The Octarepeat Domain of the Prion Protein Binds Cu(II) with Three Distinct Coordination Modes at pH 7.4

The prion protein (PrP) binds Cu2+ in its N-terminal octarepeat domain. This unusual domain is comprised of four or more tandem repeats of the fundamental sequence PHGGGWGQ. Previous work from our laboratories demonstrates that at full copper occupancy, each HGGGW segment binds a single Cu2+. However, several recent studies suggest that low copper occupancy favors different coordination modes, possibly involving imidazoles from histidines in adjacent octapeptide segments. This is investigated here using a combination of X-band EPR, S-band EPR, and ESEEM, along with a library of modified peptides designed to favor different coordination interactions. At pH 7.4, three distinct coordination modes are identified. Each mode is fully characterized to reveal a series of copper-dependent octarepeat domain structures. Multiple His coordination is clearly identified at low copper stoichiometry. In addition, EPR detected copper−copper interactions at full occupancy suggest that the octarepeat domain partially collapses, perhaps stabilizing this specific binding mode and facilitating cooperative copper uptake. This work provides the first complete characterization of all dominant copper coordination modes at pH 7.4

Engineering tyrosine-based electron flow pathways in proteins: The case of aplysia myoglobin

Tyrosine residues can act as redox cofactors that provide an electron transfer ("hole-hopping") route that enhances the rate of ferryl heme iron reduction by externally added reductants, for example, ascorbate. Aplysia fasciata myoglobin, having no naturally occurring tyrosines but 15 phenylalanines that can be selectively mutated to tyrosine residues, provides an ideal protein with which to study such through-protein electron transfer pathways and ways to manipulate them. Two surface exposed phenylalanines that are close to the heme have been mutated to tyrosines (F42Y, F98Y). In both of these, the rate of ferryl heme reduction increased by up to 3 orders of magnitude. This result cannot be explained in terms of distance or redox potential change between donor and acceptor but indicates that tyrosines, by virtue of their ability to form radicals, act as redox cofactors in a new pathway. The mechanism is discussed in terms of the Marcus theory and the specific protonation/deprotonation states of the oxoferryl iron and tyrosine. Tyrosine radicals have been observed and quantified by EPR spectroscopy in both mutants, consistent with the proposed mechanism. The location of each radical is unambiguous and allows us to validate theoretical methods that assign radical location on the basis of EPR hyperfine structure. Mutation to tyrosine decreases the lipid peroxidase activity of this myoglobin in the presence of low concentrations of reductant, and the possibility of decreasing the intrinsic toxicity of hemoglobin by introduction of these pathways is discussed. © 2012 American Chemical Society

ModelCIF: An Extension of PDBx/mmCIF Data Representation for Computed Structure Models

ModelCIF (github.com/ihmwg/ModelCIF) is a data information framework developed for and by computational structural biologists to enable delivery of Findable, Accessible, Interoperable, and Reusable (FAIR) data to users worldwide. ModelCIF describes the specific set of attributes and metadata associated with macromolecular structures modeled by solely computational methods and provides an extensible data representation for deposition, archiving, and public dissemination of predicted three-dimensional (3D) models of macromolecules. It is an extension of the Protein Data Bank Exchange / macromolecular Crystallographic Information Framework (PDBx/mmCIF), which is the global data standard for representing experimentally-determined 3D structures of macromolecules and associated metadata. The PDBx/mmCIF framework and its extensions (e.g., ModelCIF) are managed by the Worldwide Protein Data Bank partnership (wwPDB, wwpdb.org) in collaboration with relevant community stakeholders such as the wwPDB ModelCIF Working Group (wwpdb.org/task/modelcif). This semantically rich and extensible data framework for representing computed structure models (CSMs) accelerates the pace of scientific discovery. Herein, we describe the architecture, contents, and governance of ModelCIF, and tools and processes for maintaining and extending the data standard. Community tools and software libraries that support ModelCIF are also described

Effect of the distal histidine on the peroxidatic activity of monomeric cytoglobin

The reaction of hydrogen peroxide with ferric human cytoglobin and a number of distal histidine variants were studied. The peroxidase activity of the monomeric wildtype protein with an internal disulfide bond, likely to be the form of the protein in vivo, exhibits a high peroxidase-like activity above that of other globins such as myoglobin. Furthermore, the peroxidatic activity of wildtype cytoglobin shows increased resistance to radical-based degradation compared to myoglobin. The ferryl form of wildtype cytoglobin is unstable, but is able to readily oxidize substrates such as guaiacol. In contrast distal histidine mutants of cytoglobin (H81Y and H81V) show very low peroxidase activity but enhanced radical-induced degradation. Therefore, the weakly bound distal histidine appears to modulate ferryl stability and limit haem degradation. These data are consistent with a role of a peroxidase activity of cytoglobin in cell stress response mechanisms.</ns4:p

Discovery and characterization of a new family of lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs

The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4.2, which are common to one of the cytoskeletal nodes. Unusually, we showed that hemoglobin bound in this way was redox active and capable of ligand binding. It can initiate lipid peroxidation showing the potential to cause cell damage. In vivo oxidative stress studies using extreme endurance exercise challenges showed an increase in hemoglobin membrane binding, especially in older cells with lower levels of antioxidant enzymes. These are then targeted for destruction. We propose a model where mild oxidative stress initiates the binding of redox active hemoglobin to the membrane. The maximum lifetime of the erythrocyte is thus governed by the redox activity of the cell; from the moment of its release into the circulation the timer is set

Mechanism of copper(II)-induced misfolding of Parkinson's disease protein

α-synuclein (aS) is a natively unfolded pre-synaptic protein found in all Parkinson's disease patients as the major component of fibrillar plaques. Metal ions, and especially Cu(II), have been demonstrated to accelerate aggregation of aS into fibrillar plaques, the precursors to Lewy bodies. In this work, copper binding to aS is investigated by a combination of quantum and molecular mechanics simulations. Starting from the experimentally observed attachment site, several optimized structures of Cu-binding geometries are examined. The most energetically favorable attachment results in significant allosteric changes, making aS more susceptible to misfolding. Indeed, an inverse kinematics investigation of the configuration space uncovers a dynamically stable β-sheet conformation of Cu-aS that serves as a nucleation point for a second β-strand. Based on these findings, we propose an atomistic mechanism of copper-induced misfolding of aS as an initial event in the formation of Lewy bodies and thus in PD pathogenesis

A gonadotropin-releasing hormone type neuropeptide with a high affinity binding site for copper(ii) and nickel(ii).

In vertebrates gonadotropin-releasing hormone I (GnRH-I) is a key regulator of reproductive development and function. The receptor-binding activity of human GnRH-I can be modified by the presence of divalent copper. Thus, copper binding to N-terminal amino acids in GnRH-I induces structural changes that influence receptor interactions and downstream intracellular signalling cascades. It is not known if copper-binding is restricted to human GnRH-I or if it is also a feature of GnRH-type peptides that have been identified in other taxa. To investigate this, we have characterised copper binding to a recently discovered GnRH-type peptide from the starfish Asterias rubens (ArGnRH). Using a range of spectroscopic and biophysical techniques we show that this peptide can bind copper(ii) and nickel(ii). Copper(ii) is bound in a square-planar, high-affinity (Kd ∼ 10-12 M) site incorporating four nitrogen donor atoms from a histidine imidazole group, two amides and the N-terminal amine group. The ArGnRH copper affinity and geometry are quite different to GnRH-I suggesting the copper sites have evolved to suit the environment the peptides are exposed to. By comparing the copper binding sites in ArGnRH and human GnRH-I and conducting a phylogenetic analysis of GnRH-type peptide sequences from a range of species, we predict that copper-binding is an evolutionarily ancient feature of GnRH-type peptides that has been retained, modified or lost in different lineages

Preferential Cu2+ coordination by His(96) and His(111) induces beta-sheet formation in the unstructured amyloidogenic region of the prion protein

The prion protein (PrP) is a Cu2+ binding cell surface glycoprotein that can misfold into a beta-sheet-rich conformation to cause prion diseases. The majority of copper binding studies have concentrated on the octarepeat region of PrP. However, using a range of spectroscopic techniques, we show that copper binds preferentially to an unstructured region of PrP between residues 90 and 115, outside of the octarepeat domain. Comparison of recombinant PrP with PrP-(91-115) indicates that this prion fragment is a good model for Cu2+ binding to the full-length protein. In contrast to previous reports we show that Cu2+ binds to this region of PrP with a nano-molar dissociation constant. NMR and EPR spectroscopy indicate a square-planar or square-pyramidal Cu2+ coordination utilizing histidine residues. Studies with PrP analogues show that the high affinity site requires both His(96) and His(111) as Cu2+ ligands, rather than a complex centered on His(96) as has been previously suggested. Our circular dichroism studies indicate a loss of irregular structure on copper coordination with an increase in beta-sheet conformation. It has been shown that this unstructured region, between residues 90 and 120, is vital for prion propagation and different strains of prion disease have been linked with copper binding. The role of Cu2+ in prion misfolding and disease must now be re-evaluated in the light of these findings