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Title: Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity

; ;  [1]
  1. (WU)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
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Resource Type:
Journal Article
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Journal Name: Proc. Natl. Acad. Sci. USA; Journal Volume: 103; Journal Issue: (26) ; 06, 2006
Country of Publication:
United States

Citation Formats

Koropatkin, Nicole M., Pakrasi, Himadri B., and Smith, Thomas J. Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity. United States: N. p., 2015. Web. doi:10.1073/pnas.0602517103.
Koropatkin, Nicole M., Pakrasi, Himadri B., & Smith, Thomas J. Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity. United States. doi:10.1073/pnas.0602517103.
Koropatkin, Nicole M., Pakrasi, Himadri B., and Smith, Thomas J. 2015. "Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity". United States. doi:10.1073/pnas.0602517103.
title = {Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity},
author = {Koropatkin, Nicole M. and Pakrasi, Himadri B. and Smith, Thomas J.},
abstractNote = {},
doi = {10.1073/pnas.0602517103},
journal = {Proc. Natl. Acad. Sci. USA},
number = (26) ; 06, 2006,
volume = 103,
place = {United States},
year = 2015,
month = 2
  • Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of all aquatic food chains by fixing carbon and nitrogen into cellular biomass. The single most important nutrient for photosynthesis and growth is nitrate, which is severely limiting in many aquatic environments particularly the open ocean (1, 2). It is therefore not surprising that NrtA, the solute-binding component of the high-affinity nitrate ABC transporter, is the single-most abundant protein in the plasma membrane of these bacteria (3). Here we describe the first structure of a nitratespecific receptor, NrtA from Synechocystis sp. PCC 6803, complexed withmore » nitrate and determined to a resolution of 1.5Å. NrtA is significantly larger than other oxyanionbinding proteins, representing a new class of transport proteins. From sequence alignments, the only other solute-binding protein in this class is CmpA, a bicarbonatebinding protein. Therefore, these organisms created a novel solute-binding protein for two of the most important nutrients; inorganic nitrogen and carbon. The electrostatic charge distribution of NrtA appears to force the protein off of the membrane while the flexible tether facilitates the delivery of nitrate to the membrane pore. The structure not only details the determinants for nitrate selectivity in NrtA, but also the bicarbonate specificity in CmpA. Nitrate and bicarbonate transport are regulated by the cytoplasmic proteins NrtC and CmpC, respectively. Interestingly, the residues lining the ligand binding pockets suggest that they both bind nitrate. This implies that the nitrogen and carbon uptake pathways are synchronized by intracellular nitrate and nitrite.3 The nitrate ABC transporter of cyanobacteria is composed of four polypeptides (Figure 1): a high-affinity periplasmic solute-binding lipoprotein (NrtA), an integral membrane permease (NrtB), a cytoplasmic ATPase (NrtD), and a unique ATPase/solute-binding fusion protein (NrtC) that regulates transport (4). NrtA binds both nitrate and nitrite (Kd = 0.3 mM) and is necessary for cell survival when nitrate is the primary nitrogen source (5). The role of NrtA is to scavenge nitrate/nitrite from the periplasm for delivery to the membrane permease, NrtB. The passage of solute through the transmembrane pore is linked to ATP hydrolysis by NrtC and NrtD. NrtD consists of a single ATPase domain. In contrast, NrtC contains both an ATPase domain and a Cterminal solute-binding domain that shares 50% amino acid sequence similarity with NrtA, and is required for the ammonium-mediated inhibition of nitrate transport (6, 7). Aside from the homologous transporter for bicarbonate, CmpABCD, there are no other known examples of ABC transporters that have an ATPase/solute-binding fusion component. The specificity of the nitrate transporter is conferred by NrtA (4). NrtA is ~49% identical (60% similar) in amino acid sequence to the bicarbonate receptor CmpA. In its entirety, it does not have significant homology to any other known protein. To elucidate the molecular determinants of nitrate specificity, we determined the crystal structure of the Synechocystis 6803 NrtA to 1.5 Å. While the general shape of NrtA is akin to that of other solute binding proteins, NrtA clearly represents a new and unique structural variant of these ‘C clamp’ proteins. From this structure and sequence alignments of other bicarbonate and nitrate transporters, the molecular basis for solute selectivity is clear and suggests that regulatory domains of both icarbonate and nitrate transport systems bind nitrate. Based on these findings, a model is presented that 4 demonstrates how such synergistic regulation of bicarbonate and nitrate transport is important in conserving energy during the process of carbon fixation and nitrogen assimilation.« less
  • An atomic resolution structure of the copper-binding domain of the Alzheimer’s disease amyloid precursor protein is presented. Amyloid precursor protein (APP) plays a central role in the pathogenesis of Alzheimer’s disease, as its cleavage generates the Aβ peptide that is toxic to cells. APP is able to bind Cu{sup 2+} and reduce it to Cu{sup +} through its copper-binding domain (CuBD). The interaction between Cu{sup 2+} and APP leads to a decrease in Aβ production and to alleviation of the symptoms of the disease in mouse models. Structural studies of CuBD have been undertaken in order to better understand themore » mechanism behind the process. Here, the crystal structure of CuBD in the metal-free form determined to ultrahigh resolution (0.85 Å) is reported. The structure shows that the copper-binding residues of CuBD are rather rigid but that Met170, which is thought to be the electron source for Cu{sup 2+} reduction, adopts two different side-chain conformations. These observations shed light on the copper-binding and redox mechanisms of CuBD. The structure of CuBD at atomic resolution provides an accurate framework for structure-based design of molecules that will deplete Aβ production.« less
  • The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) was used to investigate membrane protein assembly in the phototrophic bacterium Rhodobacter capsulatus. As found for Escherichia coli and mitochondrial proteins, assembly across the bacterial photosynthetic membranes was sensitive to CCCP. At uncoupler concentrations which were sufficient to block the export of the periplasmic cytochrome c/sub 2/ and an outer membrane protein, the integration of pigment-binding protein into the photosynthetic apparatus was abolished. The unassembled protein was detected on the inner surface of the intracytoplasmic membrane. After inactivation of CCCP, accumulated protein continued insertion into the membrane. The data suggest that after binding to themore » cytoplasmic face of the membrane (i), translocation of protein into a transmembrane orientation takes place (ii), which is a prerequisite for the formation of a functional pigment-protein complex (iii).« less
  • 2-Acetoxymethyl-1,4-naphthoquinone (2-AcOMeNQ) binds with rapid kinetics and high affinity to the primary quinone Q/sub A/ site of reaction centers from Rhodopseudomonas capsulata. Binding of 2-AcOMeNQ fully restores electron-transfer activity with kinetics that is similar, but not identical, to that seen with ubiquinone-50. When bound at the Q/sub A/ site, 2-AcOMeNQ preferentially labels the L subunit. This preference suggests that 2-AcOMeNQ labels primarily the region of a quinone-binding site that is close to the first isoprenoid unit of the side chain, which is expected from the location and structure of the reaction region of the molecule. In photosystem II particles frommore » Synechococcus sp., 2-AcOMeNQ primarily labels two polypeptides with apparent molecular masses of 38 and 19 kDa. Labeling of only the 38-kDa polypeptide is sufficiently sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) to conclude that it is involved in binding quinones on the acceptor side of photosystem II. Although the authors have not yet identified the 38-kDa protein, its properties suggest that it is the D2 protein. From the DCMU-sensitive labeling and from homologies to functionally important regions of the bacterial reaction-center subunits, they propose that the 38-kDa protein is intimately involved in binding the cofactors that mediate primary photochemistry.« less