Improvement of biochemical methods of polyP quantification

Bru, Samuel; Jiménez, Javier; Canadell, David; Ariño, Joaquı́n; Clotet, Josep

doi:10.15698/mic2017.01.551

Cited by 45 publications

(65 citation statements)

References 40 publications

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“…A set of highly conserved basic amino-acids contributes to the formation of a large basic surface area, which has evolved to sense long-chain polyPs, but not DNA or other P i containing ligands. Our plant, archaeal and bacterial CHAD proteins bind polyPs with dissociation constants in the μM to nM range, in good agreement with the cellular concentrations reported for polyPs in different organisms (28,41,42). Our quantitative biochemical experiments show that GCI can be employed to quantify polyP binding to CHAD domains.…”

Section: Discussionsupporting

confidence: 85%

Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

Lorenzo‐Orts

Hohmann

Zhu

et al. 2019

Preprint

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Inorganic polyphosphates (polyPs) are long polymers of orthophosphate units (P i ), linked by energyrich phosphoanhydride bonds. Conserved histidine α-helical (CHAD) domains of unknown biochemical function are often located at the C-terminus of polyP-metabolizing triphosphate tunnel metalloenzymes (TTMs), or can be found as stand-alone proteins in bacterial operons harboring polyP kinases or phosphatases. Here we report that bacterial, archaeal and eukaryotic CHAD domains are specific polyP binding modules. Crystal structures reveal that CHAD domains are formed by two fourhelix bundles, giving rise to a central cavity surrounded by two conserved basic surface patches.Different CHAD domains bind polyPs with dissociation constants ranging from the nano-to midmicromolar range, but not DNA or other P i -containing ligands. A 2.1 Å CHAD -polyP complex structure reveals the phosphate polymer binding across a central pore and along the two basic patches.Mutational analysis of CHAD -polyP interface residues validates the complex structure and reveals that CHAD domains evolved to bind long-chain polyPs. The presence of a CHAD domain in the polyPase ygiF enhances its enzymatic activity. In plants, CHAD domains bind polyP in vivo and localize to the nucleus and nucleolus, suggesting that plants harbor polyP stores in these compartments.We propose that CHAD domains may be used to engineer the properties of polyP-metabolizing enzymes and to specifically localize polyP stores in eukaryotic cells and tissues. (221 words)Significance A domain of unknown function termed CHAD, present in all kingdoms of life, is characterized as a specific inorganic polyphosphate binding domain. The small size of the domain and its high specificity for inorganic polyphosphates suggest that it could be used as a tool to locate inorganic polyphosphate stores in pro-and eukaryotic cells and tissues. operons expressing polyP metabolic enzymes (39). Recently, it was found that CHAD domaincontaining proteins specifically localize to polyP granules in the bacterium Ralstonia eutropha (40). In this study, we combine structural biology and quantitative biochemistry to define CHAD domains as polyP binding modules. ResultsWe located CHAD domains in the different kingdoms of life. According to Interpro (https://www.ebi.ac.uk/interpro), ~ 99 % of the annotated CHAD proteins correspond to bacteria, while only ~ 1 % (129 proteins) and 0.1 % (10 proteins) belong to archaea and eukaryota, respectively (Fig. 1A). We selected CHAD domain-containing proteins belonging to the three kingdoms of life: archaea (Sulfolobus solfataricus; termed SsCHAD hereafter), bacteria (Chlorobium tepidum; CtCHAD) and eukaryota (Ricinus communis or castor bean; RcCHAD) ( Fig 1A) (SI Appendix Fig. S1). Several of these CHAD proteins form part of gene clusters encoding polyP metabolizing enzymes, with the exception of RcCHAD (Fig. 1B).To confirm if indeed RcCHAD is expressed in Ricinus, we performed RT-PCR experiments using Ricinus cDNA prepared from leave extracts. We detected a transcri...

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Section: Discussionsupporting

confidence: 85%

Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

Lorenzo‐Orts

Hohmann

Zhu

et al. 2019

Preprint

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“…We find that polyP is able to displace tubulin from tau (Fig. 8) at concentrations much lower than the reported cytosolic concentrations of polyP (41). The cellular cytoplasm is much more complex than the tertiary mixture explored here and thus whether polyP could compete for binding to tau under physiological conditions remains to be tested.…”

Section: Discussioncontrasting

confidence: 46%

Polyphosphate initiates tau aggregation through intra- and intermolecular scaffolding

Wickramasinghe

Lempart

Merens

et al. 2019

Preprint

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23The aggregation and deposition of tau is a hallmark of a class of neurodegenerative diseases 24 called tauopathies. Despite intensive study, cellular and molecular factors that trigger tau 25 aggregation are not well understood. Here we provide evidence for two mechanisms relevant to 26 the initiation of tau aggregation in the presence of cytoplasmic polyphosphates (polyP): changes 27 in the conformational ensemble of monomer tau and noncovalent cross-linking of multiple tau 28 monomers. We identified conformational changes throughout full-length tau, most notably 29 diminishment of long-range interactions between the termini coupled with compaction of the 30 microtubule binding and proline rich regions. We found that while the proline rich and 31 microtubule binding regions both contain polyP binding sites, the proline rich region is a 32 requisite for compaction of the microtubule binding region upon binding. Additionally, both the 33 magnitude of the conformational change and the aggregation of tau are dependent on the chain 34 length of the polyP polymer. Longer polyP chains are more effective at intermolecular, 35 noncovalent cross-linking of tau. These observations provide an understanding of the initial steps 36 of tau aggregation through interaction with a physiologically relevant aggregation inducer.

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“…L −1 . Efforts to improve polyP extraction and quantification methods are ongoing in several research disciplines (Bru et al ; Solesio and Pavlov ; Lee et al ), suggesting that fully quantitative polyP analysis of environmental samples may soon become feasible.…”

Section: Methodsmentioning

confidence: 99%

Particulate polyphosphate and alkaline phosphatase activity across a latitudinal transect in the tropical Indian Ocean

Martin

Lauro

Sarkar

et al. 2018

Limnology & Oceanography

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Polyphosphate (polyP) is an essential chemical constituent of microbial cells, and is hypothesized to play important roles in the marine biogeochemistry of phosphorus. However, polyP has only rarely been measured in the oceans. Here, we present data on the distribution of polyP across a latitudinal transect in the tropical Indian Ocean. PolyP concentrations (quantified as molar equivalents of a synthetic polyP standard) and ratios of polyP to total particulate phosphorus (TPP) along the transect ranged between 3-7 nmol eq. L 21 (polyP concentration) and 0.2-0.4 nmol eq. nmol 21 (polyP : TPP ratio), which is very similar to values reported from the North Pacific Subtropical Gyre. Yet unlike in the North Pacific, soluble reactive phosphorus was depleted to low concentrations ( 0.03 lmol L 21 ), and alkaline phosphatase activity was relatively high (1-4 nmol P L 21 h 21 ) along our cruise track. We attribute these results to the unique seasonal changes in iron and macronutrient supply in the Indian Ocean, which are caused by the monsoonal reversal in ocean currents. PolyP concentrations and polyP : TPP ratios decreased sharply with depth down to 150 m, suggesting that polyP was preferentially recycled relative to TPP, unlike in the North Pacific Subtropical Gyre. We hypothesize that alkaline phosphatase exerts a significant control over marine polyP biogeochemistry.

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Improvement of biochemical methods of polyP quantification

Cited by 45 publications

References 40 publications

Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

Molecular characterization of CHAD domains as inorganic polyphosphate binding modules

Polyphosphate initiates tau aggregation through intra- and intermolecular scaffolding

Particulate polyphosphate and alkaline phosphatase activity across a latitudinal transect in the tropical Indian Ocean

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