phosphate derivatives play a crucial role in biological phenomena such as apoptosis and cell signaling and act as the energy currency of the cell. Although their interactions with various proteins and enzymes have been described, the focus of this work is to demonstrate the effect of the phosphate group on the activity and stability of the native heme metalloprotein cytochrome c (Cyt c), which is important from both biological and industrial aspects. In situ and in silico characterizations are used to correlate the relationship between the binding affinity of adenosine and its phosphate groups with unfolding behavior, corresponding peroxidase activities, and stability factors. Interaction of adenosine (ADN), adenosine monophosphate (AMP), adenosine 5′-diphosphate (ADP), and adenosine 5′-triphosphate (ATP) with Cyt c increases peroxidase-like activity by up to 1.8–6.5-fold compared to native Cyt c. This activity is significantly maintained even after multiple stress conditions such as oxidative stress and the presence of a chaotropic agent such as guanidine hydrochloride (GuHCl). With binding affinities on the order of ADN < AMP < ADP < ATP, adenosine derivatives were found to stabilize Cyt c by varying the secondary structural features of the protein. Thus, in addition to being a fundamental study, the current work also proposes a way of stabilizing protein systems to be used for real-time biocatalytic applications.

Bharadwaj P,* Sarkar DK,* Bisht M, Shet SM, Nataraj SK, Lokesh V, Franklin G,^# Brezovsky J,^# Mondal D,^# 2023: Nano-structured hydrotrope-caged cytochrome c with boosted stability in harsh environments: a molecular insight. Green Chemistry 25: 6666-6676. full text dataset

Green and nano-structured catalytic media are vital for biocatalysis to attenuate the denaturation tendency of biocatalysts under severe reaction conditions. Hydrotropes with multi-faceted physiochemical properties represent promising systems for sustainable protein packaging. Herein, the ability of adenosine-5′-triphosphate (ATP) and cholinium salicylate ([Cho][Sal]) ionic liquid (IL) to form nano-structures and to nano-confine Cytochrome c (Cyt c) enhanced the stability and activity under multiple stresses. Experimental and computational analyses were undertaken to explain the nano-structured phenomenon of ATP and IL, structural organizations of nano-confined Cyt c, and site-specific interactions that stabilize the protein structure. Both ATP and IL form nano-structures in aqueous media and could cage Cyt c via multiple nonspecific soft interactions. Remarkably, the engineered molecular nano-cages of ATP (5–10 mM), IL (300 mg mL⁻¹), and ATP + IL surrounding Cyt c resulted in 9-to-72-fold higher peroxidase activity than native Cyt c with exceptionally high thermal tolerance (110 °C). The polar interactions with the cardiolipin binding site of Cyt c, mediated by hydrotropes, were well correlated with the increased peroxidase activity. Furthermore, higher activity trends were observed in the presence of urea, GuHCl, and trypsin without any protein degradation. Specific binding of hydrotropes in highly mobile regions of Cyt c (Ω 40–54 residues) and enhanced H-bonding with Lys and Arg offered excellent stability under extreme conditions. Additionally, ATP effectively counteracted reactive oxygen species (ROS)-induced denaturation of Cyt c, which was enhanced by the [Sal] counterpart of IL. Overall, this study explored the robustness of nano-structured hydrotropes to have a higher potential for protein packaging with improved stability and activity under extreme conditions. Thus, the present work highlights a novel strategy for real-time industrial biocatalysis to protect mitochondrial cells from ROS-instigated apoptosis.

Pakuła K,* Sequeiros-Borja C,* Biała-Leonhard W,* Pawela A, Banasiak J, Bailly A, Radom M, Geisler M, Brezovsky J,^# Jasiński M,^# 2023: Restriction of access to the central cavity is a major contributor to substrate selectivity in plant ABCG transporters. Cellular and Molecular Life Sciences 80: 105. full text

ABCG46 of the legume Medicago truncatula is an ABC-type transporter responsible for highly selective translocation of the phenylpropanoids, 4-coumarate, and liquiritigenin, over the plasma membrane. To investigate molecular determinants of the observed substrate selectivity, we applied a combination of phylogenetic and biochemical analyses, AlphaFold2 structure prediction, molecular dynamics simulations, and mutagenesis. We discovered an unusually narrow transient access path to the central cavity of MtABCG46 that constitutes an initial filter responsible for the selective translocation of phenylpropanoids through a lipid bilayer. Furthermore, we identified remote residue F562 as pivotal for maintaining the stability of this filter. The determination of individual amino acids that impact the selective transport of specialized metabolites may provide new opportunities associated with ABCGs being of interest, in many biological scenarios.