Structural Determinants of Metal Specificity in the Zinc Transport Protein ZnuA from Synechocystis 6803

Banerjee, Soojay; Wei, Baoxian; Bhattacharyya-Pakrasi, Maitrayee; Pakrasi, Himadri B.; Smith, Thomas J.

doi:10.1016/j.jmb.2003.09.008

Cited by 112 publications

(178 citation statements)

References 36 publications

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“…Indeed, this has been observed in a number of high affinity metal-binding ABC transporters (e.g. the zinc-binding ZnuA (20) and the iron-binding FutA1. 3 Bicarbonate is bound adjacent to Ca 2ϩ in a more solventexposed position in the binding pocket.…”

Section: Resultsmentioning

confidence: 84%

“…However, it is also possible that the position of carbonic acid represents an initial interaction between bicarbonate and the exterior loops of the C-terminal domain to facilitate delivery of solute to the binding pocket. This kind of "preloading" of ligand to exterior loops of the transport protein was suggested in the case of the zinc-binding protein ZnuA (20).…”

Section: Resultsmentioning

confidence: 90%

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The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

Koropatkin

Koppenaal

Pakrasi

et al. 2007

Journal of Biological Chemistry

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Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO 2 over O 2 , cyanobacteria have developed highly efficient CO 2 -concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here, we have described the structure of the bicarbonatebinding protein CmpA in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein NrtA. CmpA binds carbonic acid at the entrance to the ligand-binding pocket, whereas bicarbonate binds in nearly an identical location compared with nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca 2؉ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal appears to be highly cooperative and suggests that CmpA may co-transport bicarbonate and calcium or that calcium acts a cofactor in bicarbonate transport.Cyanobacteria are the most abundant microorganisms in aquatic environments and play a key role in the global carbon cycle (1). It is estimated that these photosynthetic microbes are responsible for ϳ50% of carbon fixation in the oceans. Over their 2.7-billion-year existence, cyanobacteria had to adapt to a changing gaseous environment where the levels of CO 2 declined and O 2 increased (2). Because O 2 can compete with CO 2 for binding to the carbon-fixing enzyme Rubisco (3), cyanobacteria evolved the most effective CO 2 -concentrating mechanism (CCM) 2 that allows them to concentrate CO 2 levels around Rubisco up to 1000-fold. The CCM involves the import and accumulation of inorganic carbon as HCO 3 Ϫ in the cytoplasm and subsequent conversion to CO 2 in the protein microcompartment called the "carboxysome" via carbonic anhydrase.One component of this CCM machinery in Synechocystis PCC 6803 is the cmpABCD operon that encodes a high affinity bicarbonate ABC transporter that is induced under low CO 2 conditions (4). This transporter is composed of four polypeptides, a high affinity solute-binding lipoprotein (CmpA), an integral membrane permease (CmpB), a cytoplasmic ATPase (CmpD), and an ATPase/solute-binding fusion protein (CmpC) that regulates transport (Fig. 1). The CmpABCD transporter is the highest affinity bicarbonate transporter of cyanobacteria (4). This affinity is predominantly conferred by the binding of bicarbonate to CmpA (K d ϭ 5 M) (5). CmpA is anchored to the periplasmic face of the cytoplasmic membrane via a lipid anchor attached to a conserved cysteine (6). The closest known homologue of CmpA is NrtA, the solute-binding protein of the nitrate-specific NrtABCD transporter that is 48% identical and 61% similar in amino acid sequence (7). We recently published an analysis (8) of the 1.6-Å structure of NrtA complexed with nitrate to elucidate the molecular determinants of nitrate specificity. From ...

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Section: Resultsmentioning

confidence: 84%

Section: Resultsmentioning

confidence: 90%

The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

Koropatkin

Koppenaal

Pakrasi

et al. 2007

Journal of Biological Chemistry

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“…In contrast to the ZinT proteins, the X-ray structures of several members of the ZnuA family are known, namely those of the proteins from E. coli (EcZnuA, PDB codes: 2OGW; 2OSV; 2PRS) [17][18][19], Synechocystis 6803 (PDB code: 1PQ4) [20] and S. enterica (SeZnuA, PDB code: 2XQV) [13]. The ZnuA family belongs to the so-called cluster 9 of periplasmic solute-binding proteins (PBPs) and displays their well conserved architecture comprising a pair of (α/β) 4 sandwich domains and a connecting long, tightly packed α-helix.…”

Section: Introductionmentioning

confidence: 99%

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

Ilari

Alaleona

Tria

et al. 2014

Biochimica et Biophysica Acta (BBA) - General Subjects

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This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues.Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. Background: In Gram-negative bacteria the ZnuABC transporter ensures adequate zinc import in Zn(II)-poor environments, like those encountered by pathogens within the infected host. Recently, the metal-binding protein ZinT was suggested to operate as an accessory component of ZnuABC in periplasmic zinc recruitment. Since ZinT is known to form a ZinT-ZnuA complex in the presence of Zn(II) it was proposed to transfer Zn(II) to ZnuA. The present work was undertaken to test this claim. Methods: ZinT and its structural relationship with ZnuA have been characterized by multiple biophysical techniques (X-ray crystallography, SAXS, analytical ultracentrifugation, fluorescence spectroscopy). Results: The metal-free and metal-bound crystal structures of Salmonella enterica ZinT show one Zn(II) binding site and limited structural changes upon metal removal. Spectroscopic titrations with Zn(II) yield a K D value of 22 ± 2 nM for ZinT, while those with ZnuA point to one high affinity (K D b 20 nM) and one low affinity Zn(II) binding site (K D in the micromolar range). Sedimentation velocity experiments established that Zn(II)-bound ZinT interacts with ZnuA, whereas apo-ZinT does not. The model of the ZinT-ZnuA complex derived from small angle X-ray scattering experiments points to a disposition that favors metal transfer as the metal binding cavities of the two proteins face each other. Conclusions: ZinT acts as a Zn(II)-buffering protein that delivers Zn(II) to ZnuA. General significance: Knowledge of the ZinT-ZnuA relationship is crucial for understanding bacterial Zn(II) uptake.

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“…It is anticipated that these proteins assist in loading metal ions onto the correct importers. There is similarity in the metal-binding sites of several substrate binding proteins that contribute toward the import of distinct metals, such as manganese versus zinc, and subtle differences in second coordination spheres may be crucial for metal selectivity (5). It has also been noted that the nature of interactions between substrate binding proteins and their respective metal transporters may make a key contribution to metal specificity (5).…”

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confidence: 99%

A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

Waldron¹,

Tottey²,

Yanagisawa

et al. 2007

Journal of Biological Chemistry

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Periplasmic substrate binding proteins are known for iron, zinc, manganese, nickel, and molybdenum but not copper. Synechocystis PCC 6803 requires copper for thylakoid-localized plastocyanin and cytochrome oxidase. Here we show that mutants deficient in a periplasmic substrate binding protein FutA2 have low cytochrome oxidase activity and produce cytochrome c 6 when grown under copper conditions (150 nM) in which wild-type cells use plastocyanin rather than cytochrome c 6 . Anaerobic separation of extracts by two-dimensional native liquid chromatography followed by metal analysis and peptide mass-fingerprinting establish that accumulation of copperplastocyanin is impaired, but iron-ferredoxin is unaffected in ⌬futA2 grown in 150 nM copper. However, recombinant FutA2 binds iron in preference to copper in vitro with an apparent Fe(III) affinity similar to that of its paralog FutA1, the principal substrate binding protein for iron import. FutA2 is also associated with iron and not copper in periplasm extracts, and this Fe(III)-protein complex is absent in ⌬futA2. There are differences in the soluble protein and small-molecule complexes of copper and iron, and the total amount of both elements increases in periplasm extracts of ⌬futA2 relative to wild type. Changes in periplasm protein and small-molecule complexes for other metals are also observed in ⌬futA2. It is proposed that FutA2 contributes to metal partitioning in the periplasm by sequestering Fe(III), which limits aberrant Fe(III) associations with vital binding sites for other metals, including copper.Oxygenic phototrophs have exceptional requirements for metals such as iron, copper, and manganese within the photosynthetic machinery (1, 2). There is interest in understanding how metal ions partition to the correct cellular destinations in all organisms (3), including to the approximately one-third of proteins that need metals (4). The periplasm is a key location for metal partitioning. Bacterial ATP binding cassette (ABC)-type importers include substrate binding proteins which may be free within the periplasm or anchored to the outer face of the plasma membrane. It is anticipated that these proteins assist in loading metal ions onto the correct importers. There is similarity in the metal-binding sites of several substrate binding proteins that contribute toward the import of distinct metals, such as manganese versus zinc, and subtle differences in second coordination spheres may be crucial for metal selectivity (5). It has also been noted that the nature of interactions between substrate binding proteins and their respective metal transporters may make a key contribution to metal specificity (5

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Structural Determinants of Metal Specificity in the Zinc Transport Protein ZnuA from Synechocystis 6803

Cited by 112 publications

References 36 publications

The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

The Salmonella enterica ZinT structure, zinc affinity and interaction with the high-affinity uptake protein ZnuA provide insight into the management of periplasmic zinc

A Periplasmic Iron-binding Protein Contributes toward Inward Copper Supply

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