Investigating Cu(I) binding to model peptides of N-terminal Aß isoforms.

Amyloid beta (Aß) peptides and copper (Cu) ions are each involved in critical biological processes including antimicrobial activity, regulation of synaptic function, angiogenesis, and others. Aß binds to Cu and may play a role in Cu trafficking. Aß peptides exist in isoforms that vary at their C-and N-termini; variation at the N-terminal sequence affects Cu binding affinity, structure, and redox activity by providing different sets of coordinating groups to the metal ion. Several N-terminal isoforms have been detected in human brain tissues including Aß1-40/42, Aß3-42, pEAß3-42, Aß4-42, Aß11-40 and pEAß11-40 (where pE denotes an N-terminal pyroglutamic acid). Several previous works have individually investigated the affinity and structure of Cu(I) bound to some of these isoforms' metal binding domains. However, the disparately reported values are apparent constants collected under different sets of conditions, and thus an integrated comparison cannot be made. The work presented here provides the Cu(I) coordination structure and binding affinities of these six biologically relevant Aß isoforms determined in parallel using model peptides of the Aß metal binding domains (Aß1-16, Aß3-16, pEAß3-16, Aß4-16, Aß11-16 and pEAß11-16). The binding affinities of Cu(I)-Aß complexes were measured using solution competition with ferrozine (Fz) and bicinchoninic acid (BCA), two colorimetric Cu(I) indicators in common use. The Cu(I) coordination structures were characterized by X-ray absorption spectroscopy. The data presented here facilitate comparison of the isoforms' Cu-binding interactions and contribute to our understanding of the role of Aß peptides as copper chelators in healthy and diseased brains.

Exploration of the Potential Role of Serum Albumin in the Delivery of Cu(I) to Ctr1.

Human serum albumin (HSA) is the major copper (Cu) carrier in blood. The majority of previous studies that have investigated Cu interactions with HSA have focused primarily on the Cu(II) oxidation state. Yet, cellular Cu uptake by the human copper transport protein (Ctr1), a plasma membrane-embedded protein responsible for Cu uptake into cells, requires Cu(I). Recent in vitro work has determined that reducing agents, such as the ascorbate present in blood, are sufficient to reduce the Cu(II)HSA complex to form Cu(I)HSA and that Cu(I) is bound to HSA with pM affinity. The biological accessibility of Cu(I)HSA suggests that HSA-bound Cu(I) may be an unappreciated form of Cu cargo and a key player in extracellular Cu trafficking. To better understand Cu trafficking by HSA, we sought to investigate the exchange of Cu(I) from HSA to a model peptide of the Cu-binding ectodomain of Ctr1. In this study, we used X-ray absorption near-edge spectroscopy to show that Cu(I) becomes more highly coordinated as increasing amounts of the Ctr1-14 model peptide are added to a solution of Cu(I)HSA. Extended X-ray absorption fine structure (EXAFS) spectroscopy was used to further characterize the interaction of Cu(I)HSA with Ctr1-14 by determining the ligands coordinating Cu(I) and their bond lengths. The EXAFS data support that some Cu(I) likely undergoes complete transfer from HSA to Ctr1-14. This finding of HSA interacting with and releasing Cu(I) to an ectodomain model peptide of Ctr1 suggests a mechanism by which HSA delivers Cu(I) to cells under physiological conditions.

Exploration of the Potential Role for Aß in Delivery of Extracellular Copper to Ctr1.

Amyloid beta (Aß) peptides are notorious for their involvement in Alzheimer's disease (AD), by virtue of their propensity to aggregate to form oligomers, fibrils, and eventually plaques in the brain. Nevertheless, they appear to be essential for correct neurophysiology on the synaptic level and may have additional functions including antimicrobial activity, sealing the blood-brain barrier, promotion of recovery from brain injury, and even tumor suppression. Aß peptides are also avid copper chelators, and coincidentally copper is significantly dysregulated in the AD brain. Copper (Cu) is released in significant amounts during calcium signaling at the synaptic membrane. Aß peptides may have a role in maintaining synaptic Cu homeostasis, including as a scavenger for redox-active Cu and as a chaperone for clearing Cu from the synaptic cleft. Here, we employed the Aß1-16 and Aß4-16 peptides as well-established non-aggregating models of major Aß species in healthy and AD brains, and the Ctr1-14 peptide as a model for the extracellular domain of the human cellular copper transporter protein (Ctr1). With these model peptides and a number of spectroscopic techniques, we investigated whether the Cu complexes of Aß peptides could provide Ctr1 with either Cu(II) or Cu(I). We found that Aß1-16 fully and rapidly delivered Cu(II) to Ctr1-14 along the affinity gradient. Such delivery was only partial for the Aß4-16/Ctr1-14 pair, in agreement with the higher complex stability for the former peptide. Moreover, the reaction was very slow and took ca. 40 h to reach equilibrium under the given experimental conditions. In either case of Cu(II) exchange, no intermediate (ternary) species were present in detectable amounts. In contrast, both Aß species released Cu(I) to Ctr1-14 rapidly and in a quantitative fashion, but ternary intermediate species were detected in the analysis of XAS data. The results presented here are the first direct evidence of a Cu(I) and Cu(II) transfer between the human Ctr1 and Aß model peptides. These results are discussed in terms of the fundamental difference between the peptides' Cu(II) complexes (pleiotropic ensemble of open structures of Aß1-16 vs the rigid closed-ring system of amino-terminal Cu/Ni binding Aß4-16) and the similarity of their Cu(I) complexes (both anchored at the tandem His13/His14, bis-His motif). These results indicate that Cu(I) may be more feasible than Cu(II) as the cargo for copper clearance from the synaptic cleft by Aß peptides and its delivery to Ctr1. The arguments in favor of Cu(I) include the fact that cellular Cu export and uptake proteins (ATPase7A/B and Ctr1, respectively) specifically transport Cu(I), the abundance of extracellular ascorbate reducing agent in the brain, and evidence of a potential associative (hand-off) mechanism of Cu(I) transfer that may mirror the mechanisms of intracellular Cu chaperone proteins.

Using N-Terminal Coordination of Cu(II) and Ni(II) to Isolate the Coordination Environment of Cu(I) and Cu(II) Bound to His13 and His14 in Amyloid-ß(4-16).

The amyloid-ß (Aß) peptide is a cleavage product of the amyloid precursor protein and has been implicated as a central player in Alzheimer's disease. The N-terminal end of Aß is variable, and different proportions of these variable-length Aß peptides are present in healthy individuals and those with the disease. The N-terminally truncated form of Aß starting at position 4 (Aß4-x) has a His residue as the third amino acid (His6 using the formal Aß numbering). The N-terminal sequence Xaa-Xaa-His is known as an amino terminal copper and nickel binding motif (ATCUN), which avidly binds Cu(II). This motif is not present in the commonly studied Aß1-x peptides. In addition to the ATCUN site, Aß4-x contains an additional metal binding site located at the tandem His residues (bis-His at His13 and 14) which is also found in other isoforms of Aß. Using the ATCUN and bis-His motifs, the Aß4-x peptide is capable of binding multiple metal ions simultaneously. We confirm that Cu(II) bound to this particular ATCUN site is redox silent, but the second Cu(II) site is redox active and can be readily reduced with ascorbate. We have employed surrogate metal ions to block copper coordination at the ATCUN or the tandem His site in order to isolate spectral features of the copper coordination environment for structural characterization using extended X-ray absorption fine structure (EXAFS) spectroscopy. This approach reveals that each copper coordination environment is independent in the Cu2Aß4-x state. The identification of two functionally different copper binding environments within the Aß4-x sequence may have important implications for this peptide in vivo.

The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1.

Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiological role of CuII binding to hCtr1.

Collaborating with Undergraduates To Contribute to Biochemistry Community Resources.

Course-based undergraduate research experiences (CUREs) have gained traction as effective ways to expand the impact of undergraduate research while fulfilling pedagogical goals. In this Perspective, we present innovative ways to incorporate fundamental benefits and principles of CUREs into a classroom environment through information/technology-based research projects that lead to student-generated contributions to digital community resources (CoRes). These projects represent an attractive class of CUREs because they are less resource-intensive than laboratory-based CUREs, and the projects align with the expectations of today's students to create rapid and publicly accessible contributions to society. We provide a detailed discussion of two example types of CoRe projects that can be implemented in courses to impact research and education at the chemistry-biology interface: bioinformatics annotations and development of educational tools. Finally, we present current resources available for faculty interested in incorporating CUREs or CoRe projects into their pedagogical practices. In sharing these stories and resources, we hope to lower the barrier for widespread adoption of CURE and CoRe approaches and generate discussions about how to utilize the classroom experience to make a positive impact on our students and the future of the field of biochemistry.

Structure and Affinity of Cu(I) Bound to Human Serum Albumin.

Human serum albumin (HSA) is a major Cu carrier in human blood and in cerebrospinal fluid. A major assumption is that Cu bound to HSA is in the Cu(II) oxidation state; thus, interactions between HSA and Cu(II) have been intensely investigated for over four decades. HSA has been reported previously to support the reduction of Cu(II) to the Cu(I) oxidation state in the presence of the weak reductant, ascorbate; however, the interactions between HSA and Cu(I) have not been explicitly investigated. Here, we characterize both the apparent affinity of HSA for Cu(I) using solution competition experiments and the coordination structure of Cu(I) bound to HSA using X-ray absorption spectroscopy and in silico modeling. We find that HSA binds to Cu(I) at pH 7.4 with an apparent conditional affinity of KCu(I):HSA = 1014.0 using digonal coordination in a structure that is similar to the bis-His coordination modes characterized for amyloid beta (Aß) and the prion protein. This high affinity and familiar Cu(I) coordination structure suggests that Cu(I) interaction with HSA in human extracellular fluids is unappreciated in the current scientific literature.

Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans.

The histidine-rich salivary peptides of the histatin family are known to bind copper (Cu) and other metal ions in vitro; however, the details of these interactions are poorly understood, and their implications for in vivo antifungal activity have not been established. Here, we show that the availability of Cu during exposure of Candida albicans to histatin-5 (Hist-5) modulates its antifungal activity. Antifungal susceptibility testing revealed that co-treatment of Hist-5 with Cu improved the EC50 from ∼5 to ∼1 µM, whereas co-treatment with a high-affinity Cu-specific chelator abrogated antifungal activity. Spectrophotometric titrations revealed two previously unrecognized Cu(I)-binding sites with apparent Kd values at pH 7.4, ∼20 nM, and confirmed a high-affinity Cu(II)-binding site at the Hist-5 N-terminus with an apparent Kd of ∼8 pM. Evaluation of a series of His-to-Ala full-length and truncated Hist-5 peptides identified adjacent His residues (bis-His) as critical anchors for Cu(I) binding, with the presence of a third ligand revealed by X-ray absorption spectroscopy. On their own, the truncated peptides were ineffective at inhibiting the growth of C. albicans, but treatment with supplemental Cu resulted in EC50 values down to ∼5 µM, approaching that of full-length Hist-5. The efficacy of the peptides depended on an intact bis-His site and correlated with Cu(I) affinity. Together, these results establish new structure-function relationships linking specific histidine residues with Cu binding affinity and antifungal activity and provide further evidence of the involvement of metals in modulating the biological activity of these antifungal peptides.

Sequence proximity between Cu(II) and Cu(I) binding sites of human copper transporter 1 model peptides defines reactivity with ascorbate and O2.

The critical nature of the copper transporter 1 (Ctr1) in human health has spurred investigation of Ctr1 structure and function. Ctr1 specifically transports Cu(I), the reduced form of copper, across the plasma membrane. Thus, extracellular Cu(II) must be reduced prior to transport. Unlike yeast Ctr1, mammalian Ctr1 does not rely on any known mammalian reductase. Previous spectroscopic studies of model peptides indicate that human Ctr1 could serve as both copper reductase and transporter. Ctr1 peptides bind Cu(II) at an amino terminal high-affinity Cu(II), Ni(II) ATCUN site. Ascorbate-dependent reduction of the Cu(II)-ATCUN complex is possible by virtue of an adjacent HH (bis-His), as this bis-His motif and one methionine ligand constitute a high affinity Ctr1 Cu(I) binding site. Here, we synthetically varied the distance between the ATCUN and bis-His motifs in a series of peptides based on the human Ctr1 amino terminal, with the general sequence MDHAnHHMGMSYMDS, where n=0-4. We tested the ability of each peptide to reduce Cu(II) with ascorbate and stabilize Cu(I) under ambient conditions (20% O2). This study reveals that significant differences in coordination structure and chemical behavior with ascorbate and O2 result from changes in the sequence proximity of ATCUN and bis-His. Peptides that deviate from the native Ctr1 pattern were less effective at forming stable Cu(I)-peptide complexes and/or resulted in O2-dependent oxidative damage to the peptide.

Model Peptide Studies Reveal a Mixed Histidine-Methionine Cu(I) Binding Site at the N-Terminus of Human Copper Transporter 1.

Copper is a vital metal cofactor in enzymes that are essential to myriad biological processes. Cellular acquisition of copper is primarily accomplished through the Ctr family of plasma membrane copper transport proteins. Model peptide studies indicate that the human Ctr1 N-terminus binds to Cu(II) with high affinity through an amino terminal Cu(II), Ni(II) (ATCUN) binding site. Unlike typical ATCUN-type peptides, the Ctr1 peptide facilitates the ascorbate-dependent reduction of Cu(II) bound in its ATCUN site by virtue of an adjacent HH (bis-His) sequence in the peptide. It is likely that the Cu(I) coordination environment influences the redox behavior of Cu bound to this peptide; however, the identity and coordination geometry of the Cu(I) site has not been elucidated from previous work. Here, we show data from NMR, XAS, and structural modeling that sheds light on the identity of the Cu(I) binding site of a Ctr1 model peptide. The Cu(I) site includes the same bis-His site identified in previous work to facilitate ascorbate-dependent Cu(II) reduction. The data presented here are consistent with a rational mechanism by which Ctr1 provides coordination environments that facilitate Cu(II) reduction prior to Cu(I) transport.

Role of MTL-1, MTL-2, and CDR-1 in mediating cadmium sensitivity in Caenorhabditis elegans.

Cadmium is an environmental toxicant whose exposure is associated with multiple human pathologies. To prevent cadmium-induced toxicity, organisms produce a variety of detoxification molecules. In response to cadmium, the nematode Caenorhabditis elegans increases the steady-state levels of several hundred genes, including two metallothioneins, mtl-1 and mtl-2, and the cadmium-specific response gene, cdr-1. Despite the presumed importance in metal detoxification of mtl-1 and mtl-2, knockdown of their expression does not increase cadmium hypersensitivity, which suggests that these genes are not required for resistance to metal toxicity in C. elegans. To determine whether cdr-1 is critical in metal detoxification and compensates for the loss of mtl-1 and/or mtl-2, C. elegans strains were generated in which one, two, and all three genes were deleted, and the effects of cadmium on brood size, embryonic lethality, the Bag phenotype, and growth were determined. Growth at low cadmium concentrations was the only endpoint in which the triple mutant displayed more sensitivity than the single and double mutants. A lack of hypersensitivity in these strains suggests that other factors may be involved in the response to cadmium. Caenorhabditis elegans produces phytochelatins (PCs) that are critical in the defense against cadmium toxicity. PC levels in wild type, cdr-1 single, mtl-1, mtl-2 double, and triple mutants were measured. PC levels were constitutively higher in the mtl-1, mtl-2 double, and triple mutants compared with wild type. Following cadmium exposure, PC levels increased. The lack of cadmium hypersensitivity when these genes are deleted may be attributed to the compensatory effects of increases in PCs.

Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1.

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.

Development of next-generation photolabile copper cages with improved copper binding properties.

Seven new nitrogen-donor ligands that contain a photoactive nitrophenyl group within the ligand backbone have been prepared and evaluated for their binding affinity for copper(ii) and zinc(ii). Among this series, the ligand 3Gcage (pyridine-2-carboxylic acid {1-(2-nitro-phenyl)-3-[(pyridin-2-ylmethyl)-amino]-propyl}-amide) has the best affinity for copper(ii), with an apparent dissociation constant at pH 7.4 of 0.18 fM. Exposure of buffered aqueous solutions of 3Gcage or Cu(ii)-bound 3Gcage to UV light induces bond cleavage in the ligand backbone, which reduces the denticity of the ligands. The quantum yields of photolysis for 3Gcage in the absence and presence of Cu(ii) are 0.66 and 0.43, respectively. Prior to photolysis, the 3Gcage ligand inhibits copper from generating hydroxyl radicals in the presence of hydrogen peroxide and ascorbic acid; however, hydroxyl radical formation increases by more than 300% following light activation, showing that the reactivity of the copper center can be triggered by light.

A photolabile ligand for light-activated release of caged copper.

A photosensitive caged copper complex has been prepared from a tetradentate ligand (H2cage) composed of two pyridyl-amide arms connected by a photoreactive nitrophenyl group. H2cage binds Cu2+ in aqueous solution with a stability constant (log beta) of 10.8, which corresponds to a KD of 16 pM at pH 7.4. The neutral Cu2+ complex, [Cu(OH2)(cage)], crystallizes as a distorted trigonal bipyramid coordinated by two amide and two pyridyl N atoms, with a water molecule bound in the trigonal plane. Photolysis with 350 nm UV light cleaves the ligand backbone to release photoproducts with significantly diminished affinity for Cu2+, thereby uncaging the metal ion. When coordinated as the caged complex, copper has diminished reactivity to produce hydroxyl radicals from Fenton-like reaction mixtures containing hydrogen peroxide and ascorbic acid. Postphotolysis, uncaged copper promotes hydroxyl radical formation under the same conditions. The strategy of caging copper is promising for applications where light could be used to trigger release of copper as a pro-oxidant to increase oxidative stress or as a tool to release copper intracellularly to study mechanisms of copper trafficking.