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

ZiFiT (Zinc Finger Targeter): an updated zinc finger engineering tool

ZiFiT (Zinc Finger Targeter) is a simple and intuitive web-based tool that provides an interface to identify potential binding sites for engineered zinc finger proteins (ZFPs) in user-supplied DNA sequences. In this updated version, ZiFiT identifies potential sites for ZFPs made by both the modular assembly and OPEN engineering methods. In addition, ZiFiT now integrates additional tools and resources including scoring schemes for modular assembly, an interface with the Zinc Finger Database (ZiFDB) of engineered ZFPs, and direct querying of NCBI BLAST servers for identifying potential off-target sites within a host genome. Taken together, these features facilitate design of ZFPs using reagents made available to the academic research community by the Zinc Finger Consortium. ZiFiT is freely available on the web without registration at http://bindr.gdcb.iastate.edu/ZiFiT/

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

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

Alzheimer's Aβ Peptides with Disease-Associated N-Terminal Modifications: Influence of Isomerisation, Truncation and Mutation on Cu2+ Coordination

coordination of various Aβ peptides has been widely studied. A number of disease-associated modifications involving the first 3 residues are known, including isomerisation, mutation, truncation and cyclisation, but are yet to be characterised in detail. In particular, Aβ in plaques contain a significant amount of truncated pyroglutamate species, which appear to correlate with disease progression. coordination modes between pH 6–9 with nominally the same first coordination sphere, but with a dramatically different pH dependence arising from differences in H-bonding interactions at the N-terminus. coordination of Aβ, which may be critical for alterations in aggregation propensity, redox-activity, resistance to degradation and the generation of the Aβ3–× (× = 40/42) precursor of disease-associated Aβ3[pE]–x species

Structural and Functional Diversity of the Microbial Kinome

The eukaryotic protein kinase (ePK) domain mediates the majority of signaling and coordination of complex events in eukaryotes. By contrast, most bacterial signaling is thought to occur through structurally unrelated histidine kinases, though some ePK-like kinases (ELKs) and small molecule kinases are known in bacteria. Our analysis of the Global Ocean Sampling (GOS) dataset reveals that ELKs are as prevalent as histidine kinases and may play an equally important role in prokaryotic behavior. By combining GOS and public databases, we show that the ePK is just one subset of a diverse superfamily of enzymes built on a common protein kinase–like (PKL) fold. We explored this huge phylogenetic and functional space to cast light on the ancient evolution of this superfamily, its mechanistic core, and the structural basis for its observed diversity. We cataloged 27,677 ePKs and 18,699 ELKs, and classified them into 20 highly distinct families whose known members suggest regulatory functions. GOS data more than tripled the count of ELK sequences and enabled the discovery of novel families and classification and analysis of all ELKs. Comparison between and within families revealed ten key residues that are highly conserved across families. However, all but one of the ten residues has been eliminated in one family or another, indicating great functional plasticity. We show that loss of a catalytic lysine in two families is compensated by distinct mechanisms both involving other key motifs. This diverse superfamily serves as a model for further structural and functional analysis of enzyme evolution