Small molecule inhibitors of the fosfomycin resistance enzyme FosM from Mycobacterium abscessus.

Fosfomycin is a safe broad-spectrum antibiotic that has not achieved widespread use because of the emergence of fosfomycin-modifying enzymes. Inhibition of fosfomycin-modifying enzymes could be used to help combat pathogens like Mycobacterium abscessus. Our previous work identified several inhibitors for the enzyme FosB from Staphylococcus aureus. We have tested those same compounds for inhibition of FosM, the fosfomycin-modifying enzyme from M. abscessus. The work described here will be used as the basis for more detailed studies into the inhibition of FosM.

Identification and analysis of small molecule inhibitors of FosB from Staphylococcus aureus.

Antimicrobial resistance (AMR) poses a significant threat to human health around the world. Though bacterial pathogens can develop resistance through a variety of mechanisms, one of the most prevalent is the production of antibiotic-modifying enzymes like FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. FosB enzymes are found in pathogens such as Staphylococcus aureus, one of the leading pathogens in deaths associated with AMR. fosB gene knockout experiments establish FosB as an attractive drug target, showing that the minimum inhibitory concentration (MIC) of fosfomycin is greatly reduced upon removal of the enzyme. Herein, we have identified eight potential inhibitors of the FosB enzyme from S. aureus by applying high-throughput in silico screening of the ZINC15 database with structural similarity to phosphonoformate, a known FosB inhibitor. In addition, we have obtained crystal structures of FosB complexes to each compound. Furthermore, we have kinetically characterized the compounds with respect to inhibition of FosB. Finally, we have performed synergy assays to determine if any of the new compounds lower the MIC of fosfomycin in S. aureus. Our results will inform future studies on inhibitor design for the FosB enzymes.

Inhibition of Fosfomycin Resistance Protein FosB from Gram-Positive Pathogens by Phosphonoformate.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of antimicrobial resistance related deaths worldwide. Like many pathogens with multidrug-resistant strains, S. aureus contains enzymes that confer resistance through antibiotic modification(s). One such enzyme present in S. aureus is FosB, a Mn2+-dependent l-cysteine or bacillithiol (BSH) transferase that inactivates the antibiotic fosfomycin. fosB gene knockout experiments show that the minimum inhibitory concentration (MIC) of fosfomycin is significantly reduced when the FosB enzyme is not present. This suggests that inhibition of FosB could be an effective method to restore fosfomycin activity. We used high-throughput in silico-based screening to identify small-molecule analogues of fosfomycin that inhibited thiol transferase activity. Phosphonoformate (PPF) was a top hit from our approach. Herein, we have characterized PPF as a competitive inhibitor of FosB from S. aureus (FosBSa) and Bacillus cereus (FosBBc). In addition, we have determined a crystal structure of FosBBc with PPF bound in the active site. Our results will be useful for future structure-based development of FosB inhibitors that can be delivered in combination with fosfomycin in order to increase the efficacy of this antibiotic.

Europium-151 and iron-57 nuclear resonant vibrational spectroscopy of naturally abundant KEu(III)Fe(II)(CN)6 and Eu(III)Fe(III)(CN)6 complexes.

We have performed and analyzed the first combined 151Eu and 57Fe nuclear resonant vibrational spectroscopy (NRVS) for naturally abundant KEu(III)[Fe(II)(CN)6] and Eu(III)[Fe(III)(CN)6] complexes. Comparison of the observed 151Eu vs.57Fe NRVS spectroscopic features confirms that Eu(III) in both KEu(III)[Fe(II)(CN)6] and Eu(III)[Fe(III)(CN)6] occupies a position outside the [Fe(CN)6] core and coordinates to the N atoms of the CN- ions, whereas Fe(III) or Fe(II) occupies the site inside the [Fe(CN)6]4- core and coordinates to the C atoms of the CN- ions. In addition to the spectroscopic interest, the results from this study provide invaluable insights for the design and evaluation of the nanoparticles of such complexes as potential cellular contrast agents for their use in magnetic resonance imaging. The combined 151Eu and 57Fe NRVS measurements are also among the first few explorations of bi-isotopic NRVS experiments.

Light-responsive and Protic Ruthenium Compounds Bearing Bathophenanthroline and Dihydroxybipyridine Ligands Achieve Nanomolar Toxicity towards Breast Cancer Cells.

We report new ruthenium complexes bearing the lipophilic bathophenanthroline (BPhen) ligand and dihydroxybipyridine (dhbp) ligands which differ in the placement of the OH groups ([(BPhen)2 Ru(n,n'-dhbp)]Cl2 with n = 6 and 4 in 1A and 2A , respectively). Full characterization data are reported for 1A and 2A and single crystal X-ray diffraction for 1A . Both 1A and 2A are diprotic acids. We have studied 1A , 1B , 2A , and 2B (B = deprotonated forms) by UV-vis spectroscopy and 1 photodissociates, but 2 is light stable. Luminescence studies reveal that the basic forms have lower energy 3 MLCT states relative to the acidic forms. Complexes 1A and 2A produce singlet oxygen with quantum yields of 0.05 and 0.68, respectively, in acetonitrile. Complexes 1 and 2 are both photocytotoxic toward breast cancer cells, with complex 2 showing EC50 light values as low as 0.50 µM with PI values as high as >200 vs. MCF7. Computational studies were used to predict the energies of the 3 MLCT and 3 MC states. An inaccessible 3 MC state for 2B suggests a rationale for why photodissociation does not occur with the 4,4'-dhbp ligand. Low dark toxicity combined with an accessible 3 MLCT state for 1 O2 generation explains the excellent photocytotoxicity of 2.

Structural and functional characterization of fosfomycin resistance conferred by FosB from Enterococcus faecium.

The Gram-positive pathogen Enterococcus faecium is one of the leading causes of hospital-acquired vancomycin resistant enterococci (VRE) infections. E. faecium has extensive multidrug resistance and accounts for more than two million infections in the United States each year. FosB is a fosfomycin resistance enzyme found in Gram-positive pathogens like E. faecium. Typically, the FosB enzymes are Mn2+ -dependent bacillithiol (BSH) transferases that inactivate fosfomycin through nucleophilic addition of the thiol to the antibiotic. However, our kinetic analysis of FosBEf shows that the enzyme does not utilize BSH as a thiol substrate, unlike the other well characterized FosB enzymes. Here we report that FosBEf is a Mn2+ -dependent L-cys transferase. In addition, we have determined the three-dimensional X-ray crystal structure of FosBEf in complex with fosfomycin at a resolution of 2.0 Å. A sequence similarity network (SSN) was generated for the FosB family to investigate the unexpected substrate selectivity. Three non-conserved residues were identified in the SSN that may contribute to the substrate selectivity differences in the family of enzymes. Our structural and functional characterization of FosBEf establishes the enzyme as a potential target and may prove useful for future structure-based development of FosB inhibitors to increase the efficacy of fosfomycin.

X-ray structure of chromium(III)-containing transferrin: First structure of a physiological Cr(III)-binding protein.

Transferrin, the Fe(III) transport protein in mammalian blood, has been suggested to also serve as a Cr(III) transporter and as part of a Cr(III) detoxification system; however, the structure of the metal-binding sites has never been fully elucidated with bound Cr(III). Chromium(III)-transferrin was crystallized in the presence of the synergistic anion malonate. In the crystals, the protein exists with a closed C-terminal lobe containing a Cr(III) ion and an open, unoccupied N-terminal lobe. The overall structure and the metal ion environments are extremely similar to those of Fe(III)- and Ti(IV)-containing transferrin crystallized under comparable conditions. The octahedral coordination about the Cr(III) is comprised of four ligands provided by the protein (two tyrosine residues, a histidine residue, and an aspartate residue) and a chelating malonate anion. This represents the first crystal structure of a Cr(III)-containing protein that binds Cr(III) as part of its physiological function.

Characterization of the genomically encoded fosfomycin resistance enzyme from Mycobacterium abscessus.

Mycobacterium abscessus belongs to a group of rapidly growing mycobacteria (RGM) and accounts for approximately 65-80% of lung disease caused by RGM. It is highly pathogenic and is considered the prominent Mycobacterium involved in pulmonary infection in patients with cystic fibrosis and chronic pulmonary disease (CPD). FosM is a putative 134 amino acid fosfomycin resistance enzyme from M. abscessus subsp. bolletii that shares approximately 30-55% sequence identity with other vicinal oxygen chelate (VOC) fosfomycin resistance enzymes and represents the first of its type found in any Mycobacterium species. Genes encoding VOC fosfomycin resistance enzymes have been found in both Gram-positive and Gram-negative pathogens. Given that FosA enzymes from Gram-negative bacteria have evolved optimum activity towards glutathione (GSH) and FosB enzymes from Gram-positive bacteria have evolved optimum activity towards bacillithiol (BSH), it was originally suggested that FosM might represent a fourth class of enzyme that has evolved to utilize mycothiol (MSH). However, a sequence similarity network (SSN) analysis identifies FosM as a member of the FosX subfamily, indicating that it may utilize water as a substrate. Here we have synthesized MSH and characterized FosM with respect to divalent metal ion activation and nucleophile selectivity. Our results indicate that FosM is a Mn2+-dependent FosX-type hydrase with no selectivity toward MSH or other thiols as analyzed by NMR and mass spectroscopy.

A Mechanism of Calmodulin Modulation of the Human Cardiac Sodium Channel.

The function of the human cardiac sodium channel (NaV1.5) is modulated by the Ca2+ sensor calmodulin (CaM), but the underlying mechanism(s) are controversial and poorly defined. CaM has been reported to bind in a Ca2+-dependent manner to two sites in the intracellular loop that is critical for inactivation of NaV1.5 (inactivation gate [IG]). The affinity of CaM for the complete IG was significantly stronger than that of fragments that lacked both complete binding sites. Structural analysis by nuclear magnetic resonance, crystallographic, and scattering approaches revealed that CaM simultaneously engages both IG sites using an extended configuration. Patch-clamp recordings for wild-type and mutant channels with an impaired CaM-IG interaction revealed CaM binding to the IG promotes recovery from inactivation while impeding the kinetics of inactivation. Models of full-length NaV1.5 suggest that CaM binding to the IG directly modulates channel function by destabilizing the inactivated state, which would promote resetting of the IG after channels close.

Structural Dynamics of 15-Lipoxygenase-2 via Hydrogen-Deuterium Exchange.

Eicosanoids are inflammatory signaling lipids that are biosynthesized in response to cellular injury or threat. They were originally thought to be pro-inflammatory molecules, but members of at least one subclass, the lipoxins, are able to resolve inflammation. One step in lipoxin synthesis is the oxygenation of arachidonic acid by 15-lipoxygenase (15-LOX). 15-LOX contains two domains: a Ca2+ binding PLAT domain and a catalytic domain. 15-LOX is a soluble cytosolic protein until binding of Ca2+ to the PLAT domain promotes translocation to the membrane surface. The role of 15-LOX structural dynamics in this translocation has remained unclear. We investigated the dynamics of 15-LOX isoform B (15-LOX-2) upon binding of Ca2+ and ligands, as well as upon membrane association using hydrogen-deuterium exchange mass spectrometry (HDX-MS). We used HDX-MS to probe the solvent accessibility and backbone flexibility of 15-LOX-2, revealing significant differences in deuterium incorporation between the PLAT and catalytic domains, with the PLAT domain demonstrating higher flexibility. Comparison of HDX for 15-LOX-2 in the presence and absence of Ca2+ indicates there are few differences in structural dynamics. Furthermore, our HDX results involving nanodisc-associated 15-LOX-2 suggest that significant structural and dynamic changes in 15-LOX-2 are not required for membrane association. Our results also show that a substrate lipid binding to the active site in the catalytic domain does induce changes in incorporation of deuterium into the PLAT domain. Overall, our results challenge the previous hypothesis that Ca2+ binding induces major structural changes in the PLAT domain and support the hypothesis that is interdomain communication in 15-LOX-2.

Analysis of Functional Dynamics of Modular Multidomain Proteins by SAXS and NMR.

Multiprotein machines drive virtually all primary cellular processes. Modular multidomain proteins are widely distributed within these dynamic complexes because they provide the flexibility needed to remodel structure as well as rapidly assemble and disassemble components of the machinery. Understanding the functional dynamics of modular multidomain proteins is a major challenge confronting structural biology today because their structure is not fixed in time. Small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy have proven particularly useful for the analysis of the structural dynamics of modular multidomain proteins because they provide highly complementary information for characterizing the architectural landscape accessible to these proteins. SAXS provides a global snapshot of all architectural space sampled by a molecule in solution. Furthermore, SAXS is sensitive to conformational changes, organization and oligomeric states of protein assemblies, and the existence of flexibility between globular domains in multiprotein complexes. The power of NMR to characterize dynamics provides uniquely complementary information to the global snapshot of the architectural ensemble provided by SAXS because it can directly measure domain motion. In particular, NMR parameters can be used to define the diffusion of domains within modular multidomain proteins, connecting the amplitude of interdomain motion to the architectural ensemble derived from SAXS. Our laboratory has been studying the roles of modular multidomain proteins involved in human DNA replication using SAXS and NMR. Here, we present the procedure for acquiring and analyzing SAXS and NMR data, using DNA primase and replication protein A as examples.

Response to Comments on "The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport".

Baranovskiy et al and Pellegrini argue that, based on structural data, the path for charge transfer through the [4Fe4S] domain of primase is not feasible. Our manuscript presents electrochemical data directly showing charge transport through DNA to the [4Fe4S] cluster of a primase p58C construct and a reversible switch in the DNA-bound signal with oxidation/reduction, which is inhibited by mutation of three tyrosine residues. Although the dispositions of tyrosines differ in different constructs, all are within range for microsecond electron transfer.

The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport.

DNA charge transport chemistry offers a means of long-range, rapid redox signaling. We demonstrate that the [4Fe4S] cluster in human DNA primase can make use of this chemistry to coordinate the first steps of DNA synthesis. Using DNA electrochemistry, we found that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus, redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.

Structure and function of the genomically encoded fosfomycin resistance enzyme, FosB, from Staphylococcus aureus.

The Gram-positive pathogen Staphylococcus aureus is a leading cause of global morbidity and mortality. Like many multi-drug-resistant organisms, S. aureus contains antibiotic-modifying enzymes that facilitate resistance to a multitude of antimicrobial compounds. FosB is a Mn(2+)-dependent fosfomycin-inactivating enzyme found in S. aureus that catalyzes nucleophilic addition of either l-cysteine (l-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bactericidal properties. The three-dimensional X-ray crystal structure of FosB from S. aureus (FosB(Sa)) has been determined to a resolution of 1.15 Å. Cocrystallization of FosB(Sa) with either l-Cys or BSH results in a disulfide bond between the exogenous thiol and the active site Cys9 of the enzyme. An analysis of the structures suggests that a highly conserved loop region of the FosB enzymes must change conformation to bind fosfomycin. While two crystals of FosB(Sa) contain Zn(2+) in the active site, kinetic analyses of FosB(Sa) indicated that the enzyme is inhibited by Zn(2+) for l-Cys transferase activity and only marginally active for BSH transferase activity. Fosfomycin-treated disk diffusion assays involving S. aureus Newman and the USA300 JE2 methicillin-resistant S. aureus demonstrate a marked increase in the sensitivity of the organism to the antibiotic in either the BSH or FosB null strains, indicating that both are required for survival of the organism in the presence of the antibiotic. This work identifies FosB as a primary fosfomycin-modifying pathway of S. aureus and establishes the enzyme as a potential therapeutic target for increased efficacy of fosfomycin against the pathogen.

Structural and chemical aspects of resistance to the antibiotic fosfomycin conferred by FosB from Bacillus cereus.

The fosfomycin resistance enzymes, FosB, from Gram-positive organisms, are M(2+)-dependent thiol tranferases that catalyze nucleophilic addition of either L-cysteine (L-Cys) or bacillithiol (BSH) to the antibiotic, resulting in a modified compound with no bacteriacidal properties. Here we report the structural and functional characterization of FosB from Bacillus cereus (FosB(Bc)). The overall structure of FosB(Bc), at 1.27 Å resolution, reveals that the enzyme belongs to the vicinal oxygen chelate (VOC) superfamily. Crystal structures of FosB(Bc) cocrystallized with fosfomycin and a variety of divalent metals, including Ni(2+), Mn(2+), Co(2+), and Zn(2+), indicate that the antibiotic coordinates to the active site metal center in an orientation similar to that found in the structurally homologous manganese-dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosB(Bc) structures show a well-defined binding pocket and an access channel to C1 of fosfomycin, the carbon to which nucleophilic addition of the thiol occurs. The pocket and access channel are appropriate in size and shape to accommodate L-Cys or BSH. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel, positioning the compound for direct nucleophilic attack by the thiol substrate. In addition, the structures of FosB(Bc) in complex with the L-Cys-fosfomycin product (1.55 Å resolution) and in complex with the bacillithiol-fosfomycin product (1.77 Å resolution) coordinated to a Mn(2+) metal in the active site have been determined. The L-Cys moiety of either product is located in the solvent channel, where the thiol has added to the backside of fosfomycin C1 located at the end of the channel. Concomitant kinetic analyses of FosB(Bc) indicated that the enzyme has a preference for BSH over L-Cys when activated by Mn(2+) and is inhibited by Zn(2+). The fact that Zn(2+) is an inhibitor of FosB(Bc) was used to obtain a ternary complex structure of the enzyme with both fosfomycin and L-Cys bound.

Role of polarity of the distal pocket in the control of inhibitor binding in dehaloperoxidase-hemoglobin.

Dehaloperoxidase (DHP A), a unique multifunctional enzyme, from the marine annelid Amphitrite ornata dehalogenates 2,4,6-tribromophenol to form 2,6-dibromo-1,4-benzoquinone. The catalytic cycle of DHP is similar to that of horseradish peroxidase (HRP), involving a high-valent ferryl heme and two single-electron transfers from the aromatic substrate to the enzyme. Like HRP, DHP has been investigated as a potential bioremediation enzyme. However, DHP fails as a bioremediation enzyme because, unlike HRP, it has an internal binding cavity on the distal side of the heme capable of accommodating p-bromophenols, which act as an inhibitor of peroxidase function. Blocking internal binding in DHP may be the key to allowing the enzyme to function effectively as a peroxidase on the full range of halogenated phenols. The distal cavity of DHP is surrounded by several hydrophobic amino acids that stabilize internal binding of the monohalogenated phenols, including a leucine residue near the back edge of the heme (L100). We have expressed the L100F, L100Q, L100T, and L100V mutants of DHP in an effort to prevent internal binding and thereby convert the inhibitors into substrates. Kinetic assays and resonance Raman indicate that the peroxidase activity of the L100T and L100F mutants is increased compared to that of native DHP in the presence of 4-bromophenol (4-BP), suggesting a reduction in the inhibitor binding constant. In addition, the X-ray crystal structure of L100F clearly indicates a reduced occupancy of the 4-BP inhibitor in the distal cavity of DHP. However, at the same time, the L100F structure reveals that steric interference alone is insufficient to exclude the inhibitor. Instead, the comparison of L100T and isosteric L100V reveals that an increase in polarity plays a decisive role in excluding the inhibitor from the distal binding pocket.

Catalytic efficiency of dehaloperoxidase A is controlled by electrostatics--application of the vibrational Stark effect to understand enzyme kinetics.

The vibrational Stark effect is gaining popularity as a method for probing electric fields in proteins. In this work, we employ it to explain the effect of single charge mutations in dehaloperoxidase-hemoglobin A (DHP A) on the kinetics of the enzyme. In a previous communication published in this journal (BBRC 2012, 420, 733-737) it has been shown that an increase in the overall negative charge of DHP A through mutation causes a decrease in its catalytic efficiency. Here, by labeling the protein with 4-mercaptobenzonitrile (MBN), a Stark probe molecule, we provide further evidence that the diffusion control of the catalytic process arises from the electrostatic repulsion between the enzyme and the negatively charged substrate. The linear correlation observed between the nitrile stretching frequency of the protein-bound MBN and the catalytic efficiency of the single-site mutants of the enzyme indicates that electrostatic interactions play a dominant role in determining the catalytic efficiency of DHP A.

The dehaloperoxidase paradox.

The dual functions of the dehaloperoxidase-hemoglobin of Amphitrite ornata leads to a paradox. Peroxidase and hemoglobin functions require ferric and ferrous resting states, respectively. Assuming that hemoglobin function is the dominant function, the starting point for peroxidase activation would be the oxyferrous state. Activation of that state leads to the ferryl intermediate, followed by one-electron oxidation of the substrate, which results in the ferric state. Since no exogenous reductant is known, there is no return to the ferrous form or hemoglobin function. The observation that an internal binding site for 4-bromophenol leads to inhibition leads to a further paradox that the enzyme would be inhibited immediately upon activation under ambient conditions in benthic ecosystems where the inhibitor, 4-bromophenol is present in greater concentration than the substrate, 2,4,6-tribromophenol. In this review, we explore the unresolved aspects of the reaction scheme that leads to the apparent paradox. Recent data showing activation of the oxyferrous state, an extremely high reduction potential and exogenous reduction by the 2,6-dibromoquinone product present a potential resolution of the paradox. These aspects are discussed in the context of control of reactivity radical pathways and reactivity by the motion of the distal histidine, H55, which in turn is coupled to the binding of substrate and inhibitor.

Functional consequences of the creation of an Asp-His-Fe triad in a 3/3 globin.

The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine 86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D [Protein Data Bank (PDB) entry 3MYN ] and M86E (PDB entry 3MYM ) mutants reveal the structural origins of a stable catalytic triad in DHP A. A decrease in the rate of H(2)O(2) activation as well as a lowered reduction potential versus that of the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine in binding to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is five-coordinate, and this structure resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a six-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a six-coordinate low-spin adduct, which inhibits the ligation and activation of H(2)O(2) as required for peroxidase activity. The results suggest that the proximal charge relay in peroxidases regulate the redox potential of the heme Fe but that the trans effect is a carefully balanced property that can both activate H(2)O(2) and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, we studied three M86 mutants, M86A, M86D, and M86E, by spectroelectrochemistry and nuclear magnetic resonance spectroscopy of (13)C- and (15)N-labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the N(δ) hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.

Dehaloperoxidase-hemoglobin from Amphitrite ornata is primarily a monomer in solution.

The crystal structures of the dehaloperoxidase-hemoglobin from A. ornata (DHP A) each report a crystallographic dimer in the unit cell. Yet, the largest dimer interface observed is 450 Å(2), an area significantly smaller than the typical value of 1200-2000 Å(2) and in contrast to the extensive interface region of other known dimeric hemoglobins. To examine the oligomerization state of DHP A in solution, we used gel permeation by fast protein liquid chromatography and small-angle X-ray scattering (SAXS). Gel permeation experiments demonstrate that DHP A elutes as a monomer (15.5 kDa) and can be separated from green fluorescent protein, which has a molar mass of 27 kDa, near the 31 kDa expected for the DHP A dimer. By SAXS, we found that DHP A is primarily monomeric in solution, but with a detectable level of dimer (~10%), under all conditions studied up to a protein concentration of 3.0 mM. These concentrations are likely 10-100-fold lower than the K(d) for dimer formation. Additionally, there was no significant effect either on the overall conformation of DHP A or its monomer-dimer equilibrium upon addition of the DHP A inhibitor, 4-iodophenol.