Cytochrome P450 17A1 Interactions with the FMN Domain of Its Reductase as Characterized by NMR.

To accomplish key physiological processes ranging from drug metabolism to steroidogenesis, human microsomal cytochrome P450 enzymes require the sequential input of two electrons delivered by the FMN domain of NADPH-cytochrome P450 reductase. Although some human microsomal P450 enzymes can instead accept the second electron from cytochrome b5, for human steroidogenic CYP17A1, the cytochrome P450 reductase FMN domain delivers both electrons, and b5 is an allosteric modulator. The structural basis of these key but poorly understood protein interactions was probed by solution NMR using the catalytically competent soluble domains of each protein. Formation of the CYP17A1·FMN domain complex induced differential line broadening of the NMR signal for each protein. Alterations in the exchange dynamics generally occurred for residues near the surface of the flavin mononucleotide, including 87-90 (loop 1), and for key CYP17A1 active site residues. These interactions were modulated by the identity of the substrate in the buried CYP17A1 active site and by b5. The FMN domain outcompetes b5 for binding to CYP17A1 in the three-component system. These results and comparison with previous NMR studies of the CYP17A1·b5 complex suggest a model of CYP17A1 enzyme regulation.

The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function.

This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to "helicopter" above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function.

Human cytochrome P450 17A1 conformational selection: modulation by ligand and cytochrome b5.

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b5 (b5). Notably, titration of b5 to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b5, providing structural evidence consistent with the reported ability of b5 to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.

Do ASARM peptides play a role in nephrogenic systemic fibrosis?

Nephrogenic systemic fibrosis (NSF) is a devastating condition associated with gadolinium (Gd3+)-based contrast agents (GBCAs) in patients with kidney disease. The release of toxic Gd3+ from GBCAs likely plays a major role in NSF pathophysiology. The cause and etiology of Gd3+ release from GBCAs is unknown. Increased Acidic Serine Aspartate Rich MEPE-associated peptides (ASARM peptides) induce bone mineralization abnormalities and contribute to renal phosphate-handling defects in inherited hypophosphatemic rickets and tumor-induced osteomalacia. The proteolytic cleavage of related bone matrix proteins with ASARM motifs results in release of ASARM peptide into bone and circulation. ASARM peptides are acidic, reactive, phosphorylated inhibitors of mineralization that bind Ca2+ and hydroxyapatite. Since the ionic radius of Gd3+ is close to that of Ca2+, we hypothesized that ASARM peptides increase the risk of NSF by inducing release of Gd3+ from GBCAs. Here, we show 1) ASARM peptides bind and induce release of Gd3+ from GBCAs in vitro and in vivo; 2) A bioengineered peptide (SPR4) stabilizes the Gd3+-GBCA complex by specifically binding to ASARM peptide in vitro and in vivo; and 3) SPR4 peptide infusion prevents GBCA-induced NSF-like pathology in a murine model with increased ASARM peptide (Hyp mouse). We conclude ASARM peptides may play a role in NSF and SPR4 peptide is a candidate adjuvant for preventing or reducing risk of disease.

Synthesis and evaluation of a novel co-initiator for dentin adhesives: polymerization kinetics and leachables study.

A new tertiary amine co-initiator (TUMA) containing three methacrylate-urethane groups was synthesized for application in dentin adhesives. The photopolymerization kinetics and leaching of unreacted components from methacrylate-based dental polymers formulated with this new co-initiator were determined. The newly synthesize co-initiator showed good chemical stability and decreased amine release from the initiator system. This study provides important information for the future development of biocompatible dentin adhesives/composites.

Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.

claMP Tag: a versatile inline metal-binding platform based on the metal abstraction peptide.

Molecularly targeted research and diagnostic tools are essential to advancing understanding and detection of many diseases. Metals often impart the desired functionality to these tools, and conjugation of high-affinity chelators to proteins is carried out to enable targeted delivery of the metal. This approach has been much more effective with large lanthanide series metals than smaller transition metals. Because chemical conjugation requires additional processing and purification steps and yields a heterogeneous mixture of products, inline incorporation of a peptide tag capable of metal binding is a highly preferable alternative. Development of a transition metal binding tag would provide opportunity to greatly expand metal-based analyses. The metal abstraction peptide (MAP) sequence was genetically engineered into recombinant protein to generate the claMP Tag. The effects of this tag on recombinant epidermal growth factor (EGF) protein expression, disulfide bond formation, tertiary structural integrity, and transition metal incorporation using nickel were examined to confirm the viability of utilizing the MAP sequence to generate linker-less metal conjugates.

Correlating structure and function of drug-metabolizing enzymes: progress and ongoing challenges.

This report summarizes a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics at Experimental Biology held April 20-24 in Boston, MA. Presentations discussed the status of cytochrome P450 (P450) knowledge, emphasizing advances and challenges in relating structure with function and in applying this information to drug design. First, at least one structure of most major human drug-metabolizing P450 enzymes is known. However, the flexibility of these active sites can limit the predictive value of one structure for other ligands. A second limitation is our coarse-grain understanding of P450 interactions with membranes, other P450 enzymes, NADPH-cytochrome P450 reductase, and cytochrome b5. Recent work has examined differential P450 interactions with reductase in mixed P450 systems and P450:P450 complexes in reconstituted systems and cells, suggesting another level of functional control. In addition, protein nuclear magnetic resonance is a new approach to probe these protein/protein interactions, identifying interacting b5 and P450 surfaces, showing that b5 and reductase binding are mutually exclusive, and demonstrating ligand modulation of CYP17A1/b5 interactions. One desired outcome is the application of such information to control drug metabolism and/or design selective P450 inhibitors. A final presentation highlighted development of a CYP3A4 inhibitor that slows clearance of human immunodeficiency virus drugs otherwise rapidly metabolized by CYP3A4. Although understanding P450 structure/function relationships is an ongoing challenge, translational advances will benefit from continued integration of existing and new biophysical approaches.

Conjugation site heterogeneity causes variable electrostatic properties in Fc conjugates.

Immunoconjugates, including antibody-drug conjugates and Fc-conjugates, represent a rapidly growing class of therapeutics undergoing clinical development. Despite their growing popularity, the high intrinsic heterogeneity of immunoconjugates often complicates the development process and limits their widespread application. In particular, immunoconjugate charge variants exhibit markedly different colloidal stabilities, solubilities, pharmacokinetics, and tissue distributions. Charge variants arise spontaneously due to degradation and, depending on the type of drug, linker, and conjugation site, through drug conjugation. Electrostatic changes in naked antibodies often result in poor performance characteristics, and therefore, charge alterations due to degradation are critical to control. Charge properties are expected to be equally important to producing well-behaved ADCs. Charge-based methods of analysis, such as isoelectric focusing and ion exchange chromatography, are capable of probing the underlying complexities within immunoconjugate drug products. Despite the utility of these methods, there are only a few published reports of charge-based assays applied to immunoconjugates. In the present study, we sought to identify the effects of chemical conjugation on the electrostatic properties of Fc-conjugates. In order to minimize the effects of post-translational modifications (e.g., deamidation), a single Fc charge variant was isolated prior to conjugation of a fluorescent probe, Alexa Fluor 350, to the side chains of lysine residues. The resulting Fc-conjugates were assessed by a variety of analytical techniques, including isoelectric focusing and ion exchange chromatography, to determine their charge properties.

Effect of pH and excipients on structure, dynamics, and long-term stability of a model IgG1 monoclonal antibody upon freeze-drying.

PURPOSE: To investigate the mechanism of IgG1 mAb stabilization after freeze-drying and the interdependence of protein structural preservation in the solid state, glassy state dynamics and long-term storage stability under different formulation conditions. METHODS: IgG1 mAb was formulated with mannitol at pH 3.0, 5.0, and 7.0 in the presence and absence of sucrose and stability was monitored over 1 year at different temperatures. Physical and covalent degradation of lyophilized formulation was monitored using SEC, CEX, and light obscuration technique. Secondary and tertiary structure of the protein in the solid state was characterized using FTIR and fluorescence spectroscopy respectively. Raman spectroscopy was also used to monitor changes in secondary and tertiary structure, while SS-NMR (1)H relaxation was used to monitor glassy state dynamics. RESULTS: IgG1 mAb underwent significant secondary structural perturbations at pH 3.0 and conditions without sucrose, while pH 5.0 condition with sucrose showed the least structural change over time. The structural changes correlated with long-term stability with respect to protein aggregate formation and SbVP counts. SS-NMR data showed reduced relaxation time at conditions that were more stable. CONCLUSIONS: Native state protein structural preservation and optimal solid-state dynamics correlate with improved long-term stability of the mAb in the different lyophilized formulations.

Embedding the Ni-SOD mimetic Ni-NCC within a polypeptide sequence alters the specificity of the reaction pathway.

The unique metal abstracting peptide asparagine-cysteine-cysteine (NCC) binds nickel in a square planar 2N:2S geometry and acts as a mimic of the enzyme nickel superoxide dismutase (Ni-SOD). The Ni-NCC tripeptide complex undergoes rapid, site-specific chiral inversion to dld-NCC in the presence of oxygen. Superoxide scavenging activity increases proportionally with the degree of chiral inversion. Characterization of the NCC sequence within longer peptides with absorption, circular dichroism (CD), and magnetic CD (MCD) spectroscopies and mass spectrometry (MS) shows that the geometry of metal coordination is maintained, though the electronic properties of the complex are varied to a small extent because of bis-amide, rather than amine/amide, coordination. In addition, both Ni-tripeptide and Ni-pentapeptide complexes have charges of -2. This study demonstrates that the chiral inversion chemistry does not occur when NCC is embedded in a longer polypeptide sequence. Nonetheless, the superoxide scavenging reactivity of the embedded Ni-NCC module is similar to that of the chirally inverted tripeptide complex, which is consistent with a minor change in the reduction potential for the Ni-pentapeptide complex. Together, this suggests that the charge of the complex could affect the SOD activity as much as a change in the primary coordination sphere. In Ni-NCC and other Ni-SOD mimics, changes in chirality, superoxide scavenging activity, and oxidation of the peptide itself all depend on the presence of dioxygen or its reduced derivatives (e.g., superoxide), and the extent to which each of these distinct reactions occurs is ruled by electronic and steric effects that emenate from the organization of ligands around the metal center.

Mapping site-specific changes that affect stability of the N-terminal domain of calmodulin.

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the ¹H-¹5N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability.

Influence of the valine zipper region on the structure and aggregation of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5).

Protein aggregation is a major problem for biopharmaceuticals. While the control of aggregation is critically important for the future of protein pharmaceuticals, mechanisms of aggregate assembly, particularly the role that structure plays, are still poorly understood. Increasing evidence indicates that partially folded intermediates critically influence the aggregation pathway. We have previously reported the use of the basic leucine zipper (bZIP) domain of activating transcription factor 5 (ATF5) as a partially folded model system to investigate protein aggregation. This domain contains three regions with differing structural propensity: a N-terminal polybasic region, a central helical leucine zipper region, and a C-terminal extended valine zipper region. Additionally, a centrally positioned cysteine residue readily forms an intermolecular disulfide bond that reduces aggregation. Computational analysis of ATF5 predicts that the valine zipper region facilitates self-association. Here we test this hypothesis using a truncated mutant lacking the C-terminal valine zipper region. We compare the structure and aggregation of this mutant to the wild-type (WT) form under both reducing and nonreducing conditions. Our data indicate that removal of this region results in a loss of α-helical structure in the leucine zipper and a change in the mechanism of self-association. The mutant form displays increased association at low temperature but improved resistance to thermally induced aggregation.

Controlling the chiral inversion reaction of the metallopeptide Ni-asparagine-cysteine-cysteine with dioxygen.

Synthetically generated metallopeptides have the potential to serve a variety of roles in biotechnology applications, but the use of such systems is often hampered by the inability to control secondary reactions. We have previously reported that the Ni(II) complex of the tripeptide LLL-asparagine-cysteine-cysteine, LLL-Ni(II)-NCC, undergoes metal-facilitated chiral inversion to dld-Ni(II)-NCC, which increases the observed superoxide scavenging activity. However, the mechanism for this process remained unexplored. Electronic absorption and circular dichroism studies of the chiral inversion reaction of Ni(II)-NCC reveal a unique dependence on dioxygen. Specifically, in the absence of dioxygen, the chiral inversion is not observed, even at elevated pH, whereas the addition of O(2) initiates this reactivity and concomitantly generates superoxide. Scavenging experiments using acetaldehyde are indicative of the formation of carbanion intermediates, demonstrating that inversion takes place by deprotonation of the alpha carbons of Asn1 and Cys3. Together, these data are consistent with the chiral inversion being dependent on the formation of a Ni(III)-NCC intermediate from Ni(II)-NCC and O(2). The data further suggest that the anionic thiolate and amide ligands in Ni(II)-NCC inhibit Cα-H deprotonation for the Ni(II) oxidation state, leading to a stable complex in the absence of O(2). Together, these results offer insights into the factors controlling reactivity in synthetic metallopeptides.

MAPping the chiral inversion and structural transformation of a metal-tripeptide complex having ni-superoxide dismutase activity.

The metal abstraction peptide (MAP) tag is a tripeptide sequence capable of abstracting a metal ion from a chelator and binding it with extremely high affinity at neutral pH. Initial studies on the nickel-bound form of the complex demonstrate that the tripeptide asparagine-cysteine-cysteine (NCC) binds metal with 2N:2S, square planar geometry and behaves as both a structural and functional mimic of Ni superoxide dismutase (Ni-SOD). Electronic absorption, circular dichroism (CD), and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic Ni(II) center. It is apparent from the CD signal of Ni-NCC that the optical activity of the complex changes over time. Mass spectrometry data show that the mass of the complex is unchanged. Combined with the CD data, this suggests that chiral rearrangement of the complex occurs. Following incubation of the nickel-containing peptide in D(2)O and back-exchange into H(2)O, incorporation of deuterium into non-exchangeable positions is observed, indicating chiral inversion occurs at two of the α carbon atoms in the peptide. Control peptides were used to further characterize the chirality of the final nickel-peptide complex, and density functional theory (DFT) calculations were performed to validate the hypothesized position of the chiral inversions. In total, these data indicate Ni-SOD activity is increased proportionally to the degree of structural change in the complex over time. Specifically, the relationship between the change in CD signal and change in SOD activity is linear.

Novel tripeptide model of nickel superoxide dismutase.

Nickel superoxide dismutase (Ni-SOD) catalyzes the disproportionation of superoxide to molecular oxygen and hydrogen peroxide, but the overall reaction mechanism has yet to be determined. Peptide-based models of the 2N:2S nickel coordination sphere of Ni-SOD have provided some insight into the mechanism of this enzyme. Here we show that the coordination sphere of Ni-SOD can be mimicked using the tripeptide asparagine-cysteine-cysteine (NCC). NCC binds nickel with extremely high affinity at physiological pH with 2N:2S geometry, as demonstrated by electronic absorption and circular dichroism (CD) data. Like Ni-SOD, Ni-NCC has mixed amine/amide ligation that favors metal-based oxidation over ligand-based oxidation. Electronic absorption, CD, and magnetic CD (MCD) data collected for Ni-NCC are consistent with a diamagnetic Ni(II) center bound in square-planar geometry. Ni-NCC is quasi-reversibly oxidized with a midpoint potential of 0.72(2) V (vs Ag/AgCl) and breaks down superoxide in an enzyme-based assay, supporting its potential use as a model for Ni-SOD chemistry.

Enzyme activity of phosphatase of regenerating liver is controlled by the redox environment and its C-terminal residues.

Phosphatase of regenerating liver-1 (PRL-1) belongs to a unique subfamily of protein tyrosine phosphatases (PTPases) associated with oncogenic and metastatic phenotypes. While considerable evidence supports a role for PRL-1 in promoting proliferation, the biological regulators and effectors of PRL-1 activity remain unknown. PRL-1 activity is inhibited by disulfide bond formation at the active site in vitro, suggesting PRL-1 may be susceptible to redox regulation in vivo. Because PRL-1 has been observed to localize to several different subcellular locations and cellular redox conditions vary with tissue type, age, stage of cell cycle, and subcellular location, we determined the reduction potential of the active site disulfide bond that controls phosphatase activity to improve our understanding of the function of PRL-1 in various cellular environments. We used high-resolution solution NMR spectroscopy to measure the potential and found it to be -364.3 +/- 1.5 mV. Because normal cellular environments range from -170 to -320 mV, we concluded that nascent PRL-1 would be primarily oxidized inside cells. Our studies show that a significant conformational change accompanies activation, suggesting a post-translational modification may alter the reduction potential, conferring activity. We further demonstrate that alteration of the C-terminus renders the protein reduced and active in vitro, implying the C-terminus is an important regulator of PRL-1 function. These data provide a basis for understanding how subcellular localization regulates the activity of PRL-1 and, with further investigation, may help reveal how PRL-1 promotes unique outcomes in different cellular systems, including proliferation in both normal and diseased states.

Degradation of MEPE, DMP1, and release of SIBLING ASARM-peptides (minhibins): ASARM-peptide(s) are directly responsible for defective mineralization in HYP.

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) and DMP1 (dentin matrix protein 1) result in X-linked hypophosphatemic rickets (HYP) and autosomal-recessive hypophosphatemic-rickets (ARHR), respectively. Specific binding of PHEX to matrix extracellular phosphoglycoprotein (MEPE) regulates the release of small protease-resistant MEPE peptides [acidic serine- and aspartate-rich MEPE-associated motif (ASARM) peptides]. ASARM peptides are potent inhibitors of mineralization (minhibins) that also occur in DMP1 [MEPE-related small integrin-binding ligand, N-linked glycoprotein (SIBLING) protein]. It is not known whether these peptides are directly responsible for the mineralization defect. We therefore used a bone marrow stromal cell (BMSC) coculture model, ASARM peptides, anti-ASARM antibodies, and a small synthetic PHEX peptide (SPR4; 4.2 kDa) to examine this. Surface plasmon resonance (SPR) and two-dimensional (1)H/(15)N nuclear magnetic resonance demonstrated specific binding of SPR4 peptide to ASARM peptide. When cultured individually for 21 d, HYP BMSCs displayed reduced mineralization compared with wild type (WT) (-87%, P < 0.05). When cocultured, both HYP and WT cells failed to mineralize. However, cocultures (HYP and WT) or monocultures of HYP BMSCs treated with SPR4 peptide or anti-ASARM neutralizing antibodies mineralized normally. WT BMSCs treated with ASARM peptide also failed to mineralize properly without SPR4 peptide or anti-ASARM neutralizing antibodies. ASARM peptide treatment decreased PHEX mRNA and protein (-80%, P < 0.05) and SPR4 peptide cotreatment reversed this by binding ASARM peptide. SPR4 peptide also reversed ASARM peptide-mediated changes in expression of key osteoclast and osteoblast differentiation genes. Western blots of HYP calvariae and BMSCs revealed massive degradation of both MEPE and DMP1 protein compared with the WT. We conclude that degradation of MEPE and DMP-1 and release of ASARM peptides are chiefly responsible for the HYP mineralization defect and changes in osteoblast-osteoclast differentiation.

High-yield expression in E. coli and refolding of the bZIP domain of activating transcription factor 5.

Activating transcription factor 5 (ATF5) recently has been demonstrated to play a critical role in promoting the survival of human glioblastoma cells. Interference with the function of ATF5 in an in vivo rat model caused glioma cell death in primary tumors but did not affect the status of normal cells surrounding the tumor, suggesting ATF5 may prove an ideal target for anti-cancer therapy. In order to examine ATF5 as a pharmaceutical target, the protein must be produced and purified to sufficient quantity to begin analyses. Here, a procedure for expressing and refolding the bZIP domain of ATF5 in sufficient yield and final concentration to permit assay development and structural characterization of this target using solution NMR is reported. Two-dimensional NMR and circular dichroism analyses indicate the protein exists in the partially alpha-helical, monomeric x-form conformation with only a small fraction of ATF5 participating in formation of higher-order structure, presumably coiled-coil homodimerization. Despite the persistence of monomers in solution even at high concentration, an electrophoretic mobility shift assay showed that ATF5 is able to bind to the cAMP response element (CRE) DNA motif. Polyacrylamide gel electrophoresis and mass spectrometry were used to confirm that ATF5 can participate in homodimer formation and that this dimerization is mediated by disulfide bond formation.