Staphylococcus aureusA bout one-third of all proteins exploit specific metal ions to assist in macromolecular folding and͞or function at the active site of metalloenzymes (1). All cells restrict the number of bioavailable metal atoms to avoid any excess that would otherwise compete with native metal ion sites that do not support biological activity (2). Essentially all cell types contain intracellular metal sensors that detect surplus metal ions and control the expression of genes encoding proteins that expel or sequester the extra ions (3). For some metals and some cell types, a complementary set of sensors detect deficiency and regulate genes encoding proteins that acquire more of the required ions (4, 5). It is currently poorly understood how such metal-sensing metalloregulators accurately discriminate between various metal ions.SmtB͞ArsR-family regulators are ubiquitous in bacterial genomes and bind to the operator͞promoter (O͞P) regions of gene(s) encoding proteins involved in metal export or sequestration, repressing transcription (for a review, see ref. 6). As the concentration of metal ion increases, the effector-binding sites of the regulators become occupied eliciting a conformational change that weakens the affinity for the O͞P region, allowing transcription to proceed. Members of the SmtB͞ArsR family include: As(III), Sb(III), Bi(III)-responsive ArsR (7), Zn(II)-responsive SmtB (8), Cd(II), Pb(II), Bi(III)-responsive CadC (9-11), Zn(II)-responsive ZiaR (12), Co(II), Zn(II)-responsive CzrA (13,14), and, most recently, Ni(II), Co(II)-responsive NmtR (15).Comparative structural and spectroscopic studies of six SmtB͞ ArsR family members reveal that individual members are characterized by one or both of two structurally distinct metal coordination sites (6, 11, 15-20). These two metal sites are designated ␣3N (or ␣3) and ␣5 (or ␣5C), named for the location of the metal-binding ligands within the known or predicted secondary structure of individual family members. The coordination environment and precise ligand set of the ␣3, ␣3N, and͞or ␣5, ␣5C sites in the different SmtB͞ArsR proteins differ and are presumed to contribute toward metal selectivity. A sequence comparison for proteins discussed herein is shown in Fig. 1 and highlights these sites.Here we report insights gained from the study of two additional family members, Staphylococcus aureus CzrA and Mycobacterium tuberculosis NmtR. CzrA and NmtR share 30% sequence identity and a high degree of similarity (60%) yet respond to distinct but partially overlapping metal profiles in vivo. S. aureus CzrA is a Co(II)͞Zn(II)-specific sensor that regulates the expression of the czr operon, which encodes a Co(II)͞ Zn(II)-facilitated pump, CzrB, that effluxes metal out of the cell (13, 14). Electromobility-shift assays and in vivo expression studies indicate that Zn(II) is the strongest inducer of CzrA regulation, with Co(II) also capable of regulation but only at higher concentrations than Zn(II). Other metals, including Ni(II), have little to no effect on derep...

Interest in the molecular structure of amyloid fibrils originates both from their association with many devastating diseases and as systems for exploring the energetics of higher order protein folding and assembly. These fibril arrays are generally viewed as rich in β-sheets, of either parallel or antiparallel orientation. [1][2][3][4][5] However, the relative arrangement of the sheets within the fibril remains poorly constrained in the existing structure models, 4,6,7 as these sheet-to-sheet arrangements are mediated predominantly by side chain packing. We now extend the use of metal ions as probes of amyloid side chain packing in simple segments of the Aβ peptide of Alzheimer's disease. By restricting the possible metal binding sites, we show that Zn 2+ can specifically control the rate of self-assembly and dramatically regulate amyloid morphology via distinct coordination environments.The histidine dyad, His13 and His14, of Aβ is implicated in metal binding 8,9 and the metalmediated toxicity of Aβ. [10][11][12][13] In a parallel, in-register β-sheet arrangement with sheet Hbonds oriented along the fibril axis (Figure 1a.), 3,4 the side chains of the His13 and His14 are spaced 5 Å apart along each surface of the β-sheets (Figure 1b). If the sheets are arrayed parallel to one another, the His13 and His14 side chains from different sheets are proximal, providing potential sites for Zn 2+ chelation along the sheets (Figure 1b), between the sheets (Figure 1c), or both. 6 Aβ(13-21), HHQKLVFFA, includes both the core segment, Aβ(17-21), known to be crucial for fibril formation, 14-18 and the metal binding dyad. To isolate His13/14 as the sole binding elements, the K16A peptide HHQALVFFA-NH 2 , Aβ(13-21)K16A, was prepared. As shown in Figure 2a, Aβ(13-21)K16A develops β-sheet secondary structure within 49 h, showing an increased mean residue molar ellipticity (MRME) by circular dichroism (CD) at 197 and 212 nm. The development of β-sheet structure was further confirmed by FTIR, showing the appearance of the amide I absorbance at 1628 cm −1 ( Figure S1a). TEM further established that Aβ(13-21)K16A assembles into fibrils (Fig. S1b), and these mature fibrils bind Congo red with the typical UV/vis absorption shift from 500 to 540 nm ( Figure S1c Figure 3). When the ribbons from the 1:1 incubation were pelleted, washed, and analyzed, the Zn 2+ to peptide ratio was 0.6-0.8 across three independent measurements. 19Wider 100-150 nm ribbons with variable twists form with longer incubation times (panels 2a, b, Figure 3). Some of the ribbons appear to coil and fuse to form tubular structures 200-300 nm in diameter (panels 2c, d, Figure 3). Therefore, Zn 2+ reduces the nucleation time of self-assembly across the entire concentration range and transforms Aβ(13-21)K16A assembly into either fibrillar or ribbon/ tubular morphology. 17 Struck by the different morphologies accessible to Aβ(13-21)-K16A, we investigated the coordination environment of Zn 2+ in the different assemblies by X-ray absorption spectroscopy (XAS). The soluble ...

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn 2؉ to modulate the assembly kinetics and morphology of congeners of the amyloid ␤ peptide (A␤) associated with Alzheimer's disease. We now reveal a correlation among A␤-Cu 2؉ coordination, peptide self-assembly, and neuronal viability. By using the central segment of A␤, HHQKLVFFA or A␤(13-21), which contains residues H13 and H14 implicated in A␤-metal ion binding, we show that Cu 2؉ forms complexes with A␤(13-21) and its K16A mutant and that the complexes, which do not selfassemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-A␤(13-21)H14A, alters metal coordination, allowing Cu 2؉ to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of A␤ can access different metal-ion-coordination environments and that different complexes can lead to profound changes in A␤ self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.copper-binding ͉ neurotoxicity ͉ self-assembly P rotein intermolecular assembly, especially formation of amyloid fibrillar structures, is correlated with a variety of human neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases (1). More recently, amyloid has been tied to many nonpathological functional roles. For example, formation and self-perpetuation of amyloids in Saccharomyces cerevisiae regulate diverse yeast phenotypic expression as a positive response to environmental fluctuations (2), and amyloid may be involved in long-term memory and synapse maintenance in the marine snail, Aplysia (3, 4). Many proteins, including archetypical globular proteins such as myoglobin, can also form amyloid fibrils, suggesting that amyloidogenesis may be an intrinsic property of any ␣-amino acid polymer (5). Accordingly, these highly ordered paracrystalline protein self-assemblies have now been recognized as useful for nanostructure fabrication and biotechnology (6-8). Fully capturing these technological opportunities and understanding the biological roles of amyloid will depend on further definition of the organized structure and assembly pathway.Increasing evidence now implicates transition metal ions, including Zn 2ϩ , Cu 2ϩ , and Fe 3ϩ , as contributors both to amyloid ␤ (A␤) assembly in vitro and to the neuropathology of Alzheimer's disease, AD (9). The obligatory region of metal ion (Zn 2ϩ /Cu 2ϩ ) binding of A␤ has been mapped to the N terminus, amino acids 1-28 (10-16). In its soluble nonamyloid conformation, the peptide contains multiple intramolecular binding sites for Zn 2ϩ and Cu 2ϩ (9, 17), and intermolecular Zn 2ϩ binding can promote A␤ aggrega...

We heterologously overproduced a hyperthermostable archaeal low potential (E m ‫؍‬ ؊62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys 42 and Cys 61 ) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Riesketype ferredoxin appear to be inequivalent as indicated by much higher stability of the His 64 Proteins containing Rieske-type [2Fe-2S] clusters are widespread in nature from hyperthermophilic Archaea and Bacteria to Eukarya and play critical electron transfer roles in various pathways such as aerobic respiration, photosynthesis, and biodegradation of various alkene and aromatic compounds (1-4). In contrast to regular plant-and vertebrate-type ferredoxins having complete cysteinyl ligations, the Rieske-type cluster has an asymmetric iron-sulfur core with the S ␥ atom of each of the two cysteine residues coordinated to one iron site and the N ␦ atom of each of the two histidine residues coordinated to the other iron site. This asymmetric ligation results in some unique redox and spectroscopic properties (for reviews, see Refs. 1 and 3-5). This cluster coordination was firmly established by recent x-ray crystal structures of several different Rieske-type protein domains (6 -11).Two different types of Rieske clusters are observed in proteins. One type displays higher reduction potentials (E m ) 1 of approximately ϩ150 to ϩ490 mV and occurs in proton-translocating respiratory complexes (cytochrome bc 1 /b 6 f complexes and their archaeal homologs without c-type cytochromes), being involved in not only electron transfer but also substrate binding and oxidation at the quinol-oxidizing Q o site (2-5, 12-15). The other type displays lower E m values of approximately Ϫ150 to Ϫ50 mV and has been found in a diverse group of bacterial multicomponent terminal oxygenases and soluble Rieske-type ferredoxins (1, 3, 8, 9, 16 -27). However, none of the latter class has been characterized in detail from any archaeal species.We recently found that the genomic DNA sequence of the thermoacidophilic archaeon Sulfolobus solfataricus strain P-1 (DSM 1616T) encodes an archaeal homolog of bacterial small Rieske-type ferredoxins with no consensus disulfide signature (DDBJ accession number AB047031 (27)). This arf gene was found by homology search against the deduced amino acid sequence of Sulfolobus tokodaii sulredoxin, a water-soluble homolog of a high potential Rieske protein (E m,low pH ϳ ϩ190 mV) with a consensus disulfide linkage (DDBJ accession number AB023295) 2 (28 -30) (Fig. 1). Subsequent cloning and heterologous overexpression in Escherichia coli of this S. solfataricus arf gene encoding the archaeal Rieske-type ferredoxin (ARF) (27) have provided an opportunity to define the influence of surrounding amino acid residues on the electronic and s...