metal binding
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2022 ◽  
Author(s):  
Krishnendu Roy ◽  
Thomas Pucadyil

Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Unlike that seen with the native mitochondrial lipid cardiolipin, metal-bound chelator lipids recruit Drp1 to the membrane but is rendered functionally inactive in membrane fission. Metal-bound chelator beads also recruit Drp1 and represents a potential strategy to deplete or purify the protein from native tissue lysates.


Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 135
Author(s):  
Yanchun Lin ◽  
Michael L. Gross

Metal ions are critical for the biological and physiological functions of many proteins. Mass spectrometry (MS)-based structural proteomics is an ever-growing field that has been adopted to study protein and metal ion interactions. Native MS offers information on metal binding and its stoichiometry. Footprinting approaches coupled with MS, including hydrogen/deuterium exchange (HDX), “fast photochemical oxidation of proteins” (FPOP) and targeted amino-acid labeling, identify binding sites and regions undergoing conformational changes. MS-based titration methods, including “protein–ligand interactions by mass spectrometry, titration and HD exchange” (PLIMSTEX) and “ligand titration, fast photochemical oxidation of proteins and mass spectrometry” (LITPOMS), afford binding stoichiometry, binding affinity, and binding order. These MS-based structural proteomics approaches, their applications to answer questions regarding metal ion protein interactions, their limitations, and recent and potential improvements are discussed here. This review serves as a demonstration of the capabilities of these tools and as an introduction to wider applications to solve other questions.


2022 ◽  
Vol 8 (2) ◽  
Author(s):  
Yana Bromberg ◽  
Ariel A. Aptekmann ◽  
Yannick Mahlich ◽  
Linda Cook ◽  
Stefan Senn ◽  
...  

Computational exploration of similarities among metal-binding protein structural motifs elucidates the origins of life.


2022 ◽  
Vol 8 ◽  
Author(s):  
Agnieszka Ruszkowska ◽  
Ya Ying Zheng ◽  
Song Mao ◽  
Milosz Ruszkowski ◽  
Jia Sheng

G•U wobble base pair frequently occurs in RNA structures. The unique chemical, thermodynamic, and structural properties of the G•U pair are widely exploited in RNA biology. In several RNA molecules, the G•U pair plays key roles in folding, ribozyme catalysis, and interactions with proteins. G•U may occur as a single pair or in tandem motifs with different geometries, electrostatics, and thermodynamics, further extending its biological functions. The metal binding affinity, which is essential for RNA folding, catalysis, and other interactions, differs with respect to the tandem motif type due to the different electrostatic potentials of the major grooves. In this work, we present the crystal structure of an RNA 8-mer duplex r[UCGUGCGA]2, providing detailed structural insights into the tandem motif I (5′UG/3′GU) complexed with Ba2+ cation. We compare the electrostatic potential of the presented motif I major groove with previously published structures of tandem motifs I, II (5′GU/3′UG), and III (5′GG/3′UU). A local patch of a strongly negative electrostatic potential in the major groove of the presented structure forms the metal binding site with the contributions of three oxygen atoms from the tandem. These results give us a better understanding of the G•U tandem motif I as a divalent metal binder, a feature essential for RNA functions.


Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 121
Author(s):  
Denise Bellotti ◽  
Adriana Miller ◽  
Magdalena Rowińska-Żyrek ◽  
Maurizio Remelli

Zrt2 is a zinc transporter of the ZIP family. It is predicted to be located in the plasma membrane and it is essential for Candida albicans zinc uptake and growth at acidic pH. Zrt2 from C. albicans is composed of 370 amino acids and contains eight putative transmembrane domains and an extra-membrane disordered loop, corresponding to the amino acid sequence 126–215. This protein region contains at least three possible metal binding motifs: HxHxHxxD (144–153), HxxHxxEHxD (181–193) and the Glu- and Asp- rich sequence DDEEEDxE (161–168). The corresponding model peptides, protected at their termini (Ac-GPHTHSHFGD-NH2, Ac-DDEEEDLE-NH2 and Ac-PSHFAHAQEHQDP-NH2), have been investigated in order to elucidate the thermodynamic and coordination properties of their Zn2+ and Cu2+ complexes, with the further aim to identify the most effective metal binding site among the three fragments. Furthermore, we extended the investigation to the peptides Ac-GPHTHAHFGD-NH2 and Ac-PAHFAHAQEHQDP-NH2, where serine residues have been substituted by alanines in order to check if the presence of a serine residue may favor the displacement of amidic protons by Cu2+. In the native Zrt2 protein, the Ac-GPHTHSHFGD-NH2 region of the Zrt2 loop has the highest metal binding affinity, showing that three alternated histidines separated by only one residue (-HxHxH-) bind Zn2+ and Cu2+ more strongly than the region in which three histidines are separated by two and three His residues (-HxxHxxxH- in Ac-PSHFAHAQEHQDP-NH2). All studied Zrt2 loop fragments have lower affinity towards Zn2+ than the zinc(II) binding site on the Zrt1 transporter; also, all three Zrt2 regions bind Zn2+ and Cu2+ with comparable affinity below pH 5 and, therefore, may equally contribute to the metal acquisition under the most acidic conditions in which the Zrt2 transporter is expressed.


2022 ◽  
Vol 12 ◽  
Author(s):  
T. V. Divya ◽  
Celin Acharya

Metallothioneins (MTs) are cysteine-rich, metal-sequestering cytosolic proteins that play a key role in maintaining metal homeostasis and detoxification. We had previously characterized NmtA, a MT from the heterocystous, nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 and demonstrated its role in providing protection against cadmium toxicity. In this study, we illustrate the regulation of Anabaena NmtA by AzuR (Alr0831) belonging to the SmtB/ArsR family of transcriptional repressors. There is currently no experimental evidence for any functional role of AzuR. It is observed that azuR is located within the znuABC operon but in the opposite orientation and remotely away from the nmtA locus. Sequence analysis of AzuR revealed a high degree of sequence identity with Synechococcus SmtB and a distinct α5 metal binding site similar to that of SmtB. In order to characterize AzuR, we overexpressed it in Escherichia coli and purified it by chitin affinity chromatography. Far-UV circular dichroism spectroscopy indicated that the recombinant AzuR protein possessed a properly folded structure. Glutaraldehyde cross-linking and size-exclusion chromatography revealed that AzuR exists as a dimer of ∼28 kDa in solution. Analysis of its putative promoter region [100 bp upstream of nmtA open reading frame (ORF)] identified the presence of a 12–2–12 imperfect inverted repeat as the cis-acting element important for repressor binding. Electrophoretic mobility shift assays (EMSAs) showed concentration-dependent binding of recombinant dimeric AzuR with the promoter indicating that NmtA is indeed a regulatory target of AzuR. Binding of AzuR to DNA was disrupted in the presence of metal ions like Zn2+, Cd2+, Cu2+, Co2+, Ni2+, Pb2+, and Mn2+. The metal-dependent dissociation of protein–DNA complexes suggested the negative regulation of metal-inducible nmtA expression by AzuR. Overexpression of azuR in its native strain Anabaena 7120 enhanced the susceptibility to cadmium stress significantly. Overall, we propose a negative regulation of Anabaena MT by an α5 SmtB/ArsR metalloregulator AzuR.


2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Emilia Dzień ◽  
Dorota Dudek ◽  
Danuta Witkowska ◽  
Magdalena Rowińska-Żyrek

AbstractMembrane environment often has an important effect on the structure, and therefore also on the coordination mode of biologically relevant metal ions. This is also true in the case of Cu(II) coordination to amylin analogues—rat amylin, amylin1–19, pramlintide and Ac-pramlintide, which offer N-terminal amine groups and/or histidine imidazoles as copper(II) anchoring sites. Complex stabilities are comparable, with the exception of the very stable Cu(II)–amylin1–19, which proves that the presence of the amylin C-terminus lowers its affinity for copper(II); although not directly involved, its appropriate arrangement sterically prevents early metal binding. Most interestingly, in membrane-mimicking solution, the Cu(II) affinities of amylin analogues are lower than the ones in water, probably due to the crowding effect of the membrane solution and the fact that amide coordination occurs at higher pH, which happens most likely because the α-helical structure, imposed by the membrane-mimicking solvent, prevents the amides from binding at lower pH, requiring a local unwinding of the α-helix.


2022 ◽  
Author(s):  
Louisa Stewart ◽  
YoungJin Hong ◽  
Isabel Holmes ◽  
Samantha Firth ◽  
Jack Bolton ◽  
...  

The family of human salivary histidine-rich peptides known as histatins bind zinc (Zn) and copper (Cu), but whether they contribute to nutritional immunity by influencing Zn and/or Cu availability has not been examined. We hypothesised that histatin-5 (Hst5) limits Zn availability (and promotes bacterial Zn starvation) and/or raises Cu availability (and promotes bacterial Cu poisoning). To test this hypothesis, Group A Streptococcus (GAS), which colonises the human oropharynx, was used as a model bacterium. Contrary to our hypothesis, Hst5 did not strongly influence Zn availability. This peptide did not induce expression of Zn uptake genes in GAS, nor did it suppress growth of an ΔadcAI mutant strain that is impaired in Zn uptake. Equilibrium competition measurements confirmed that Hst5 binds Zn weakly and does not compete with the high-affinity Zn uptake protein AdcAI for binding Zn. By contrast, Hst5 bound Cu with a high affinity and strongly influenced Cu availability. However, contrary to our hypothesis, Hst5 did not promote Cu toxicity. Instead, this peptide suppressed expression of Cu-inducible genes in GAS, stopped intracellular accumulation of Cu, and rescued growth of a ΔcopA mutant strain that is impaired in Cu efflux in the presence of added Cu. These findings led us to propose a new role for Hst5 and salivary histatins as major Cu buffers in saliva that reduce the potential negative effects of Cu exposure to microbes. We speculate that histatins promote oral and oropharyngeal health by contributing to microbial homeostasis in these host niches.


2022 ◽  
Author(s):  
Yeseul Park ◽  
Zohar Eyal ◽  
Péter Pekker ◽  
Daniel M. Chevrier ◽  
Christopher T. Lefèvre ◽  
...  

Metal sulfides are a common group of extracellular bacterial biominerals. Only few cases of intracellular biomineralization have been reported in this group, mostly limited to greigite (Fe3S4) in magnetotactic bacteria. Here, we report the intracellular but periplasmic biomineralization of copper sulfide by the magnetotactic bacterium Desulfamplus magnetovallimortis (strain BW-1) that is known to mineralize greigite and magnetite (Fe3O4) in the cytoplasm. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide that remains in a thermodynamically unstable state. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization.


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