Impact of naturally occurring serine/cysteine variations on the structure and function of <i>Pseudomonas</i> metallothioneins

Habjanič, Jelena; Chesnov, Serge; Zerbe, Oliver; Freisinger, Eva

doi:10.1039/c9mt00213h

Cited by 8 publications

(6 citation statements)

References 22 publications

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“…The difference UV-Vis spectra, where the protein contribution to the absorbance is subtracted out, clearly shows the build-up of a shoulder near λ = 270 nm ( Figure 1C ). The wavelength range is consistent with the position of thiolate-Cd 2+ charge transfer bands observed in other studies ( Busenlehner et al, 2001 ; Habjanič et al, 2020 ). Based on this information and previous work on the Zn 2+ -containing proteins with Cys-rich sites ( Wang et al, 2005 ; Chakraborty et al, 2011 ; Malgieri et al, 2011 ), we conclude that Cd 2+ forms coordination bonds with the cysteine residues of C1B, even in the presence of Cd 2+ -sequestering C2.…”

Section: Resultssupporting

confidence: 91%

“…The first step was to determine how Cd 2+ interacts with the C1B-C2 domain using UV-vis absorption spectroscopy. It is well established that thiolate-Cd 2+ charge transfer bands have characteristic wavelengths at around ∼240 nm ( Busenlehner et al, 2001 ; Habjanič et al, 2020 ). The C1B domain has six cysteine residues, all of which are involved in coordinating the structural Zn 2+ ions ( Figure 1A ).…”

Section: Resultsmentioning

confidence: 99%

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Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy

Cole

Igumenova

2021

Front. Mol. Biosci.

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Conserved homology 1 (C1) domains are peripheral zinc finger domains that are responsible for recruiting their host signaling proteins, including Protein Kinase C (PKC) isoenzymes, to diacylglycerol-containing lipid membranes. In this work, we investigated the reactivity of the C1 structural zinc sites, using the cysteine-rich C1B regulatory region of the PKCα isoform as a paradigm. The choice of Cd2+ as a probe was prompted by previous findings that xenobiotic metal ions modulate PKC activity. Using solution NMR and UV-vis spectroscopy, we found that Cd2+ spontaneously replaced Zn2+ in both structural sites of the C1B domain, with the formation of all-Cd and mixed Zn/Cd protein species. The Cd2+ substitution for Zn2+ preserved the C1B fold and function, as probed by its ability to interact with a potent tumor-promoting agent. Both Cys3His metal-ion sites of C1B have higher affinity to Cd2+ than Zn2+, but are thermodynamically and kinetically inequivalent with respect to the metal ion replacement, despite the identical coordination spheres. We find that even in the presence of the oxygen-rich sites presented by the neighboring peripheral membrane-binding C2 domain, the thiol-rich sites can successfully compete for the available Cd2+. Our results indicate that Cd2+ can target the entire membrane-binding regulatory region of PKCs, and that the competition between the thiol- and oxygen-rich sites will likely determine the activation pattern of PKCs.

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Section: Resultssupporting

confidence: 91%

Section: Resultsmentioning

confidence: 99%

Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy

Cole

Igumenova

2021

Front. Mol. Biosci.

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“…Co-occurrence of the two cysteine-rich domains, CXCXXCXC and znf, in a protein is the hallmark of a MT because together they can form a metal cluster. Several bacterial MTs have been sequenced in the last two decades, − and the most investigated are SmtA in cyanobacteria and PflQ2 in Pseudomonas sp, which each harbor 9–10 Cys residues. The NMR structure of the Zn-bound SmtA MT for the cyanobacteria Synechococcus sp and the Zn- and Cd-bound PFLQ2 MT for Pseudomonas fluorescens show that the cysteine residues form a pocket and bind four Cd atoms or three Zn atoms (Figure S7b).…”

Section: Discussionmentioning

confidence: 99%

Acute Toxicity of Divalent Mercury to Bacteria Explained by the Formation of Dicysteinate and Tetracysteinate Complexes Bound to Proteins in Escherichia coli and Bacillus subtilis

Manceau

Nagy

Glatzel

et al. 2021

Environ. Sci. Technol.

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Bacteria are the most abundant organisms on Earth and also the major life form affected by mercury (Hg) poisoning in aquatic and terrestrial food webs. For this reason, identifying the intracellular molecular forms and trafficking pathways of Hg in bacteria at environmentally relevant concentrations is critical to controlling Hg toxicity. We applied high energy-resolution X-ray absorption near edge structure (HR-XANES) spectroscopy to bacteria with intracellular concentrations of Hg as low as 0.7 ng/mg (ppm). Gram-positive Bacillus subtilis and gram-negative Escherichia coli were exposed to three Hg species: HgCl2, Hg-dicysteinate (Hg(Cys)2), and Hg-dithioglycolate (Hg(TGA)2). In all cases, Hg was transformed into new two-and four-coordinate cysteinate complexes, interpreted to be bound, respectively, to the consensus metal binding CXXC motif and zinc finger domains of proteins, with glutathione acting as a transfer ligand. Replacement of zinc cofactors essential to gene regulatory proteins with Hg would inhibit vital functions such as DNA transcription and repair and is suggested to be a main cause of Hg genotoxicity.

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“…On the other hand, specially conserved in anhydrobiotic cyanobacterial MTs, serine residues (Ser-19 and Ser-33) can provide an enhanced molecular stability (Emoto et al, 1996). Such stabilizing effect was described in Pseudomonas fluorescens (Cd4PflQ2 MT), in which Ser29 has an essential role as an N-cap for a short alpha-helix, forming two hydrogen bonds, one from its carbonyl oxygen to the amide proton of Ala33 and a second one from the side-chain oxygen to the amide proton of Ala31 (Habjanič et al, 2020). However, most of these conserved non-coordinating residues (V4, T5, M7, K8, A10, L15, V18, E21, A23, K26, D27, K29, Y32, G39, G46, and G51) do not have well characterized functions in MT.…”

Section: Primary Sequence Analysesmentioning

confidence: 99%

“…A slightly increased percentage of cysteine was observed in non-anhydrobiotic cyanobacterial MTs that can be pointed out, for example, by the amino acid substitution S33C, in which cysteine appears partially conserved (80%) in this group. This common residue substitution relies on a change of a single oxygen atom by a sulfur, which positively influences the coordination of cadmium (Cd), due to the involvement of Cys-33 in metal-binding reactions (Habjanič et al, 2020), without affecting the MT structure. However, as mentioned by (Emoto et al, 1996), this Ser-to-Cys substitution results in an overall destabilization.…”

Section: Primary Sequence Analysesmentioning

confidence: 99%

A distinct molecular signature on anhydrobiotic cyanobacterial metallothioneins

Contiliani

Moraes

Ribeiro

et al. 2021

RSD

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Anhydrobiosis refers to a state of suspended animation in which some organisms enter when exposed to extreme desiccation, ensuring them an outstanding tolerance to several physical stresses due to molecular and cellular adaptations. Metallothioneins (MTs) are short cysteine-rich metal-chelating proteins that work as a cellular protection element in metal ion-rich conditions. Here we aimed to investigate possible molecular signatures in primary and tertiary structures in anhydrobiotic cyanobacterial MTs. Anhydrobiotic and non-anhydrobiotic cyanobacterial MT amino acid sequences were retrieved from NCBI database and aligned in Clustal Omega server. Additionally, the amino acid compositions of these sequences were determined by GeneRunner. Further, we carried out homology-modeling via SWISS-MODEL, structural superposition in UCSF Chimera 1.4 Matchmaker tool and ligand-binding site prediction via COFACTOR. In silico analyses revealed specific divergences in amino acid positions between MT groups, evidencing positive and negative selections, however without affecting final protein structures. Some of these changes on polypeptide sequence potentially enhance protein stabilization during desiccation, whereas others possibly act as additional metal-ion coordinating residues. Analyses on the molecular adaptations on anhydrobiotic cyanobacterial MTs help shed light on their molecular functions and biological roles, as well as may have applications on the development of desiccation- and metal-tolerant organisms.

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Impact of naturally occurring serine/cysteine variations on the structure and function of Pseudomonas metallothioneins

Abstract: Naturally occurring Ser/Cys variations in Pseudomonas metallothioneinss affect intra-cluster dynamics rather than binding capacity.

Cited by 8 publications

References 22 publications

Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy

Reactivity of Thiol-Rich Zn Sites in Diacylglycerol-Sensing PKC C1 Domain Probed by NMR Spectroscopy

Acute Toxicity of Divalent Mercury to Bacteria Explained by the Formation of Dicysteinate and Tetracysteinate Complexes Bound to Proteins in Escherichia coli and Bacillus subtilis

A distinct molecular signature on anhydrobiotic cyanobacterial metallothioneins

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