Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activ... more Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.
Conformational preferences of a group of hexapeptides containing two dehydroamino acid residues i... more Conformational preferences of a group of hexapeptides containing two dehydroamino acid residues in Positions 2 and 5 in peptide chain were investigated by means of spectroscopic methods (NMR and CD) and theoretical calculations. In the case of dimethylsulfoxide (DMSO) solution, only peptide with free N-termini adopted rigid 3 10-helical conformation, for the rest of examined peptides extended and ''zig-zag'' conformers were predominant. CD measurements showed that only in chloroform solution the conformational freedom of investigated peptides was restricted.
Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activ... more Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.
Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participa... more Mammalian metallothioneins (MTs) are small cysteine-rich proteins whose primary role is participation in zinc and copper homeostasis. Ever since their discovery, MTs have been investigated in terms of metal-binding affinity. The initial concept of seven Zn(II) ions (Zn7MT) bound with the same, undifferentiated low-picomolar affinity in the α and β domains prevailed for many years and derived from spectroscopic studies. The application of fluorescent zinc probes has changed the perception of MTs, showing that they function in nanomolar to subnanomolar free zinc concentrations due to the presence of tight, moderate, and weak binding sites. The discovery of Zn(II)-depleted MTs in many tissues and determination of cellular free Zn(II) concentrations with differentiated zinc affinity sites revealed the critical importance of partially saturated Zn4–6MTs species in cellular zinc buffering in a wide picomolar to nanomolar range of free Zn(II) concentrations. Until today, there was no clear...
We present a molecular dynamics approach to explore the conformational landscape of proteins anal... more We present a molecular dynamics approach to explore the conformational landscape of proteins analyzed by ion mobility-mass spectrometry, providing new insights into the structural properties of metallothioneins, a cellular zinc buffer system.
Recent discoveries in zinc biology provide a new platform for discussing the primary physiologica... more Recent discoveries in zinc biology provide a new platform for discussing the primary physiological functions of mammalian metallothioneins (MTs) and their exquisite zinc-dependent regulation. It is now understood that the control of cellular zinc homeostasis includes buffering of Zn 2+ ions at picomolar concentrations, extensive subcellular redistribution of Zn 2+ , the loading of exocytotic vesicles with zinc species, and the control of Zn 2+ ion signalling. In parallel, characteristic features of human MTs became known: their graded affinities for Zn 2+ and the redox activity of their thiolate coordination environments. Unlike the single species that structural models of mammalian MTs describe with a set of seven divalent or eight to twelve monovalent metal ions, MTs are metamorphic. In vivo, they exist as many species differing in redox state and load with different metal ions. The functions of mammalian MTs should no longer be considered elusive or enigmatic because it is now evident that the reactivity and coordination dynamics of MTs with Zn 2+ and Cu + match the biological requirements for controlling-binding and delivering-these cellular metal ions, thus completing a 60-year search for their functions. MT represents a unique biological principle for buffering the most competitive essential metal ions Zn 2+ and Cu +. How this knowledge translates to the function of other families of MTs awaits further insights into the specifics of how their properties relate to zinc and copper metabolism in other organisms.
Archives of biochemistry and biophysics, Jan 23, 2016
The solution and complexation chemistry of zinc ions is the basis for zinc biology. In living org... more The solution and complexation chemistry of zinc ions is the basis for zinc biology. In living organisms, zinc is redox-inert and has only one valence state: Zn(II). Its coordination environment in proteins is limited by oxygen, nitrogen, and sulfur donors from the side chains of a few amino acids. In an estimated 10% of all human proteins, zinc has a catalytic or structural function and remains bound during the lifetime of the protein. However, in other proteins zinc ions bind reversibly with dissociation and association rates commensurate with the requirements in regulation, transport, transfer, sensing, signalling, and storage. In contrast to the extensive knowledge about zinc proteins, the coordination chemistry of the "mobile" zinc ions in these processes, i.e. when not bound to proteins, is virtually unexplored and the mechanisms of ligand exchange are poorly understood. Knowledge of the biological inorganic chemistry of zinc ions is essential for understanding its ce...
The metal binding at protein–protein interfaces is still uncharted territory in intermolecular in... more The metal binding at protein–protein interfaces is still uncharted territory in intermolecular interactions. To date, only a few protein complexes binding Zn(II) in an intermolecular manner have been deeply investigated. The most notable example of such interfaces is located in the highly conserved Rad50 protein, part of the Mre11-Rad50-Nbs1 (MRN) complex, where Zn(II) is required for homodimerization (Zn(Rad50)2). The high stability of Zn(Rad50)2 is conserved not only for the protein derived from the thermophilic archaeon Pyrococcus furiosus (logK12 = 20.95 for 130-amino-acid-long fragment), which was the first one studied, but also for the human paralog studied here (logK12 = 19.52 for a 183-amino-acid-long fragment). As we reported previously, the extremely high stability results from the metal-coupled folding process where particular Rad50 protein fragments play a critical role. The sequence–structure–stability analysis based on human Rad50 presented here separates the individua...
The binding of metal ions at the interface of protein complexes presents a unique and poorly unde... more The binding of metal ions at the interface of protein complexes presents a unique and poorly understood mechanism of molecular assembly. A remarkable example is the Rad50 zinc hook domain, which is highly conserved and facilitates the Zn 2+-mediated homodimerization of Rad50 proteins. Here, we present a detailed analysis of the structural and thermodynamic effects governing the formation and stability (logK 12 = 20.74) of this evolutionarily conserved protein assembly. We have dissected the determinants of the stability contributed by the small β-hairpin of the domain surrounding the zinc binding motif and the coiled-coiled regions using peptides of various lengths from 4 to 45 amino acid residues, alanine substitutions and peptide bond-to-ester perturbations. In the studied series of peptides, an >650 000-fold increase of the formation constant of the dimeric complex arises from favorable enthalpy because of the increased acidity of the cysteine thiols in metal-free form and the structural properties of the dimer. The dependence of the enthalpy on the domain fragment length is partially compensated by the entropic penalty of domain folding, indicating enthalpy-entropy compensation. This study facilitates understanding of the metal-mediated protein-protein interactions in which the metal ion is critical for the tight association of protein subunits.
Phytochelatins (PCs) are short Cys-rich peptides with repeating γ-Glu-Cys motifs found in plants,... more Phytochelatins (PCs) are short Cys-rich peptides with repeating γ-Glu-Cys motifs found in plants, algae, certain fungi, and worms. Their biosynthesis has been found to be induced by heavy metalsboth biogenic and toxic. Among all metal inducers, Cd(II) has been the most explored from a biological and chemical point of view. Although Cd(II)-induced PC biosynthesis has been widely examined, still little is known about the structure of Cd(II) complexes and their thermodynamic stability. Here, we systematically investigated glutathione (GSH) and PC2−PC6 systems, with regard to their complex stoichiometries and spectroscopic and thermodynamic properties. We paid special attention to the determination of stability constants using several complementary techniques. All peptides form CdL complexes, but CdL 2 was found for GSH, PC2, and partially for PC3. Moreover, binuclear species Cd x L y were identified for the series PC3−PC6 in an excess of Cd(II). Potentiometric and competition spectroscopic studies showed that the affinity of Cd(II) complexes increases from GSH to PC4 almost linearly from micromolar (log K 7.4 GSH = 5.93) to the femtomolar range (log K 7.4 PC4 = 13.39) and additional chain elongation does not increase the stability significantly. Data show that PCs form an efficient system which buffers free Cd(II) ions in the pico-to femtomolar range under cellular conditions, avoiding significant interference with Zn(II) complexes. Our study confirms that the favorable entropy change is the factor governing the elevation of phytochelatins' stability and illuminates the importance of the chelate effect in shifting the free Gibbs energy.
Cover Feature: Metal Exchange in the Interprotein Zn II ‐Binding Site of the Rad50 Hook Domain: Structural Insights into Cd II ‐Induced DNA‐Repair Inhibition (Chem. Eur. J. 15/2020)
InterMetalDB is a free-of-charge database and browser of intermolecular metal binding sites that ... more InterMetalDB is a free-of-charge database and browser of intermolecular metal binding sites that are present on the interfaces of macromolecules forming larger assemblies based on structural information deposited in Protein Data Bank (PDB). It can be found and freely used at https://intermetaldb.biotech.uni. wroc.pl/. InterMetalDB collects the interfacial binding sites with involvement of metal ions and clusters them on the basis of 50% sequence similarity and the nearest metal environment (5 Å radius). The data are available through the web interface where they can be queried, viewed, and downloaded. Complexity of the query depends on the user, because the questions in the query are connected with each other by a logical AND. InterMetalDB offers several useful options for filtering records including searching for structures by particular parameters such as structure resolution, structure description, and date of deposition. Records can be filtered by coordinated metal ion, number of bound amino acid residues, coordination sphere, and other features. InterMetalDB is regularly updated and will continue to be regularly updated with new content in the future. InterMetalDB is a useful tool for all researchers interested in metalloproteins, protein engineering, and metal-driven oligomerization.
Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which... more Macroautophagy/autophagy plays an important role in cellular copper clearance. The means by which the copper metabolism and autophagy pathways interact mechanistically is vastly unexplored. Dysfunctional ATP7B, a copper-transporting ATPase, is involved in the development of monogenic Wilson disease, a disorder characterized by disturbed copper transport. Using in silico prediction, we found that ATP7B contains a number of potential binding sites for LC3, a central protein in the autophagy pathway, the so-called LC3 interaction regions (LIRs). The conserved LIR3, located at the C-terminal end of ATP7B, was found to directly interact with LC3B in vitro. Replacing the two conserved hydrophobic residues W1452 and L1455 of LIR3 significantly reduced interaction. Furthermore, autophagy was induced in normal human hepatocellular carcinoma cells (HepG2) leading to enhanced colocalization of ATP7B and LC3B on the autophagosome membranes. By contrast, HepG2 cells deficient of ATP7B (HepG2 ATP...
HP1021 is a redox switch protein identified in Helicobacter pylori
Nucleic Acids Research
Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread co... more Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicat...
The functions, purposes, and roles of metallothioneins have been the subject of speculations sinc... more The functions, purposes, and roles of metallothioneins have been the subject of speculations since the discovery of the protein over 60 years ago. This article guides through the history of investigations and resolves multiple contentions by providing new interpretations of the structure-stability-function relationship. It challenges the dogma that the biologically relevant structure of the mammalian proteins is only the one determined by X-ray diffraction and NMR spectroscopy. The terms metallothionein and thionein are ambiguous and insufficient to understand biological function. The proteins need to be seen in their biological context, which limits and defines the chemistry possible. They exist in multiple forms with different degrees of metalation and types of metal ions. The homoleptic thiolate coordination of mammalian metallothioneins is important for their molecular mechanism. It endows the proteins with redox activity and a specific pH dependence of their metal affinities. The proteins, therefore, also exist in different redox states of the sulfur donor ligands. Their coordination dynamics allows a vast conformational landscape for interactions with other proteins and ligands. Many fundamental signal transduction pathways regulate the expression of the dozen of human metallothionein genes. Recent advances in understanding the control of cellular zinc and copper homeostasis are the foundation for suggesting that mammalian metallothioneins provide a highly dynamic, regulated, and uniquely biological metal buffer to control the availability, fluctuations, and signaling transients of the most competitive Zn(II) and Cu(I) ions in cellular space and time.
Identification of metal-binding sites in proteins and understanding metalcoupled protein folding ... more Identification of metal-binding sites in proteins and understanding metalcoupled protein folding mechanisms are aspects of high importance for the structure-tofunction relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal−protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low-to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.
Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in tw... more Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two αand β-domains and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. At cellular free Zn(II) concentrations (10 −11 −10 −9 M), MTs do not exist in fully loaded forms with seven Zn(II)-bound ions (Zn 7 MTs). Instead, MTs exist as partially metal-depleted species (Zn 4−6 MT) because their Zn(II) binding affinities are on the nano-to picomolar range comparable to the concentrations of cellular Zn(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn(II) (un)binding to MTs, the thermodynamic properties of the Zn 1−6 MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quantitative bottom-up and topdown MS in combination with steered molecular dynamics simulations, welltempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5 μs) were integrated to unravel the chemical coordination of Zn(II) in all Zn 1−6 MT2 species and to explain the differences in binding affinities of Zn(II) ions to MTs. Differences are found to be the result of the degree of water participation in MT (un)folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodynamics properties of Zn(II) (un)binding to MT2 are found to differ from those of Cd(II), justifying their distinctive roles. The potential of this integrated strategy in the investigation of numerous unexplored metalloproteins is attested by the results highlighted in the present study.
Biarsenical fluorescent probes for multifunctional site-specific modification of proteins applicable in life sciences: an overview and future outlook
Metallomics
Though arsenic alone is acutely toxic, its incorporation into fluorescent probes renders it less ... more Though arsenic alone is acutely toxic, its incorporation into fluorescent probes renders it less harmful and enables multiple applications in life sciences. See how your research can be improved using biarsenical probes.
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Papers by Artur Krężel