Zinc-binding Site Research Articles

Search for potential Alzheimer’s disease therapeutics: Identification of inhibitors of amyloid oligomerization with high affinity for the zinc-binding site

The progression of Aβ peptide aggregation in the brain has been suggested to play a significant role in the pathogenesis and development of Alzheimer’s disease. This study is intended to provide insight into the interactions between the zinc-binding site of beta-amyloids and the zinc ion itself. The absence of zinc bonded to the beta-amyloid has been shown to potentially slow down the progression of Alzheimer's disease, so the goal is to provide an analysis of available drugs that can be repurposed and could profoundly impact Alzheimer's disease treatment. We address how and with what strength the existing compounds bind with beta-amyloid, potentially replacing or blocking zinc and preventing it from attaching to the amyloid. The analysis was performed using molecular operating environment software, which, starting from a filtered database, identified the drugs most likely to bind to the zinc-binding site on beta-amyloid.

Natural zinc-rich oyster peptides ameliorate DSS-induced colitis via antioxidation, anti-inflammation, intestinal barrier repair, microbiota modulation, and SCFA accumulation

Natural oyster zinc-rich peptide (OZP) was extracted and purified. The higher zinc concentration in natural OZP may be closely related to the high zinc affinity of amino acids containing Lys, His, Cys, Val, Arg and Ser residues. FTIR analysis indicated that amino, amide, carboxyl and carbonyl groups may contribute to the binding sites for zinc and peptides. Administration of OZP ameliorated the symptoms of DSS-induced ulcerative colitis (UC) in mice, including weight loss, colonic damage, mucosal edema, goblet cell exhaustion and crypt disappearance. Natural OZP suppressed oxidative stress by significantly reducing the level of NO, MDA and MPO as well as increasing SOD activity. The anti-inflammatory effects of OZP were associated with the inhibition of TLR4/NF-κB pathways by diminishing NF-κB, TLR4, COX-2, iNOS, TNF-α, IL-6 and IFN-γ and elevating IL-10. By promoting mucus secretion, repairing goblet cells and enhancing the expression of tight junction proteins including occludin, claudin-1 and ZO-1, natural OZP maintained the intestinal barrier function. Furthermore, natural OZP regulated the microbial composition by remarkedly enhancing the beneficial microbial abundance like Faecalibaculum, Odoribacter and Anaerostipes, which in turn led to a significant increase in SCFA production. Collectively, natural OZP exerted the synergistic capacity to protect against DSS-induced UC by improving antioxidation and anti-inflammation, repairing intestinal barrier and modulating intestinal microbiota and SCFA.

PIAS3 acts as a zinc sensor under zinc deficiency and plays an important role in myocardial ischemia/reperfusion injury

Alterations in zinc transporter expression in response to zinc loss protect cardiac cells from ischemia/reperfusion (I/R) injury. However, the underlying molecular mechanisms how cardiac cells sense zinc loss remains unclear. Here, we found that zinc deficiency induced ubiquitination and degradation of the protein inhibitor of activated STAT3 (PIAS3), which can alleviate myocardial I/R injury by activating STAT3 to promote the expression of ZIP family zinc transporter genes. The RING finger domain within PIAS3 is vital for PIAS3 degradation, as PIAS3-dRing (missing the RING domain) and PIAS3-Mut (zinc-binding site mutation) were resistant to degradation in the setting of zinc deficiency. Meanwhile, the RING finger domain within PIAS3 is critical for the inhibition of STAT3 activation. Moreover, PIAS3 knockdown increased cardiac Zn2+ levels and reduced myocardial infarction in mouse hearts subjected to I/R, whereas wild-type PIAS3 overexpression, but not PIAS3-Mut, reduced cardiac Zn2+ levels, and exacerbated myocardial infarction. These findings elucidate a unique mechanism of zinc sensing, showing that fast degradation of the zinc-binding regulatory protein PIAS3 during zinc deficiency can correct zinc dyshomeostasis and alleviate reperfusion injury.

Cotton plants overexpressing the Bacillus thuringiensis Cry23Aa and Cry37Aa binary-like toxins exhibit high resistance to the cotton boll weevil (Anthonomus grandis)

The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin’s mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV.

ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.

Genome-wide identification and analysis of SPL gene family in chickpea (Cicer arietinum L.).

A transcription factor in plants encodes SQUAMOSA promoter binding protein-like (SPL) serves a broad spectrum of important roles for the plant, like, growth, flowering, and signal transduction. A gene family that encodes SPL proteins is documented in various model plant species, including Arabidopsis thaliana and Oryza sativa. Chickpea (Cicer arietinum), a leguminous crop, has not been thoroughly explored with regard to the SPL protein-encoding gene family. Chickpea SPL family genes were located and characterized computationally using a genomic database. Gene data of chickpea were obtained from the phytozome repository and was examined using bioinformatics methods. For investigating the possible roles of SPLs in chickpea, genome-wide characterization, expression, as well as structural analyses of this SPL gene family were performed. Cicer arietinum genome had 19 SPL genes, whereas, according to phylogenetic analysis, the SPLs in chickpea are segregated among four categories: Group-I has 2 introns, Group-II and IV have 1-2 introns (except CaSPL13 and CaSPL15 having 3 introns), and Group-III has 9 introns (except CaSPL1 and CaSPL11 with 1 and 8 introns, respectively). The SBP domain revealed that SPL proteins featured two zinc-binding sites, i.e., C3H and C2HC and one nuclear localization signal. All CaSPL proteins are found to contain highly conserved motifs, i.e., Motifs 1, 2, and 4, except CaSPL10 in which Motifs 1 and 4 were absent. Following analysis, it was found that Motifs 1 and 2 of the chickpea SBP domain are Zinc finger motifs, and Motif 4 includes a nuclear localization signal. All pairs of CaSPL paralogs developed by purifying selection. The CaSPL promoter investigation discovered cis-elements that are responsive to stress, light, and phytohormones. Examination of their expression patterns highlighted major CaSPLs to be evinced primarily among younger pods and flowers. Indicating their involvement in the plant's growth as well as development, along with their capacity to react as per different situations by handling the regulation of target gene's expression, several CaSPL genes are also expressed under certain stress conditions, namely, cold, salt, and drought. The majority of the CaSPL genes are widely expressed and play crucial roles in terms of the plant's growth, development, and responses to the environmental-stress conditions. Our work provides extensive insight into the gene family CaSPL, which might facilitate further studies related to the evolution and functions of the SPL genes for chickpea and other plant species.

Manganese- and zinc-coordinated interaction of Schistosoma japonicum glutathione S-transferase with neurotransmitter transporters GlyT1 and GAT3 in vitro

Glutathione S-transferases (GSTs) are a family of multifunctional isoenzymes involved in the neutralization of toxic compounds, drug resistance and several other cellular functions. The glutathione S-transferase enzyme of Schistosoma japonicum (SjGST-26) plays a role in human schistosomiasis and is also a frequently used fusion partner in mammalian and bacterial expression and pull-down systems. GSTs seem not to be naturally associated with metal ions. Exceptionally, in vitro, metal binding sites have been previously described in some schistosome GSTs; however, their possible physiological role is unclear. Molecules of several neurotransmitter transporters also contain a regulatory zinc binding site, which affects their transport cycle. Here we show that among several metals, manganese and zinc are able to induce a specific protein interaction of SjGST-26 with the glycine transporter GlyT1 and the GABA transporter GAT3 in vitro. The results suggest that metal-binding sites on SjGST-26 and neurotransmitter transporters might function in metal-coordinated interactions with other metalloproteins. Our results additionally indicate that the presence of metal ions in SjGST-26-based GST protein pull-down assays may lead to a false-positive interaction if the potential interacting target is the metalloprotein.

Activation of zinc uptake regulator by zinc binding to three regulatory sites.

Zur is a Fur-family metalloregulator that is widely used to control zinc homeostasis in bacteria. In Streptomyces coelicolor, Zur (ScZur) acts as both a repressor for zinc uptake (znuA) gene and an activator for zinc exporter (zitB) gene. Previous structural studies revealed three zinc ions specifically bound per ScZur monomer; a structural one to allow dimeric architecture and two regulatory ones for DNA-binding activity. In this study, we present evidence that Zur contains a fourth specific zinc-binding site with a key histidine residue (H36), widely conserved among actinobacteria, for regulatory function. Biochemical, genetic, and calorimetric data revealed that H36 is critical for hexameric binding of Zur to the zitB zurbox and further binding to its upstream region required for full activation. A comprehensive thermodynamic model demonstrated that the DNA-binding affinity of Zur to both znuA and zitB zurboxes is remarkably enhanced upon saturation of all three regulatory zinc sites. The model also predicts that the strong coupling between zinc binding and DNA binding equilibria of Zur drives a biphasic activation of the zitB gene in response to a wide concentration change of zinc. Similar mechanisms may be pertinent to other metalloproteins, expanding their response spectrum through binding multiple regulatory metals.

Dynamic changes of zinc chemical speciation and zinc-containing peptides release in oysters (Crassostrea hongkongensis) during enzymatic hydrolysis

Oyster hydrolysates exhibit promising potential as a zinc supplement for its significant amounts of zinc, however, the zinc chemical speciation which determines zinc nutrition in oyster hydrolysates has not been investigated. This study optimized the oyster hydrolysis conditions using the Response surface methodology (RSM) method, and the zinc chemical speciation, the sequence of zinc-containing peptides, and the binding sites of zinc and peptides were further investigated. Results showed that inorganic and organic zinc in oysters were 60.15% and 36.53% of total zinc respectively, while organic zinc in oysters aqueous extract was 4.54% of which 3.56% was present in the polysaccharide fraction. The zinc yield was 84.20% of total zinc under optimal hydrolysis conditions, while the organic zinc increased to 52.93% of which 38.14% existed in the protein/peptides fraction. Zinc in protein/peptide fraction primarily was bound to low-molecular-weight peptides (Mw < 1000 Da). Further, 42 shared zinc-containing peptides (mainly 3–4 peptides) were identified. Molecular docking indicated that Glu, Leu, and Tyr might be the binding sites for zinc and peptides. These findings provided a theoretical basis for the development of novel and safe zinc dietary supplements.

OR02-01 Somatic Mutations In The Zinc Transporter ZnT1 Cause Primary Aldosteronism

Abstract Disclosure: J. Rege: None. S. Bandulik: None. K. Nanba: None. C. Kosmann: None. A. Blinder: None. A. Plain: None. P. Vats: None. C. Kumar-Sinha: None. A.M. Lerario: None. T. Else: None. Y. Yamazaki: None. F. Satoh: None. H. Sasano: None. T.J. Giordano: None. T.A. Williams: None. M. Reincke: None. A. Turcu: None. A. Udager: None. R. Warth: None. W.E. Rainey: None. Primary aldosteronism (PA) is the most common form of endocrine hypertension and affects approximately one in 50 adults. PA is characterized by inappropriately elevated adrenal aldosterone production via renin-independent mechanisms. Aldosterone-driver somatic mutations in a G protein-coupled inwardly rectifying potassium channel (KCNJ5), Na+/K+ and Ca2+ ATPases (ATP1A1 and ATP2B3, respectively), L-type and T-type voltage-gated calcium channels (CACNA1D and CACNA1H, respectively), and chloride voltage-gated channel (CLCN2) have been found in approximately 90% of adrenal aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n=3; p.L49_L55del, n=2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated in close proximity to the zinc-binding site (His43 and Asp47) in transmembrane domain II and likely cause abnormal ion transport. PA cases with the unique SLC30A1 mutations showed male dominance and demonstrated increased serum aldosterone and 18-oxo-cortisol concentrations. Functional studies of the mutant SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathologic Na+ influx caused by the mutant. Aberrant Na+ current led to the depolarization of the resting membrane potential, and thus to the opening of voltage-gated calcium channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate the first-in-field zinc transporter mutations as a dominant driver of aldosterone excess in PA. Presentation: Thursday, June 15, 2023

Phosphatase and Pseudo-Phosphatase Functions of Phosphatase of Regenerating Liver 3 (PRL-3) Are Insensitive to Divalent Metals In Vitro.

Phosphatase of regenerating liver 3 (PRL-3) is associated with cancer metastasis and has been shown to interact with the cyclin and CBS domain divalent metal cation transport mediator (CNNM) family of proteins to regulate the intracellular concentration of magnesium and other divalent metals. Despite PRL-3's importance in cancer, factors that regulate PRL-3's phosphatase activity and its interactions with CNNM proteins remain unknown. Here, we show that divalent metal ions, including magnesium, calcium, and manganese, have no impact on PRL-3's structure, stability, phosphatase activity, or CNNM binding capacity, indicating that PRL-3 does not act as a metal sensor, despite its interaction with CNNM metal transporters. In vitro approaches found that PRL-3 is a broad but not indiscriminate phosphatase, with activity toward di- and tri-nucleotides, phosphoinositols, and NADPH but not other common metabolites. Although calcium, magnesium, manganese, and zinc-binding sites were predicted near the PRL-3 active site, these divalent metals did not specifically alter PRL-3's phosphatase activity toward a generic substrate, its transition from an inactive phospho-cysteine intermediate state, or its direct binding with the CBS domain of CNNM. PRL-3's insensitivity to metal cations negates the possibility of its role as an intracellular metal content sensor for regulating CNNM activity. Further investigation is warranted to define the regulatory mechanisms governing PRL-3's phosphatase activity and CNNM interactions, as these findings could hold potential therapeutic implications in cancer treatment.

Rhizobium etli has two L-asparaginases with low sequence identity but similar structure and catalytic center.

The genome of Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes two L-asparaginases, ReAIV and ReAV, that have no similarity to the well characterized enzymes of class 1 (bacterial type) and class 2 (plant type). It has been hypothesized that ReAIV and ReAV might belong to the same structural class 3 despite their low level of sequence identity. When the crystal structure of the inducible and thermolabile protein ReAV was solved, this hypothesis gained a stronger footing because the key residues of ReAV are also present in the sequence of the constitutive and thermostable ReAIV protein. High-resolution crystal structures of ReAIV now confirm that it is a class 3 L-asparaginase that is structurally similar to ReAV but with important differences. The most striking differences concern the peculiar hydration patterns of the two proteins, the presence of three internal cavities in ReAIV and the behavior of the zinc-binding site. ReAIV has a high pH optimum (9-11) and a substrate affinity of ∼1.3 mM at pH 9.0. These parameters are not suitable for the direct application of ReAIV as an antileukemic drug, although its thermal stability and lack of glutaminase activity would be of considerable advantage. The five crystal structures of ReAIV presented in this work allow a possible enzymatic scenario to be postulated in which the zinc ion coordinated in the active site is a dispensable element. The catalytic nucleophile seems to be Ser47, which is part of two Ser-Lys tandems in the active site. The structures of ReAIV presented here may provide a basis for future enzyme-engineering experiments to improve the kinetic parameters for medicinal applications.

Catalytic mechanism of the zinc-dependent MutL endonuclease reaction.

DNA mismatch repair endonuclease MutL binds two zinc ions. However, the endonuclease activity of MutL is drastically enhanced by other divalent metals such as manganese, implying that MutL binds another catalytic metal at some site other than the zinc-binding sites. Here, we solved the crystal structure of the endonuclease domain of Aquifex aeolicus MutL in the manganese- or cadmium-bound form, revealing that these metals compete with zinc at the same sites. Mass spectrometry revealed that the MutL yielded 5'-phosphate and 3'-OH products, which is characteristic of the two-metal-ion mechanism. Crystallographic analyses also showed that the position and flexibility of a highly conserved Arg of A. aeolicus MutL altered depending on the presence of zinc/manganese or the specific inhibitor cadmium. Site-directed mutagenesis revealed that the Arg was critical for the catalysis. We propose that zinc ion and its binding sites are physiologically of catalytic importance and that the two-metal-ion mechanism works in the reaction, where the Arg plays a catalytic role. Our results also provide a mechanistic insight into the inhibitory effect of a mutagen/carcinogen, cadmium, on MutL.

Conformational exchange at a C2H2 zinc-binding site facilitates redox sensing by the PML protein

The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using invitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the invitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.

Disruption of zinc (II) binding and dimeric protein structure of the XIAP-RING domain by copper (I) ions.

Modulation of metalloprotein structure and function via metal ion substitution may constitute a molecular basis for metal ion toxicity and/or metal-mediated functional control. The X-linked Inhibitor of Apoptosis Protein (XIAP) is a metalloprotein that requires zinc for proper structure and function. In addition to its role as a modulator of apoptosis, XIAP has been implicated in copper homeostasis. Given the similar coordination preferences of copper and zinc, investigation of XIAP structure and function upon interaction with copper is relevant. The Really Interesting New Gene (RING) domain of XIAP is representative of a class of zinc finger proteins that utilize a bi-nuclear zinc-binding motif to maintain proper structure and ubiquitin ligase function. Herein, we report the characterization of copper (I) binding to the Zn2-RING domain of XIAP. Electronic absorption studies that monitor copper-thiolate interactions demonstrate that the RING domain of XIAP binds 5-6 Cu(I) ions and that copper is thermodynamically preferred relative to zinc. Repetition of the experiments in the presence of the Zn(II)-specific dye Mag-Fura2 shows that Cu(I) addition results in Zn(II) ejection from the protein, even in the presence of glutathione. Loss of dimeric structure of the RING domain, which is a requirement for its ubiquitin ligase activity, upon copper substitution at the zinc-binding sites, was readily observed via size exclusion chromatography. These results provide a molecular basis for the modulation of RING function by copper and add to the growing body of literature that describe the impact of Cu(I) on zinc metalloprotein structure and function.

The structural basis of tRNA recognition by arginyl-tRNA-protein transferase

Arginyl-tRNA-protein transferase 1 (ATE1) is a master regulator of protein homeostasis, stress response, cytoskeleton maintenance, and cell migration. The diverse functions of ATE1 arise from its unique enzymatic activity to covalently attach an arginine onto its protein substrates in a tRNA-dependent manner. However, how ATE1 (and other aminoacyl-tRNA transferases) hijacks tRNA from the highly efficient ribosomal protein synthesis pathways and catalyzes the arginylation reaction remains a mystery. Here, we describe the three-dimensional structures of Saccharomyces cerevisiae ATE1 with and without its tRNA cofactor. Importantly, the putative substrate binding domain of ATE1 adopts a previously uncharacterized fold that contains an atypical zinc-binding site critical for ATE1 stability and function. The unique recognition of tRNAArg by ATE1 is coordinated through interactions with the major groove of the acceptor arm of tRNA. Binding of tRNA induces conformational changes in ATE1 that helps explain the mechanism of substrate arginylation.

Zn(II) to Ag(I) Swap in Rad50 Zinc Hook Domain Leads to Interprotein Complex Disruption through the Formation of Highly Stable Agx(Cys)y Cores.

The widespread application of silver nanoparticles in medicinal and daily life products increases the exposure to Ag(I) of thiol-rich biological environments, which help control the cellular metallome. A displacement of native metal cofactors from their cognate protein sites is a known phenomenon for carcinogenic and otherwise toxic metal ions. Here, we examined the interaction of Ag(I) with the peptide model of the interprotein zinc hook (Hk) domain of Rad50 protein from Pyrococcus furiosus, a key player in DNA double-strand break (DSB) repair. The binding of Ag(I) to 14 and 45 amino acid long peptide models of apo- and Zn(Hk)2 was experimentally investigated by UV-vis spectroscopy, circular dichroism, isothermal titration calorimetry, and mass spectrometry. The Ag(I) binding to the Hk domain was found to disrupt its structure via the replacement of the structural Zn(II) ion by multinuclear Agx(Cys)y complexes. The ITC analysis indicated that the formed Ag(I)-Hk species are at least 5 orders of magnitude stronger than the otherwise extremely stable native Zn(Hk)2 domain. These results show that Ag(I) ions may easily disrupt the interprotein zinc binding sites as an element of silver toxicity at the cellular level.

Novel SLC30A2 mutations in the pathogenesis of transient neonatal zinc deficiency.

Transient neonatal zinc deficiency (TNZD) occurs in breastfed infants due to abnormally low breast milk zinc levels. Mutations in the solute carrier family 30 member 2 (SLC30A2) gene, which encodes the zinc transporter ZNT2, cause low zinc concentration in breast milk. This study aimed to provide further insights into TNZD pathophysiology. SLC30A2 sequencing was performed in three unrelated Japanese mothers, whose infants developed TNZD due to low-zinc milk consumption. The effects of the identified mutations were examined using cell-based assays and luciferase reporter analysis. Novel SLC30A2 mutations were identified in each mother. One harbored a heterozygous missense mutation in the ZNT2 zinc-binding site, which resulted in defective zinc transport. The other two mothers exhibited multiple heterozygous mutations in the SLC30A2 promoter, the first mutations in the SLC30A2 regulatory region reported to date. This report provides new genetic insights into TNZD pathogenesis in breastfed infants.

A conserved zinc-binding site in Acinetobacter baumannii PBP2 required for elongasome-directed bacterial cell shape

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. β-lactam targeting of penicillin-binding protein (PBP)-mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii, an effect resembling that caused by deficiency in the RodA-PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA-PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to β-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii. Furthermore, the Zn-coordinating residues are conserved in various β- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-β-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore β-lactam potency against gram-negative bacteria.

Sequence adaptation in various environments across organisms; similar yet different superoxide dismutase.

Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium that attack the host immune system and killed millions of people annually. The body first defense mechanism is through phagocytosis where an outburst of radical oxygen species (ROS) is released and potentially kill the bacterium. M. tuberculosis superoxide dismutase (MtSOD) survives the phagocytosis and suppresses the host immune system by converting reactive oxygen species to H2O2 and O2. SOD proteins are important for cell survival to prevent the buildup of reactive ROS in the cell that may cause damage to DNA, RNA, proteins, and even cell death. MtSOD (oxidized) is homologous to human SOD1 (hSOD1, reduced) which contains both zinc and copper metals along with the disulfide bond. Though, MtSOD lacks the zinc binding site which proposed to play a role in stability, dimerization, and function of SOD. Utilizing Circular Dichroism, an apparent correlation of MtSOD stabilization with the copper and disulfide bond as more stable secondary structure is observed for oxidized apoprotein compared to the reduced apo and oxidized holoprotein. To further investigate the novel role of zinc in SOD (ZnMtSOD), a mutation of zinc binding site consists of T119H, A123H, T192K (modeled from hSOD1) is introduced in the wild type MtSOD. With the low sequence identity of 19.7% but similar in protein structures, the thermodynamic and kinetics results are compared for hSOD1, ZnMtSOD, and wt MtSOD.