Potential Metal-binding Site Research Articles

In Silico Simulations Reveal Molecular Mechanism of Uranyl Ion Toxicity towards DNA-Binding Domain of PARP-1 Protein.

The molecular toxicity of the uranyl ion (UO22+) in living cells is primarily determined by its high affinity to both native and potential metal-binding sites that commonly occur in the structure of biomolecules. Recent advances in computational and experimental research have shed light on the structural properties and functional impacts of uranyl binding to proteins, organic ligands, nucleic acids, and their complexes. In the present work, we report the results of the computational investigation of the uranyl-mediated loss of DNA-binding activity of PARP-1, a eukaryotic enzyme that participates in DNA repair, cell differentiation, and the induction of inflammation. The latest experimental studies have shown that the uranyl ion directly interacts with its DNA-binding subdomains, zinc fingers Zn1 and Zn2, and alters their tertiary structure. Here, we propose an atomistic mechanism underlying this process and compute the free energy change along the suggested pathway. Our Quantum Mechanics/Molecular Mechanics (QM/MM) simulations of the Zn2-UO22+ complex indicate that the uranyl ion replaces zinc in its native binding site. However, the resulting state is destroyed due to the spontaneous internal hydrolysis of the U-Cys162 coordination bond. Despite the enthalpy of hydrolysis being +2.8 kcal/mol, the overall reaction free energy change is -0.6 kcal/mol, which is attributed to the loss of domain's native tertiary structure originally maintained by a zinc ion. The subsequent reorganization of the binding site includes the association of the uranyl ion with the Glu190/Asp191 acidic cluster and significant perturbations in the domain's tertiary structure driven by a further decrease in the free energy by 6.8 kcal/mol. The disruption of the DNA-binding interface revealed in our study is consistent with previous experimental findings and explains the loss of PARP-like zinc fingers' affinity for nucleic acids.

Immobilization of silver ions onto casein

In this study, the physicochemical and biological properties of silver-casein complexes were investigated. The casein modification was carried out by a novel method, i.e., at basic conditions (pH 8) using silver precursor in a form of ammonia complex [Ag(NH3)2]+. The selected experimental conditions allowed us to obtain high colloidal stability of protein suspension and also minimized the risk of metal secondary reduction. Subsequently, the mechanism of silver immobilization was evaluated by using a batch sorption study (Freundlich, Langmuir isotherm modeling). Moreover, the influence of accompanying the protein ions on the silver binding process was studied (using ultrafiltration). Results revealed, that casein after the desalting step has much higher silver-binding capacity (258.2 mg/g) compared to untreated protein (157.1 mg/g). The nature of casein-silver interactions was comprehensively studied by multiple instrumental techniques, namely, spectrometric (ICP-MS), spectroscopic (DLS, ATR-FTIR, Fluorescence Spectroscopy), electron microscopy (STEM, SEM-EDX), and separation (SDS-PAGE). Results indicated, that silver sorption had a complex character. The heterogeneous character of metal binding processes was discovered. The DLS studies pointed out the great role of phosphoserine residues in silver-casein interactions and underlined their impact on the micelle size and structure. Moreover, STEM images confirmed the growth of silver nanoparticles upon metal binding to casein. According to FTIR analysis, carboxylic groups of aspartic and glutamic acid were identified as potential metal-binding sites which also could participate in silver reduction. Peptic digestion studies showed a relevant influence of silver incorporation on the structural stability of caseins. Finally, the biological activity of the 600Ag-CN complex was evaluated. Results indicated that the complex effectively inhibited the growth of both Gram-positive (S. aureus) and Gram-negative (K. pneumoniae) bacteria. Still, the treatment of mouse fibroblast cell line L929 with analyzed complex (0.32 mg/mL of 600Ag-CN corresponding to 7.5 µg/mL of silver) decrease cell viability up to 60 %. The toxic activity of the complex may be connected to the Ag+ release. The silver desorption study showed that 600Ag-CN complex released 21.5 %, 49.3 %, and 68.7 % in pH 6.8, 4.5, and 1.2 respectively.

Structural and Functional Insights into the Roles of Potential Metal-Binding Sites in Apostichopus japonicus Ferritin.

Ferritin is widely acknowledged as a conservative iron storage protein found in almost all living kingdoms. Apostichopus japonicus (Selenka) is among the oldest echinoderm fauna and has unique regenerative potential, but the catalytic mechanism of iron oxidation in A. japonicus ferritin (AjFER) remains elusive. We previously identified several potential metal-binding sites at the ferroxidase center, the three- and four-fold channels in AjFER. Herein, we prepared AjFER, AjFER-E25A/E60A/E105A, AjFER-D129A/E132A, and AjFER-E168A mutants, investigated their structures, and functionally characterized these ferritins with respect to Fe2+ uptake using X-ray techniques together with biochemical analytical methods. A crystallographic model of the AjFER-D129A/E132A mutant, which was solved to a resolution of 1.98 Å, suggested that the substitutions had a significant influence on the quaternary structure of the three-fold channel compared to that of AjFER. The structures of these ferritins in solution were determined based on the molecular envelopes of AjFER and its variants by small-angle X-ray scattering, and the structures were almost consistent with the characteristics of well-folded and globular-shaped proteins. Comparative biochemical analyses indicated that site-directed mutagenesis of metal-binding sites in AjFER presented relatively low rates of iron oxidation and thermostability, as well as weak iron-binding affinity, suggesting that these potential metal-binding sites play critical roles in the catalytic activity of ferritin. These findings provide profound insight into the structure-function relationships related to marine invertebrate ferritins.

Metal-Ion Interactions with Dodecapeptide Fragments of Human Cationic Antimicrobial Protein LL-37 [hCAP(134-170)

Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were used to determine potential metal binding sites in human cationic antimicrobial protein (hCAP) corresponding to overlapping the dodecapeptide sequences of hCAP(134–170) referred to as LL-37. The correct antibacterial action of LL-37 is closely related to its established unique structure. Disturbances in the LL-37 structure (e.g., unwanted presence of metal ions) lead to a radical change in its biological functions. Five fragments of the LL-37 [hCAP(134–170)], namely, hCAP(134–145) (A1), hCAP(140–151) (A2), hCAP(146–157) (A3), hCAP(152–163) (A4), and hCAP(159–170) (A5), were taken into account and their affinity to Mn(II) and Zn(II) ions was rigorously assessed. We prove that only three of the investigated peptides (A1, A2, and A5) are capable of forming thermodynamically stable complexes with metal ions. Additionally, based on density functional theory (DFT) calculations, we propose the most likely coordination modes of metal(II) to peptides as well as discuss the chemical nature of the interactions. Finally, we present the structural features of the strongest binding peptide, hCAP(159–170), responsible for the metal binding. The presented results provide important structural and thermodynamic information to understand the influence of some metal ions on the activity of hCAP(134–170).

Poly-Gly Region Regulates the Accessibility of Metal Binding Sites in Snake Venom Peptides.

It is supposed that the presence of poly-His regions in close proximity to poly-Gly domains in snake venoms is related to their biological activity; poly-His/poly-Gly (pHpG) peptides inhibit the activity of metalloproteinases during venom storage via the chelation metal ions, necessary for their proper functioning. This work shows that only the histidyl residues from the N-terminal VDHDHDH motif (but not from the poly-His tag) were the primary Zn(II) binding sites and that the poly-Gly domain situated in the proximity of a central proline residue may play a regulatory role in venom gland protection. The proline induces a kink of the peptide, resulting in steric hindrance, which may modulate the accessibility of potential metal binding sites in the poly-His domain and may, in turn, be one of the regulators of Zn(II) accessibility in the venom gland and therefore a modulator of metalloproteinase activity during venom storage.

A redox-sensitive iron-sulfur cluster in murine FAM72A controls its ability to degrade the nuclear form of uracil-DNA glycosylase

Murine FAM72A, mFAM72A, binds the nuclear form of uracil-DNA glycosylase, mUNG2, inhibits its activity and causes its degradation. In immunoprecipitation assays the human paralog, hFAM72A, binds hUNG2 and is a potential anti-cancer drug target because of its high expression in many cancers. Using purified mFAM72A, and mUNG2 proteins we show that mFAM72A binds mUNG2, and the N-terminal 25 amino acids of mUNG2 bind mFAM72A at a nanomolar dissociation constant. We also show that mFAM72A is present throughout the cells, and mUNG2 helps localize it to nuclei. Based on in silico models of mFAM72A-mUNG2 interactions, we constructed several mutants of mFAM72A and found that while they have reduced ability to deplete mUNG2, the mutations also destabilized the former protein. We confirmed that Withaferin A, a predicted lead molecule for the design of FAM72A inhibitors, binds mFAM72A with micromolar affinity but has little affinity to mUNG2. We identified two potential metal-binding sites in mFAM72A and show that one of the sites contains an Fe-S cluster. This redox-sensitive cluster is involved in the mFAM72A-mUNG2 interaction and modulates mFAM72A activity. Hydrogen peroxide treatment of cells increases mUNG2 depletion in a FAM72A-dependent fashion suggesting that mFAM72A activity is redox-sensitive.

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes using Ion Mobility-Mass Spectrometry.

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids' Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (ΔH0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (ΔH298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical and conformational studies of Ni(II) and Zn(II) ternary complexes of alternative metal binding peptides with nitrilotriacetic acid

Energy-resolved threshold collision-induced dissociation (TCID) of negatively-charged [amb5-H+M(II)+NTA-2H]− ternary complexes were studied for how they competitively dissociated into the products [amb5-3H+M(II)]− + NTA or [NTA-3H+M(II)]− + amb5, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contained one of the alternative metal binding-5 (amb5) heptapeptides with primary structures acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where the amino acids Aa1,2,6,7 included the potential metal binding sites of Asp, His, and/or Cys. The study included heptahistidine which represents the His affinity tag commonly used in immobilized metal affinity chromatography (IMAC) separation of recombinant proteins. Molecular modeling using PM6 located low energy, geometry-optimized conformers of the ternary complexes and their products which had collision cross sections that agreed with those measured by ion mobility. Using the molecular parameters, including vibrational and rotational frequencies, of the reactant and products from the molecular modeling, the energy-dependent intensities of the two product channels were modeled using a competitive TCID method to determine their threshold energies which equated to the two product channels 0 K dissociation enthalpies (ΔH0). Statistical mechanics thermal corrections provided the 298 K enthalpies (ΔH298) and Gibbs free energies (ΔG298) for the association reactions [amb5-3H+M(II)]− + NTA ↔ [amb5-H+M(II)+NTA-2H]− and [NTA-3H+M(II)]− + amb5 ↔ [amb5-H+M(II)+NTA-2H]−. These results were compared to determine which amb5 were effective as alternative tags for IMAC purification of recombinant proteins and is a routine that can be generally applied to compare the thermochemistry of metal ion ternary complexes.

Formation of Co(II), Ni(II), Zn(II) complexes of alternative metal binding heptapeptides and nitrilotriacetic acid: Discovering new potential affinity tags

Using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS) a new series of alternative metal binding-5 (amb5) heptapeptides that provide various combinations of His, Cys and Asp as potential metal binding sites are compared for their relative formation of complexes with the metal ions Co(II), Ni(II) and Zn(II). These amb5 peptides have the general primary structure acetyl-Aa1-Aa2-Gly3-Pro4-Tyr5-Aa6-Aa7, where Aa1,2,6,7 are the four amino acids that include the His, Cys, and/or Asp potential metal binding sites. The study includes two polyhistidine heptapeptides; the His-tag and His-tag with Pro4, because they represent the widely applied His affinity tag used in expression cloning and purification of recombinant proteins with immobilized metal affinity chromatography (IMAC). We compare these amb5 heptapeptides for their formation of the amb5+M(II) and amb5+M(II)+NTA complexes, where M = Co, Ni, or Zn, and NTA is nitrilotriacetic acid as a model for the binding of amb5 in a IMAC column. Overall, the results show that the inclusion of Asp at positions 1, 2, and 7; i.e., ac-Asp1-Asp2-Gly3-Pro4-Tyr5-Aa6-Asp7, generally favors the formation of amb5+Ni(II) and amb5+Ni(II)+NTA complexes, while the inclusion of Cys at positions 2 and 6; i.e., ac-Aa1-Cys2-Gly3-Pro4-Tyr5-Aa6-Cys7, with Aa1 = His, Cys or Asp, favors the formation of amb5+Zn(II) complexes. The comparison with the His-tag and competition between Ni(II) and Zn(II) for forming the amb5+M(II)+NTA complexes, showed the structure ac-Asp1-Asp2-Gly3-Pro4-Tyr5-His6-Aa7, with Aa7 = Cys or Asp, were the most selective for Ni(II) and forming the amb5+Ni(II)+NTA complex. Collision-induced dissociation of the single negatively-charged [amb5+Ni(II)+NTA]− shows it competitively dissociates into [amb5−3H+Ni(II)]− or [NTA−3H+Ni(II)]− while the doubly-charged [amb5+Ni(II)+NTA]2− complex dissociates into [NTA−3H+Ni(II)]− and [amb5J−H]−, indicating these complexes are realistic mimics for the complex inside an IMAC column.

BioMetAll: Identifying Metal-Binding Sites in Proteins from Backbone Preorganization.

With a large amount of research dedicated to decoding how metallic species bind to proteins, in silico methods are interesting allies for experimental procedures. To date, computational predictors mostly work by identifying the best possible sequence or structural match of the target protein with metal-binding templates. These approaches are fundamentally focused on the first coordination sphere of the metal. Here, we present the BioMetAll predictor that is based on a different postulate: the formation of a potential metal-binding site is related to the geometric organization of the protein backbone. We first report the set of convenient geometric descriptors of the backbone needed for the algorithm and their parameterization from a statistical analysis. Then, the successful benchmark of BioMetAll on a set of more than 90 metal-binding X-ray structures is presented. Because BioMetAll allows structural predictions regardless of the exact geometry of the side chains, it appears extremely valuable for systems whose structures (either experimental or theoretical) are not optimal for metal-binding sites. We report here its application on three different challenging cases: (i) the modulation of metal-binding sites during conformational transition in human serum albumin, (ii) the identification of possible routes of metal migration in hemocyanins, and (iii) the prediction of mutations to generate convenient metal-binding sites for de novo biocatalysts. This study shows that BioMetAll offers a versatile platform for numerous fields of research at the interface between inorganic chemistry and biology and allows to highlight the role of the preorganization of the protein backbone as a marker for metal binding. BioMetAll is an open-source application available at https://github.com/insilichem/biometall.

Cytotoxic activity of copper(ii), nickel(ii) and platinum(ii) thiosemicarbazone derivatives: interaction with DNA and the H2A histone peptide.

Metal complexes still represent promising pharmacological tools in the development of new anticancer drugs. Bis(citronellalthiosemicarbazonate)nickel(ii) is a metal compound extremely effective against leukemic and NCS cancer cell lines. Preliminary experiments performed with this compound and with its Cu(ii) and Pt(ii) analogues evidenced alterations, detectable by comet assay, in the DNA of treated U937 cells. In addition, [Cu(tcitr)2] and [Pt(tcitr)2] were also able to induce gene mutations and produce frameshift events. To gain further insights into the mechanism of action of these metal compounds, we carried out a multidisciplinary study to investigate whether their biological activity can be ascribed to the direct interaction with DNA or with chromatin. The DNA interaction was investigated by means of CD and UV-Vis spectroscopic techniques and by AFM, whereas the chromatin interaction was studied by analyzing the effects of the compounds on the structure of a peptide that mimicks the potential metal binding site in the "C-tail" region of histone H2A by means of NMR, CD, UV-Vis and MS. The intensities of the effects induced by the metal compounds on the peptide follow the order [Ni(tcitr)2] > [Pt(tcitr)2] ≫ [Cu(tcitr)2]. From the AFM data, a remarkable DNA compaction was observed in the presence of [Pt(tcitr)2], while [Ni(tcitr)2] causes the formation of large interlaced DNA aggregates.

First studies on the interactions of the C-terminal cystatin C fragment 85–94 with Cu(II) ions

The hypothesis connecting dyshomeostasis of selected metal ions in the brain with increased probability of the development of severe neurodegenerative diseases is gaining increasing attention. The mechanisms of metal toxicity can be studied using different methodologies. One of the approaches is based on peptides as mimetics of the metal binding sites in proteins. In our studies, we have focused on human cystatin C (hCC) as one of the proteins involved in neurodegeneration. Physiologically, it is an important inhibitor of cysteine proteases and modulator of many physiological and pathological states. Native hCC is present at particularly high concentrations in cerebrospinal fluid (CSF) and was shown to possess both neurodegenerative and neuroprotective propensities. We have studied the copper(II) binding to its C-terminal fragment containing two histidine residues as potential site(s) for metal coordination. Our data indicate that the hCC fragment is fully capable of effective metal binding. Moreover, two potential metal binding sites can be proposed for the studied ligand. At the physiological range of pH formation of two types of complexes was observed: with imidazole and one/two amide donors in the coordination sphere of the metal ion and the final species characterized by the 4N-type of coordination.

Structure Guided Functional Studies of a Lipoprotein Involved in Salmonella Copper Homeostasis

Cells from aerobic organisms require copper ions to generate biochemical energy and for certain enzyme‐catalyzed redox reactions. However, recent studies suggest that when bacteria are engulfed by phagosomes within macrophages, Cu2+ is used in high concentrations, among other molecules, to kill the bacterium. To infect a host, bacteria such as Salmonella enterica must survive this toxic environment containing high Cu2+ levels. A recently discovered protein of unknown function, DcrB, plays a role in copper homeostasis within Salmonella enterica, a major cause of food‐borne illness. DcrB is a small lipoprotein anchored to the periplasmic face of the cytoplasmic membrane. Currently, Salmonella phenotypic assays have shown DcrB is necessary for growth in high levels of Cu2+. The hypothesis of this project is that DcrB binds copper and possibly other divalent cations. To test this hypothesis, we used thermal stability assays paired with structure‐based functional studies. We examined the effect of divalent cations on the stability of purified DcrB protein using a fluorescence‐based thermal shift assay and circular dichroism. Furthermore, we are using our recent crystallographic structure of DcrB to identify possible metal ion binding sites and to test whether each hypothesized metal ion binding site is required for the ability of DcrB to confer resistance to Cu2+. For each site, we used site‐directed mutagenesis to generate an allele of dcrB that encodes a DcrB variant lacking the functional side‐chains at the site. Then, we determined whether a multicopy plasmid that contains the mutant allele of dcrB can complement the copper sensitivity of a dcrB knockout mutant of Salmonella enterica. Our results have established the specificity of DcrB for binding divalent cations and have led to the identification of potential metal binding sites, which we anticipate will shed light on the role of DcrB in metal homeostasis in pathogenic bacteria.Support or Funding InformationThis research was supported by a UW‐La Crosse Faculty Research Grant (JFM), a UW‐La Crosse Undergraduate Research Grant (RWS), and the Dean's Distinguished Fellowship (RWS). This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC0206CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Copper-assisted interaction between amyloid-β and prion: Ternary metal complexes with Aβ N-terminus and octarepeat

Alzheimer and prion diseases are neurodegenerative disorders in which amyloid-beta (Aβ) and prion protein (PrP), respectively, failed their normal functions inducing cellular apoptosis. Histidine-based high-affinity metal-binding sites in the N-terminus domains, which favor the binding of transition metals such as copper and zinc, feature both Aβ and PrP. Recently, the biological role of copper(II) on the two protein interaction has been revealed. Many studies have been focused on copper(II) binding affinity for both Aβ or prion protein while ternary complexes formed by this metal with both proteins have not been investigated. Here we report a combined ESI MS, potentiometric and spectroscopic (CD, UV-Vis) study on ternary complexes formed by copper(II) ion with Aβ and PrP peptides fragments containing potential metal binding sites. In particular, Aβ(1-4), Aβ(1-6), Ac-Aβ(1-6), Aβ(1-16)PEG and the octarepeat (PHGGGWGQ) of prion protein were studied. ESI-MS results indicate that the formation of 1:1:1 ternary complexes occurred at the physiological pH range. Potentiometric and spectroscopic data demonstrate that in a large pH range, ternary species prevail over binary species with the histidine-driven amide deprotonation.

Biophysical characterisation of the novel zinc binding property in Suppressor of Fused

Suppressor of Fused (SUFU) is a highly conserved protein that acts as a negative regulator of the Hedgehog (HH) signalling pathway, a major determinant of cell differentiation and proliferation. Therefore, SUFU deletion in mammals has devastating effects on embryo development. SUFU is part of a multi-protein cytoplasmic signal-transducing complex. Its partners include the Gli family of transcription factors that function either as repressors, or as transcription activators according to the HH activation state. The crystal structure of SUFU revealed a two-domain arrangement, which undergoes a closing movement upon binding a peptide from Gli1. There remains however, much to be discovered about SUFU’s behaviour. To this end, we expressed recombinant, full-length SUFU from Drosophila, Zebrafish and Human. Guided by a sequence analysis that revealed a conserved potential metal binding site, we discovered that SUFU binds zinc. This binding was found to occur with a nanomolar affinity to SUFU from all three species. Mutation of one histidine from the conserved motif induces a moderate decrease in affinity for zinc, while circular dichroism indicates that the mutant remains structured. Our results reveal new metal binding affinity characteristics about SUFU that could be of importance for its regulatory function in HH.

Complex Nature of Protein Carbonylation Specificity After Metal-Catalyzed Oxidation.

Protein carbonylation is an irreversible modification of Lys, Arg, Thr and Pro amino acids under conditions of oxidative stress. Previous studies have reported specific carbonylated residues in purified recombinant albumins, albeit with a lack of agreement between the studies. Currently, structural factors that determine site-specific protein carbonylation are not well understood. In this study, we utilized metal-catalyzed oxidizing conditions to generate carbonylation in recombinant human serum albumin (HSA) and granulocyte-colony stimulating factor (G-CSF), two proteins with distinct metal-binding abilities. To estimate predictability of HSA carbonylation sites, the same oxidative reaction was repeated based on the previously reported conditions. For G-CSF, oxidative conditions were gradually adjusted to achieve substantial levels of protein carbonylation. Corresponding accumulation of specific oxidized residues was identified and confirmed with high-resolution mass spectrometry. Our HSA dataset contained 55 carbonylated residues and showed a significant overlap with the previously published pooled data, indicating a certain level of carbonylation site specificity for albumins. Oxidation of G-CSF under multiple oxidative conditions consistently showed a highly specific carbonylation at position Pro45. We also detected a previously unreported, oxidation-induced cleavage site in G-CSF between His44 and Pro45, which might be attributed to a presence of a potential metal-binding site near residue Pro45. Our results show distinct patterns of protein carbonylation for HSA and G-CSF. Thus, specificity of protein carbonylation induced by metal-catalyzed oxidation is protein dependent and might be predicted by availability of transition metal binding site(s) within the protein.

Structural Basis for the Magnesium-Dependent Activation and Hexamerization of the Lon AAA+ Protease

The Lon AAA+ protease (LonA) plays important roles in protein homeostasis and regulation of diverse biological processes. LonA behaves as a homomeric hexamer in the presence of magnesium (Mg(2+)) and performs ATP-dependent proteolysis. However, it is also found that LonA can carry out Mg(2+)-dependent degradation of unfolded protein substrate in an ATP-independent manner. Here we show that in the presence of Mg(2+) LonA forms a non-secluded hexameric barrel with prominent openings, which explains why Mg(2+)-activated LonA can operate as a diffusion-based chambered protease to degrade unstructured protein and peptide substrates efficiently in the absence of ATP. A 1.85Å crystal structure ofMg(2+)-activated protease domain reveals Mg(2+)-dependent remodeling of a substrate-binding loop and a potential metal-binding site near the Ser-Lys catalytic dyad, supported by biophysical binding assays and molecular dynamics simulations. Together, these findings reveal the specific roles of Mg(2+) in the molecular assembly and activation of LonA.

Structure and Interaction of Type II Cas1 from Sterptococcus Pyogenes

Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form a microbial immune system against invading foreign nucleic acids. Cas1 is one of the two universal Cas proteins present in nearly all types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated immunity. Here we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and study its interaction with other S. pyogenes Cas proteins. Despite conserved domain folds, the SpCas1 structure exhibits a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of a potential metal-binding site for catalysis. By using analytical size-exclusion chromatography and isothermal titration calorimetry, we demonstrate that SpCas1 directly interacts with SpCsn2 to form a complex. Taken together, our results provide structural information for a type II Cas1 protein, a previously uncharacterized Cas1 homologue, and lay a foundation for studying multiprotein Cas complexes functioning in the adaptive immunity of type II CRISPR-Cas systems.

Comparative and phylogenetic analysis of zinc transporter genes/proteins in plants

Zinc is an important catalytic element for more than 300 enzymes and plays a structural role in the stabilization of many proteins. Protein domain analysis showed that identified Zn transporter proteins belong to the ZIP protein family (PF02535). Zn transporter sequences were found to have similar molecular weights (33.1-51.4 kDa) and amino acid lengths (306-478 amino acids) with 5.31-8.92 pI values. Subcellular localization of Zn transporters was predicted as the plasma membrane. They had 6-9 putative transmembrane domains with a variable region between TM-3 and TM-4, which could contain a potential histidine-rich metal-binding domain. Moreover, alignment analysis showed that the TM-2, -4, and -5 domains could be potential metal-binding sites because they contain highly conserved His residues. Based on a homology search, the retrieved sequences were identified as corresponding homologs of either Arabidopsis thaliana or Oryza sativa. Phylogenetic analysis also supported that A. thaliana and O. sativa sequences could be used as a reference/benchmark to identify Zn transporter homologs in various plant species. The findings of this study will be valuable theoretical knowledge for feature studies in terms of understanding the gene and protein features of Zn transporters in various plants.

Crystal Structure of Streptococcus pyogenes Cas1 and Its Interaction with Csn2 in the Type II CRISPR-Cas System

CRISPRs and Cas proteins constitute an RNA-guided microbial immune system against invading nucleic acids. Cas1 is a universal Cas protein found in all three types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated adaptive immunity. Here, we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and characterize its interaction with S. pyogenes Csn2 (SpCsn2). The SpCas1 structure reveals a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of potential metal-binding sites for catalysis. We demonstrate that SpCas1 directly interacts with SpCsn2, and identify the binding interface and key residues for Cas complex formation. These results provide structural information for a type II Cas1 protein, and lay a foundation for studying multiprotein Cas complexes functioning in type II CRISPR-Cas systems.