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.

Surpeta B, Grulich M, Palyzová A, Marešová H, Brezovsky J, 2022: Common Dynamic Determinants Govern Quorum Quenching Activity in N-Terminal Serine Hydrolases. ACS Catalysis 12: 6359-6374. full text

Growing concerns about microbial antibiotic resistance have motivated extensive research into ways of overcoming antibiotic resistance. Quorum quenching (QQ) processes disrupt bacterial communication via quorum sensing, which enables bacteria to sense the surrounding bacterial cell density and markedly affects their virulence. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and may thus avoid inducing resistance. Although many enzymes display QQ activity against various bacterial signaling molecules, their mechanisms of action are poorly understood, limiting their potential optimization as QQ agents. Here, we evaluate the capacity of three N-terminal serine hydrolases to degrade N-acyl-homoserine lactones (HSLs) that serve as signaling compounds for Gram-negative bacteria. Using molecular dynamics (MD) simulations of the free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics MD simulations of two catalytic steps, we clarify the molecular processes underpinning their QQ activity. We conclude that all three enzymes degrade HSLs via similar reaction mechanisms. Moreover, we experimentally confirmed the activity of two penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), adding these industrially optimized enzymes to the QQ toolbox. We also observed substrate-dependent differences in the catalytic actions of these enzymes, arising primarily from the distinct structures of their acyl-binding cavities and the dynamics of their molecular gates. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate had an elevated energy barrier compared to its complex with a shorter substrate because its shallow acyl-binding site could not accommodate a productive substrate-binding configuration of the former one. Conversely, aPGA in complex with the shorter substrate exhibited unfavorable energetics in the first step, while the longer substrate was penalized in the second step, both due to the dynamics of the residues gating the acyl-binding cavity entrance. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to the unique positioning of Arg297β in this enzyme. The discovery of these dynamic determinants will guide future efforts to design robust QQ agents capable of selectively controlling virulence in resistant bacterial species.

Sequeiros-Borja CE, Surpeta B, Brezovsky J, 2020: Recent advances in user-friendly computational tools to engineer protein function. Briefings in Bioinformatics. (Advance article DOI: 10.1093/bib/bbaa150) full text

Progress in technology and algorithms throughout the past decade has transformed the field of protein design and engineering. Computational approaches have become well-engrained in the processes of tailoring proteins for various biotechnological applications. Many tools and methods are developed and upgraded each year to satisfy the increasing demands and challenges of protein engineering. To help protein engineers and bioinformaticians navigate this emerging wave of dedicated software, we have critically evaluated recent additions to the toolbox regarding their application for semi-rational and rational protein engineering. These newly developed tools identify and prioritize hotspots and analyze the effects of mutations for a variety of properties, comprising ligand binding, protein–protein and protein–nucleic acid interactions, and electrostatic potential. We also discuss notable progress to target elusive protein dynamics and associated properties like ligand-transport processes and allosteric communication. Finally, we discuss several challenges these tools face and provide our perspectives on the further development of readily applicable methods to guide protein engineering efforts.