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.

Sequeiros-Borja C, Surpeta B, Marchlewski I,  Brezovsky J, 2022: Divide-and-conquer approach to study protein tunnels in long molecular dynamics simulations. MethodsX (in press – DOI: 10.1016/j.mex.2022.101968). full text

Nowadays, molecular dynamics (MD) simulations of proteins with hundreds of thousands of snapshots are commonly produced using modern GPUs. However, due to the abundance of data, analyzing transport tunnels present in the internal voids of these molecules, in all generated snapshots, has become challenging. Here, we propose to combine the usage of CAVER3, the most popular tool for tunnel calculation, and the TransportTools Python3 library into a divide-and-conquer approach to speed up tunnel calculation and reduce the hardware resources required to analyze long MD simulations in detail. By slicing an MD trajectory into smaller pieces and performing a tunnel analysis on these pieces by CAVER3, the runtime and resources are considerably reduced. Next, the TransportTools library merges the smaller pieces and gives an overall view of the tunnel network for the complete trajectory without quality loss.