Mapping Cu(II) Binding Sites in Prion Proteins by Diethyl Pyrocarbonate Modification and Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometric Footprinting

Qin, Ken; Yang, Ying; Mastrangelo, Peter; Westaway, David

doi:10.1074/jbc.m108744200

Cited by 150 publications

(148 citation statements)

References 58 publications

Supporting

Mentioning

133

Contrasting

Order By: Relevance

“…As shown in our previous work, EPR spectra from a copper titration of PrP (23)(24)(25)(26)(27)(28) show a progression of different spectra and these are assigned to individual binding components (17). Component 3 dominates at low copper occupancy (≤1.0 equiv).…”

Section: Cooperativity Of Cu 2+ Bindingsupporting

confidence: 56%

“…Clearly, the results reported here require not only confirmation in full-length PrP but also an assessment of the influence of non-octarepeat coordination sites (20,28) and other features that may affect Cu 2+ uptake. With regard to octarepeat coordination, however, our previously published experiments on full-length PrP Since the discovery of the physiological connection of PrP to copper, there have been intensive efforts to determine the precise Cu 2+ affinity (8,10,(21)(22)(23)(24)(25).…”

Section: Discussionmentioning

confidence: 82%

See 1 more Smart Citation

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

2006

View full text Add to dashboard Cite

The prion protein (PrP) binds Cu 2+ in its N-terminal octarepeat domain, composed of four or more tandem PHGGGWGQ segments. Previous work from our laboratory demonstrates that copper interacts with the octarepeat domain through three distinct coordination modes at pH 7.4, depending upon the precise ratio of Cu 2+ to protein. Here, we apply both electron paramagnetic resonance (EPR) and fluorescence quenching to determine the copper affinity for each of these modes. At low copper occupancy, which favors multiple His coordination, the octarepeat domain binds Cu 2+ with a dissociation constant of 0.10 (±0.08) nM. In contrast, high copper occupancy, involving coordination through deprotonated amide nitrogens, exhibits a weaker affinity characterized by dissociation constants in the range of 7.0-12.0 μM. Decomposition of the EPR spectra reveals the proportions of all coordination species throughout the copper concentration range and identifies significant populations of intermediates, consistent with negative cooperativity. At most copper concentrations, the Hill coefficient is less than 1.0 and approximately 0.7 at half copper occupancy. These findings demonstrate that the octarepeat domain is responsive to a remarkably wide copper concentration range covering approximately 5 orders of magnitude. Consideration of these findings, along with the demonstrated ability of the protein to quench copper redox activity at high occupancy, suggests that PrP may function to protect cells by scavenging excess copper.The prion protein (PrP) 1 is responsible for a novel class of infectious, neurodegenerative diseases collectively known as the transmissible spongiform encephalopathies (TSEs) (1-3). The TSEs include scrapie in sheep, mad cow disease (bovine spongiform encephalopathy, BSE), chronic wasting disease (CWD) in deer and elk, and Creutzfeldt-Jakob disease (CJD) in humans. The normal cellular form of the prion protein, referred to as PrP C , is found in a wide range of tissues of all mammalian and avian species. A misfolding event converts the protein to the infectious, β-sheet-rich scrapie form (PrP Sc ) responsible for the TSEs.PrP C is a GPI-anchored glycoprotein expressed in abundance on the surface of neurons, predominantly on presynaptic membranes (4,5). The mature form of PrP, consisting of residues 23-231 in hamster, has a globular C-terminal domain and a largely unstructured N-terminal domain (6) (Figure 1). The C-terminal domain (residues 125-231) contains three α helices, two of which are stabilized by a single interhelical disulfide bond, and a small antiparallel β sheet. The flexible N-terminal domain is glycine-rich and highly flexible. Within the flexible N-terminal region of PrP is the octarepeat domain, composed of four or five highly conserved, contiguous repeats of the eight-residue sequence PHGGGWGQ. The physiological function

show abstract

Section: Cooperativity Of Cu 2+ Bindingsupporting

confidence: 56%

Section: Discussionmentioning

confidence: 82%

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

2006

View full text Add to dashboard Cite

show abstract

“…Therefore, the irreversible carboethoxylation by DEPC has been used for the identification of essential His residues in many different enzymes [31,32] among which is castor plant i-PGAM [33]. It has also been used for the characterization of histidinecontaining metal-binding sites [34]. As DEPC also hydrolyses spontaneously in water, some enzyme activity may be retained when such residues are not easily accessible for the compound.…”

Section: Chemical Modification Of Histidinesmentioning

confidence: 99%

Characterization of the cofactor‐independent phosphoglycerate mutase from Leishmania mexicana mexicana

Guerra

Vertommen

Fothergill‐Gilmore

et al. 2004

European Journal of Biochemistry

View full text Add to dashboard Cite

Phosphoglycerate mutase (PGAM) activity in promastigotes of the protozoan parasite Leishmania mexicana is found only in the cytosol. It corresponds to a cofactor-independent PGAM as it is not stimulated by 2,3-bisphosphoglycerate and is susceptible to EDTA and resistant to vanadate. We have cloned and sequenced the gene and developed a convenient bacterial expression system and a high-yield purification protocol. Kinetic properties of the bacterially produced protein have been determined (3-phosphoglycerate:The activity is inhibited by phosphate but is resistant to Cland SO 4 2-. Inactivation by EDTA is almost fully reversed by incubation with CoCl 2 but not with MnCl 2 , FeSO 4 , CuSO 4 , NiCl 2 or ZnCl 2 . Alkylation by diethyl pyrocarbonate resulted in irreversible inhibition, but saturating concentrations of substrate provided full protection. Kinetics of the inhibitory reaction showed the modification of a new group of essential residues only after removal of metal ions by EDTA. The modified residues were identified by MS analysis of peptides generated by trypsin digestion. Two substrate-protected histidines in the proximity of the active site were identified (His136, His467) and, unexpectedly, also a distant one (His160), suggesting a conformational change in its environment. Partial protection of His467 was observed by the addition of 25 lM CoCl 2 to the EDTA treated enzyme but not of 125 lM MnCl 2 , suggesting that the latter metal ion cannot be accommodated in the active site of Leishmania PGAM.Keywords: chemical modification; kinetics; Leishmania mexicana; metal dependence; phosphoglycerate mutase.The reversible isomerization of 2-phosphoglycerate (2PGA) and 3-phosphoglycerate (3PGA) is an obligate step for both glycolysis and gluconeogenesis. This step is carried out in two different ways in nature, by two different types of evolutionarily unrelated enzymes (although both EC 5.4.2.1). The better documented enzyme is the cofactor-dependent phosphoglycerate mutase (d-PGAM) due to its requirement for 2,3-bisphophoglycerate. It is present in some eubacteria, yeast and all vertebrates most frequently as a dimer or tetramer of 23-30-kDa subunits [1]. The second enzyme, called cofactor-independent phosphoglycerate mutase (i-PGAM), is a monomeric protein of 60 kDa. Upon comparative sequence and structure analysis, the former enzyme has been classified as a member of the phospho-histidine acid phosphatase superfamily [2] and the latter as a member of the metal-dependent alkaline phosphatase superfamily [3,4]. Whereas d-PGAM is the enzyme present in all vertebrates, i-PGAM is found in all plants and archaebacteria [5] and, together with d-PGAMs, in lower eukaryotes and eubacteria [1,6].We have previously shown that an i-PGAM participates in glycolysis in the protist Trypanosoma brucei [7], a human pathogen. The completely distinct structures and catalytic mechanisms of trypanosomal and human PGAM offer great promise for the design of inhibitors with high selectivity for the parasite's enzyme. Therefore, this fi...

show abstract

“…These studies have been successfully applied to determining metal binding stoichiometry (3-9), metal-binding sites (10, 11), and metal-dependent structure/conformation changes (12,13). A number of metalbinding proteins, such as cytochrome c oxidase (14), albumin (3, 7), metallothionein (12,15,16), prion protein (PrP) (8,9,11,13,17), matrilysin (4, 5), and non-heme iron-containing metalloproteins (6), have been individually well characterized by either overall mass measurements on the intact metal-protein complexes or peptide sequencing on the protein digest with tandem mass spectrometry.Rapid developments in proteomic technology and bioinformatics have permitted identification of proteomes of cell lines and tissue by using mass spectrometry instrumentation (for review, see . The specific advances include the use of two-dimensional gel electrophoresis (2DE),…”

mentioning

confidence: 99%

Identification of Metal-binding Proteins in Human Hepatoma Lines by Immobilized Metal Affinity Chromatography and Mass Spectrometry

She

Narindrasorasak

Yang

et al. 2003

Molecular & Cellular Proteomics

100

View full text Add to dashboard Cite

The metalloproteome is defined as the set of proteins that have metal-binding capacity by being metalloproteins or having metal-binding sites. A different metalloproteome may exist for each metal. Mass spectrometric characterization of metalloproteomes provides valuable information relating to cellular disposition of metals physiologically and in metal-associated diseases. We examined the Cu and Zn metalloproteomes in three human hepatoma lines: Hep G2 and Mz-Hep-1, which retain many functional characteristics of normal human hepatocytes, and SKHep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep Metals play a pivotal role in cellular metabolism. They function as the catalytic centers in many biochemical reactions and serve as structural elements for a large number of regulatory proteins (1, 2). Characterization of metal-binding proteins is important for understanding the structure and biological functions of such proteins in metal-associated diseases. In the past few years, a variety of mass spectrometric techniques has been used to probe the metal-protein interactions in metal-containing proteins. These studies have been successfully applied to determining metal binding stoichiometry (3-9), metal-binding sites (10, 11), and metal-dependent structure/conformation changes (12,13). A number of metalbinding proteins, such as cytochrome c oxidase (14), albumin (3, 7), metallothionein (12,15,16), prion protein (PrP) (8,9,11,13,17), matrilysin (4, 5), and non-heme iron-containing metalloproteins (6), have been individually well characterized by either overall mass measurements on the intact metal-protein complexes or peptide sequencing on the protein digest with tandem mass spectrometry.Rapid developments in proteomic technology and bioinformatics have permitted identification of proteomes of cell lines and tissue by using mass spectrometry instrumentation (for review, see . The specific advances include the use of two-dimensional gel electrophoresis (2DE),

show abstract

Mapping Cu(II) Binding Sites in Prion Proteins by Diethyl Pyrocarbonate Modification and Matrix-assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) Mass Spectrometric Footprinting

Cited by 150 publications

References 58 publications

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

The Affinity of Copper Binding to the Prion Protein Octarepeat Domain: Evidence for Negative Cooperativity

Characterization of the cofactor‐independent phosphoglycerate mutase from Leishmania mexicana mexicana

Identification of Metal-binding Proteins in Human Hepatoma Lines by Immobilized Metal Affinity Chromatography and Mass Spectrometry

Contact Info

Product

Resources

About