Order out of disorder: working cycle of an intrinsically unfolded chaperone.

The redox-regulated chaperone Hsp33 protects organisms against oxidative stress that leads to protein unfolding. Activation of Hsp33 is triggered by the oxidative unfolding of its own redox-sensor domain, making Hsp33 a member of a recently discovered class of chaperones that require partial unfolding for full chaperone activity. Here we address the long-standing question of how chaperones recognize client proteins. We show that Hsp33 uses its own intrinsically disordered regions to discriminate between unfolded and partially structured folding intermediates. Binding to secondary structure elements in client proteins stabilizes Hsp33's intrinsically disordered regions, and this stabilization appears to mediate Hsp33's high affinity for structured folding intermediates. Return to nonstress conditions reduces Hsp33's disulfide bonds, which then significantly destabilizes the bound client proteins and in doing so converts them into less-structured, folding-competent client proteins of ATP-dependent foldases. We propose a model in which energy-independent chaperones use internal order-to-disorder transitions to control substrate binding and release.

Uncoupling the Hsp90 and DnaK chaperone activities revealed the in vivo relevance of their collaboration in bacteria.

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major adenosine triphosphate (ATP)-dependent chaperone highly conserved from bacteria to eukaryotes. Recent studies have shown that bacterial Hsp90 is essential in some bacteria in stress conditions and that it participates in the virulence of pathogenic bacteria. In vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins; however, it is still unknown whether this collaboration is relevant in vivo with physiological substrates. Here, we used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the chaperone activities. We tested the mutants in vivo in two bacterial models in which Hsp90 has known physiological functions. We found that the Hsp90 point mutants were defective to support (1) growth under heat stress and activation of an essential Hsp90 client in the aquatic bacterium Shewanella oneidensis and (2) biosynthesis of the colibactin toxin involved in the virulence of pathogenic Escherichia coli. Our study therefore demonstrates the essentiality of the direct collaboration between Hsp90 and DnaK in vivo in bacteria to support client folding. It also suggests that this collaboration already functional in bacteria has served as an evolutionary basis for a more complex Hsp70-Hsp90 collaboration found in eukaryotes.

Bacterial adaptation to cold: Conservation of a short J-domain co-chaperone and its protein partners in environmental proteobacteria.

Bacterial genomes are a huge reservoir of genes encoding J-domain protein co-chaperones that recruit the molecular chaperone DnaK to assist protein substrates involved in survival, adaptation, or fitness. The atc operon of the aquatic mesophilic bacterium Shewanella oneidensis encodes the proteins AtcJ, AtcA, AtcB, and AtcC, and all of them, except AtcA, are required for growth at low temperatures. AtcJ is a short J-domain protein that interacts with DnaK, but also with AtcC through its 21 amino acid C-terminal domain. This interaction network is critical for cold growth. Here, we show that AtcJ represents a subfamily of short J-domain proteins that (i) are found in several environmental, mostly aquatic, ß- or É£-proteobacteria and (ii) contain a conserved PX7 W motif in their C-terminal extension. Using a combination of NMR, biochemical and genetic approaches, we show that the hydrophobic nature of the tryptophan of the S. oneidensis AtcJ PX7 W motif determines the strong AtcJ-AtcC interaction essential for cold growth. The AtcJ homologues are encoded by operons containing at least the S. oneidensis atcA, atcB, and atcC homologues. These findings suggest a conserved network of DnaK and Atc proteins necessary for low-temperature growth and, given the variation in the atc operons, possibly for other biological functions.

Interplay between Orientation at Electrodes and Copper Activation of Thermus thermophilus Laccase for O2 Reduction.

Multicopper oxidases (MCOs) catalyze the oxidation of a variety of substrates while reducing oxygen into water through four copper atoms. As an additional feature, some MCOs display an enhanced activity in solution in the presence of Cu2+. This is the case of the hyperthermophilic laccase HB27 from Thermus thermophilus, the physiologic role of which is unknown. As a particular feature, this enzyme presents a methionine rich domain proposed to be involved in copper interaction. In this work, laccase from T. thermophilus was produced in E. coli, and the effect of Cu2+ on its electroactivity at carbon nanotube modified electrodes was investigated. Direct O2 electroreduction is strongly dictated by carbon nanotube surface chemistry in accordance with the enzyme dipole moment. In the presence of Cu2+, an additional low potential cathodic wave occurs, which was never described earlier. Analysis of this wave as a function of Cu2+ availability allows us to attribute this wave to a cuprous oxidase activity displayed by the laccase and induced by copper binding close to the Cu T1 center. A mutant lacking the methionine-rich hairpin domain characteristic of this laccase conserves its copper activity suggesting a different site of copper binding. This study provides new insight into the copper effect in methionine rich MCOs and highlights the utility of the electrochemical method to investigate cuprous oxidase activity and to understand the physiological role of these MCOs.

Bacterial Respiratory Chain Diversity Reveals a Cytochrome c Oxidase Reducing O2 at Low Overpotentials.

Cytochrome c oxidases (CcOs) are the terminal enzymes in energy-converting chains of microorganisms, where they reduce oxygen into water. Their affinity for O2 makes them attractive biocatalysts for technological devices in which O2 concentration is limited, but the high overpotentials they display on electrodes severely limit their applicative use. Here, the CcO of the acidophilic bacterium Acidithiobacillus ferrooxidans is studied on various carbon materials by direct protein electrochemistry and mediated one with redox mediators either diffusing or co-immobilized at the electrode surface. The entrapment of the CcO in a network of hydrophobic carbon nanofibers permits a direct electrochemical communication between the enzyme and the electrode. We demonstrate that the CcO displays a µM affinity for O2 and reduces O2 at exceptionally high electrode potentials in the range of +700 to +540 mV vs NHE over a pH range of 4-6. The kinetics of interactions between the enzyme and its physiological partners are fully quantified. Based on these results, an electron transfer pathway allowing O2 reduction in the acidic metabolic chain is proposed.

Impact of copper ligand mutations on a cupredoxin with a green copper center.

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential.

Insight into the evolution of the iron oxidation pathways.

Iron is a ubiquitous element in the universe. Ferrous iron (Fe(II)) was abundant in the primordial ocean until the oxygenation of the Earth's atmosphere led to its widespread oxidation and precipitation. This change of iron bioavailability likely put selective pressure on the evolution of life. This element is essential to most extant life forms and is an important cofactor in many redox-active proteins involved in a number of vital pathways. In addition, iron plays a central role in many environments as an energy source for some microorganisms. This review is focused on Fe(II) oxidation. The fact that the ability to oxidize Fe(II) is widely distributed in Bacteria and Archaea and in a number of quite different biotopes suggests that the dissimilatory Fe(II) oxidation is an ancient energy metabolism. Based on what is known today about Fe(II) oxidation pathways, we propose that they arose independently more than once in evolution and evolved convergently. The iron paleochemistry, the phylogeny, the physiology of the iron oxidizers, and the nature of the cofactors of the redox proteins involved in these pathways suggest a possible scenario for the timescale in which each type of Fe(II) oxidation pathways evolved. The nitrate dependent anoxic iron oxidizers are likely the most ancient iron oxidizers. We suggest that the phototrophic anoxic iron oxidizers arose in surface waters after the Archaea/Bacteria-split but before the Great Oxidation Event. The neutrophilic oxic iron oxidizers possibly appeared in microaerobic marine environments prior to the Great Oxidation Event while the acidophilic ones emerged likely after the advent of atmospheric O(2). This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features.

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated.

Quest for a stable Cu-ligand complex with a high catalytic activity to produce reactive oxygen species.

Metal ion-catalyzed overproduction of reactive oxygen species (ROS) is believed to contribute significantly to oxidative stress and be involved in several biological processes, from immune defense to development of diseases. Among the essential metal ions, copper is one of the most efficient catalysts in ROS production in the presence of O2 and a physiological reducing agent such as ascorbate. To control this chemistry, Cu ions are tightly coordinated to biomolecules. Free or loosely bound Cu ions are generally avoided to prevent their toxicity. In the present report, we aim to find stable Cu-ligand complexes (Cu-L) that can efficiently catalyze the production of ROS in the presence of ascorbate under aerobic conditions. Thermodynamic stability would be needed to avoid dissociation in the biological environment, and high ROS catalysis is of interest for applications as antimicrobial or anticancer agents. A series of Cu complexes with the well-known tripodal and tetradentate ligands containing a central amine linked to three pyridyl-alkyl arms of different lengths were investigated. Two of them with mixed arm length showed a higher catalytic activity in the oxidation of ascorbate and subsequent ROS production than Cu salts in buffer, which is an unprecedented result. Despite these high catalytic activities, no increased antimicrobial activity toward Escherichia coli or cytotoxicity against eukaryotic AGS cells in culture related to Cu-L-based ROS production could be observed. The potential reasons for discrepancy between in vitro and in cell data are discussed.

DnaJ (Hsp40 protein) binding to folded substrate impacts KplE1 prophage excision efficiency.

Temperate phages mediate gene transfer and can modify the properties of their host organisms through the acquisition of novel genes, a process called lysogeny. The KplE1 prophage is one of the 10 prophage regions in Escherichia coli K12 MG1655. KplE1 is defective for lysis but fully competent for site-specific recombination. The TorI recombination directionality factor is strictly required for prophage excision from the host genome. We have previously shown that DnaJ promotes KplE1 excision by increasing the affinity of TorI for its site-specific recombination DNA target. Here, we provide evidence of a direct association between TorI and DnaJ using in vitro cross-linking assays and limited proteolysis experiments that show that this interaction allows both proteins to be transiently protected from trypsin digestion. Interestingly, NMR titration experiments showed that binding of DnaJ involves specific regions of the TorI structure. These regions, mainly composed of α-helices, are located on a surface opposite the DNA-binding site. Taken together, we propose that DnaJ, without the aid of DnaK/GrpE, is capable of increasing the efficiency of KplE1 excision by causing a conformational stabilization that allows TorI to adopt a more favorable conformation for binding to its specific DNA target.

Rhodanese functions as sulfur supplier for key enzymes in sulfur energy metabolism.

How microorganisms obtain energy is a challenging topic, and there have been numerous studies on the mechanisms involved. Here, we focus on the energy substrate traffic in the hyperthermophilic bacterium Aquifex aeolicus. This bacterium can use insoluble sulfur as an energy substrate and has an intricate sulfur energy metabolism involving several sulfur-reducing and -oxidizing supercomplexes and enzymes. We demonstrate that the cytoplasmic rhodanese SbdP participates in this sulfur energy metabolism. Rhodaneses are a widespread family of proteins known to transfer sulfur atoms. We show that SbdP has also some unusual characteristics compared with other rhodaneses; it can load a long sulfur chain, and it can interact with more than one partner. Its partners (sulfur reductase and sulfur oxygenase reductase) are key enzymes of the sulfur energy metabolism of A. aeolicus and share the capacity to use long sulfur chains as substrate. We demonstrate a positive effect of SbdP, once loaded with sulfur chains, on sulfur reductase activity, most likely by optimizing substrate uptake. Taken together, these results lead us to propose a physiological role for SbdP as a carrier and sulfur chain donor to these key enzymes, therefore enabling channeling of sulfur substrate in the cell as well as greater efficiency of the sulfur energy metabolism of A. aeolicus.

Revisiting the pro-oxidant activity of copper: interplay of ascorbate, cysteine, and glutathione.

Copper (Cu) is essential for most organisms, but it can be poisonous in excess, through mechanisms such as protein aggregation, trans-metallation, and oxidative stress. The latter could implicate the formation of potentially harmful reactive oxygen species (O2•-, H2O2, and HO•) via the redox cycling between Cu(II)/Cu(I) states in the presence of dioxygen and physiological reducing agents such as ascorbate (AscH), cysteine (Cys), and the tripeptide glutathione (GSH). Although the reactivity of Cu with these reductants has been previously investigated, the reactions taking place in a more physiologically relevant mixture of these biomolecules are not known. Hence, we report here on the reactivity of Cu with binary and ternary mixtures of AscH, Cys, and GSH. By measuring AscH and thiol oxidation, as well as HO• formation, we show that Cu reacts preferentially with GSH and Cys, halting AscH oxidation and also HO• release. This could be explained by the formation of Cu-thiolate clusters with both GSH and, as we first demonstrate here, Cys. Moreover, we observed a remarkable acceleration of Cu-catalyzed GSH oxidation in the presence of Cys. We provide evidence that both thiol-disulfide exchange and the generated H2O2 contribute to this effect. Based on these findings, we speculate that Cu-induced oxidative stress may be mainly driven by GSH depletion and/or protein disulfide formation rather than by HO• and envision a synergistic effect of Cys on Cu toxicity.

The tip protein PAAR is required for the function of the type VI secretion system.

IMPORTANCE: The type VI secretion system (T6SS) is a bacterial contractile injection system involved in bacterial competition by the delivery of antibacterial toxins. The T6SS consists of an envelope-spanning complex that recruits the baseplate, allowing the polymerization of a contractile tail structure. The tail is a tube wrapped by a sheath and topped by the tip of the system, the VgrG spike/PAAR complex. Effectors loaded onto the puncturing tip or into the tube are propelled in the target cells upon sheath contraction. The PAAR protein tips and sharpens the VgrG spike. However, the importance and the function of this protein remain unclear. Here, we provide evidence for association of PAAR at the tip of the VgrG spike. We also found that the PAAR protein is a T6SS critical component required for baseplate and sheath assembly.

Mineral respiration under extreme acidic conditions: from a supramolecular organization to a molecular adaptation in Acidithiobacillus ferrooxidans.

Acidithiobacillus ferrooxidans is an acidophilic chemolithoautotrophic Gram-negative bacterium that can derive energy from the oxidation of ferrous iron at pH 2 using oxygen as electron acceptor. The study of this bacterium has economic and fundamental biological interest because of its use in the industrial extraction of copper and uranium from ores. For this reason, its respiratory chain has been analysed in detail in recent years. Studies have shown the presence of a functional supercomplex that spans the outer and the inner membranes and allows a direct electron transfer from the extracellular Fe2+ ions to the inner membrane cytochrome c oxidase. Iron induces the expression of two operons encoding proteins implicated in this complex as well as in the regeneration of the reducing power. Most of these are metalloproteins that have been characterized biochemically, structurally and biophysically. For some of them, the molecular basis of their adaptation to the periplasmic acidic environment has been described. Modifications in the metal surroundings have been highlighted for cytochrome c and rusticyanin, whereas, for the cytochrome c oxidase, an additional partner that maintains its stability and activity has been demonstrated recently.

The redox-switch domain of Hsp33 functions as dual stress sensor.

The redox-regulated chaperone Hsp33 is specifically activated upon exposure of cells to peroxide stress at elevated temperatures. Here we show that Hsp33 harbors two interdependent stress-sensing regions located in the C-terminal redox-switch domain of Hsp33: a zinc center sensing peroxide stress conditions and an adjacent linker region responding to unfolding conditions. Neither of these sensors works sufficiently in the absence of the other, making the simultaneous presence of both stress conditions a necessary requirement for Hsp33's full activation. Upon activation, Hsp33's redox-switch domain adopts a natively unfolded conformation, thereby exposing hydrophobic surfaces in its N-terminal substrate-binding domain. The specific activation of Hsp33 by the oxidative unfolding of its redox-switch domain makes this chaperone optimally suited to quickly respond to oxidative stress conditions that lead to protein unfolding.

Copper Induces Protein Aggregation, a Toxic Process Compensated by Molecular Chaperones.

Copper is well known for its antimicrobial and antiviral properties. Under aerobic conditions, copper toxicity relies in part on the production of reactive oxygen species (ROS), especially in the periplasmic compartment. However, copper is significantly more toxic under anaerobic conditions, in which ROS cannot be produced. This toxicity has been proposed to arise from the inactivation of proteins through mismetallations. Here, using the bacterium Escherichia coli, we discovered that copper treatment under anaerobic conditions leads to a significant increase in protein aggregation. In vitro experiments using E. coli lysates and tightly controlled redox conditions confirmed that treatment with Cu+ under anaerobic conditions leads to severe ROS-independent protein aggregation. Proteomic analysis of aggregated proteins revealed an enrichment of cysteine- and histidine-containing proteins in the Cu+-treated samples, suggesting that nonspecific interactions of Cu+ with these residues are likely responsible for the observed protein aggregation. In addition, E. coli strains lacking the cytosolic chaperone DnaK or trigger factor are highly sensitive to copper stress. These results reveal that bacteria rely on these chaperone systems to protect themselves against Cu-mediated protein aggregation and further support our finding that Cu toxicity is related to Cu-induced protein aggregation. Overall, our work provides new insights into the mechanism of Cu toxicity and the defense mechanisms that bacteria employ to survive. IMPORTANCE With the increase of antibiotic drug resistance, alternative antibacterial treatment strategies are needed. Copper is a well-known antimicrobial and antiviral agent; however, the underlying molecular mechanisms by which copper causes cell death are not yet fully understood. Herein, we report the finding that Cu+, the physiologically relevant copper species in bacteria, causes widespread protein aggregation. We demonstrate that the molecular chaperones DnaK and trigger factor protect bacteria against Cu-induced cell death, highlighting, for the first time, the central role of these chaperones under Cu+ stress. Our studies reveal Cu-induced protein aggregation to be a central mechanism of Cu toxicity, a finding that will serve to guide future mechanistic studies and drug development.

Unfolding of metastable linker region is at the core of Hsp33 activation as a redox-regulated chaperone.

Hsp33, a molecular chaperone specifically activated by oxidative stress conditions that lead to protein unfolding, protects cells against oxidative protein aggregation. Stress sensing in Hsp33 occurs via its C-terminal redox switch domain, which consists of a zinc center that responds to the presence of oxidants and an adjacent metastable linker region, which responds to unfolding conditions. Here we show that single mutations in the N terminus of Hsp33 are sufficient to either partially (Hsp33-M172S) or completely (Hsp33-Y12E) abolish this post-translational regulation of Hsp33 chaperone function. Both mutations appear to work predominantly via the destabilization of the Hsp33 linker region without affecting zinc coordination, redox sensitivity, or substrate binding of Hsp33. We found that the M172S substitution causes moderate destabilization of the Hsp33 linker region, which seems sufficient to convert the redox-regulated Hsp33 into a temperature-controlled chaperone. The Y12E mutation leads to the constitutive unfolding of the Hsp33 linker region thereby turning Hsp33 into a constitutively active chaperone. These results demonstrate that the redox-controlled unfolding of the Hsp33 linker region plays the central role in the activation process of Hsp33. The zinc center of Hsp33 appears to act as the redox-sensitive toggle that adjusts the thermostability of the linker region to the cell redox status. In vivo studies confirmed that even mild overexpression of the Hsp33-Y12E mutant protein inhibits bacterial growth, providing important evidence that the tight functional regulation of Hsp33 chaperone activity plays a vital role in bacterial survival.

An unconventional copper protein required for cytochrome c oxidase respiratory function under extreme acidic conditions.

Very little is known about the processes used by acidophile organisms to preserve stability and function of respiratory pathways. Here, we reveal a potential strategy of these organisms for protecting and keeping functional key enzymes under extreme conditions. Using Acidithiobacillus ferrooxidans, we have identified a protein belonging to a new cupredoxin subfamily, AcoP, for "acidophile CcO partner," which is required for the cytochrome c oxidase (CcO) function. We show that it is a multifunctional copper protein with at least two roles as follows: (i) as a chaperone-like protein involved in the protection of the Cu(A) center of the CcO complex and (ii) as a linker between the periplasmic cytochrome c and the inner membrane cytochrome c oxidase. It could represent an interesting model for investigating the multifunctionality of proteins known to be crucial in pathways of energy metabolism.

Quantifying changes in the thiol redox proteome upon oxidative stress in vivo.

Antimicrobial levels of reactive oxygen species (ROS) are produced by the mammalian host defense to kill invading bacteria and limit bacterial colonization. One main in vivo target of ROS is the thiol group of proteins. We have developed a quantitative thiol trapping technique termed OxICAT to identify physiologically important target proteins of hydrogen peroxide (H(2)O(2)) and hypochlorite (NaOCl) stress in vivo. OxICAT allows the precise quantification of oxidative thiol modifications in hundreds of different proteins in a single experiment. It also identifies the affected proteins and defines their redox-sensitive cysteine(s). Using this technique, we identified a group of Escherichia coli proteins with significantly (30-90%) oxidatively modified thiol groups, which appear to be specifically sensitive to either H(2)O(2) or NaOCl stress. These results indicate that individual oxidants target distinct proteins in vivo. Conditionally essential E. coli genes encode one-third of redox-sensitive proteins, a finding that might explain the bacteriostatic effect of oxidative stress treatment. We identified a select group of redox-regulated proteins, which protect E. coli against oxidative stress conditions. These experiments illustrate that OxICAT, which can be used in a variety of different cell types and organisms, is a powerful tool to identify, quantify, and monitor oxidative thiol modifications in vivo.

The Central Role of Redox-Regulated Switch Proteins in Bacteria.

Bacteria possess the ability to adapt to changing environments. To enable this, cells use reversible post-translational modifications on key proteins to modulate their behavior, metabolism, defense mechanisms and adaptation of bacteria to stress. In this review, we focus on bacterial protein switches that are activated during exposure to oxidative stress. Such protein switches are triggered by either exogenous reactive oxygen species (ROS) or endogenous ROS generated as by-products of the aerobic lifestyle. Both thiol switches and metal centers have been shown to be the primary targets of ROS. Cells take advantage of such reactivity to use these reactive sites as redox sensors to detect and combat oxidative stress conditions. This in turn may induce expression of genes involved in antioxidant strategies and thus protect the proteome against stress conditions. We further describe the well-characterized mechanism of selected proteins that are regulated by redox switches. We highlight the diversity of mechanisms and functions (as well as common features) across different switches, while also presenting integrative methodologies used in discovering new members of this family. Finally, we point to future challenges in this field, both in uncovering new types of switches, as well as defining novel additional functions.