Molecular Insights of an Avian Species with Low Oxygen Affinity, the Crystal Structure of Duck T-State Methemoglobin.

Hemoglobin (Hb) is the key metalloprotein within red blood cells involved in oxygen transportation from lungs to body cells. The heme-iron atom inherent within Hb effectuates the mechanism of oxygen transportation and carbon dioxide removal. Structural investigations on avian Hb are limited when compared with the enormous work has been carried out on mammalian Hb. Here, the crystal structure of T-state methemoglobin (T-metHb) from domestic duck (Anas platyrhynchos), a low oxygen affinity avian species, determined to 2.1Å resolution is presented. Duck T-metHb crystallized in the orthorhombic space group C2221 with unit cell parameters a = 59.89, b = 109.42 and c = 92.07Å. The final refined model with R-factor: 19.5% and Rfree: 25.2% was obtained. The structural analysis reveals that duck T-metHb adopts a unique quaternary structure that is distinct from any of the avian liganded Hb structures. Moreover, it closely resembles the deoxy Hb of bar-headed goose, a high oxygen-affinity species. Besides the amino acid αPro119 located in the α1ß1 interface, a unique quaternary structure with a constrained heme environment is attributed for the intrinsic low oxygen-affinity of duck Hb. This study reports the first protein crystal structure of low oxygen-affinity avian T-metHb from Anas platyrhynchos.

Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening.

Target-based discovery of first-in-class therapeutics demands an in-depth understanding of the molecular mechanisms underlying human diseases. Precise measurements of cellular and biochemical activities are critical to gain mechanistic knowledge of biomolecules and their altered function in disease conditions. Such measurements enable the development of intervention strategies for preventing or treating diseases by modulation of desired molecular processes. Fluorescence-based techniques are routinely employed for accurate and robust measurements of in-vitro activity of molecular targets and for discovering novel chemical molecules that modulate the activity of molecular targets. In the current review, the authors focus on the applications of fluorescence-based high throughput screening (HTS) and fragment-based ligand discovery (FBLD) techniques such as fluorescence polarization (FP), Förster resonance energy transfer (FRET), fluorescence thermal shift assay (FTSA) and microscale thermophoresis (MST) for the discovery of chemical probe to exploring target's role in disease biology and ultimately, serve as a foundation for drug discovery. Some recent advancements in these techniques for compound library screening against important classes of drug targets, such as G-protein-coupled receptors (GPCRs) and GTPases, as well as phosphorylation- and acetylation-mediated protein-protein interactions, are discussed. Overall, this review presents a landscape of how these techniques paved the way for the discovery of small-molecule modulators and biologics against these targets for therapeutic benefits.

High-affinity binding of celastrol to monomeric α-synuclein mitigates in vitro aggregation.

α-Synuclein (αSyn) aggregation is associated with Parkinson's disease (PD). The region αSyn36-42 acts as the nucleation 'master controller' and αSyn1-12 as a 'secondary nucleation site'. They drive monomeric αSyn to aggregation. Small molecules targeting these motifs are promising for disease-modifying therapy. Using computational techniques, we screened thirty phytochemicals for αSyn binding. The top three compounds were experimentally validated for their binding affinity. Amongst them, celastrol showed high binding affinity. NMR analysis confirmed stable αSyn-celastrol interactions involving several residues in the N-terminus and NAC regions but not in the C-terminal tail. Importantly, celastrol interacted extensively with the key motifs that drive αSyn aggregation. Thioflavin-T assay indicated that celastrol reduced αSyn aggregation. Thus, celastrol holds promise as a potent drug candidate for PD.Communicated by Ramaswamy H. Sarma.

Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum.

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties.

Structure of a 14-3-3ε:FOXO3apS253 Phosphopeptide Complex Reveals 14-3-3 Isoform-Specific Binding of Forkhead Box Class O Transcription Factor (FOXO) Phosphoproteins.

The transcriptional activity of Forkhead Box O3 (FOXO3a) is inactivated by AKT-mediated phosphorylation on Serine 253 (S253), which enables FOXO3a binding to 14-3-3. Phosphorylated FOXO3a binding to 14-3-3 facilitates the nuclear exclusion of FOXO3a, causing cancer cell proliferation. The FOXO3a/14-3-3 interaction has, therefore, emerged as an important therapeutic target. Here, we report a comprehensive analysis using fluorescence polarization, isothermal titration calorimetry, small-angle X-ray scattering, X-ray crystallography, and molecular dynamics simulations to gain molecular-level insights into the interaction of FOXO3apS253 phosphopeptide with 14-3-3ε. A high-resolution structure of the fluorophore-labeled FOXO3apS253:14-3-3ε complex revealed a distinct mode of interaction compared to other 14-3-3 phosphopeptide complexes. FOXO3apS253 phosphopeptide showed significant structural difference in the positions of the -3 and -4 Arg residues relative to pSer, compared to that of a similar phosphopeptide, FOXO1pS256 bound to 14-3-3σ. Moreover, molecular dynamics studies show that the significant structural changes and molecular interactions noticed in the crystal structure of FOXO3apS253:14-3-3ε are preserved over the course of the simulation. Thus, this study reveals structural differences between the binding to 14-3-3 isoforms of FOXO1pS256 versus FOXO3apS253, providing a framework for the rational design of isoform-specific FOXO/14-3-3 protein-protein interaction inhibitors for therapy.

Molecular insights into α-synuclein interaction with individual human core histones, linker histone, and dsDNA.

α-Synuclein (αS) plays a key role in Parkinson's disease (PD). The αS nuclear role, its binding affinity and specificity to histones and dsDNA remains unknown. Here, we have measured the binding affinity ( Kd ) between αS wild-type (wt) and PD-specific αS S129-phosphorylation mimicking (S129E) mutant with full-length and flexible tail truncated individual core histones (H2a, H2b, H3, and H4), linker histone (H1), and carried out αS-dsDNA interaction studies. This study revealed that αS(wt) interacts specifically with N-terminal flexible tails of histone H3, H4, and flexible tails of H1. The αS(S129E) mutant recognizes histones similar to αS(wt) but binds with higher affinity. Intriguingly, αS(S129E) showed a binding affinity for control proteins (bovine serum albumin and lysozyme), while no interaction was seen for αS(wt). Based on our above observation, we contemplate that the physio-chemical properties of αS with S129-phosphorylation has changed compared to αS(wt), resulting in interaction for other proteins, which is the basis for Lewy body formation. Besides, this study showed αS binding to dsDNA is weak and nonspecific. Overall, αS specificity for histone binding suggests that its nuclear role is possibly driven through histone interaction.

Target identification for small-molecule discovery in the FOXO3a tumor-suppressor pathway using a biodiverse peptide library.

Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.

Residues of helix ɑ2 are critical for catalytic efficiency of mycobacterial alkylhydroperoxide reductase subunit C.

The ability of Mycobacteria to overcome oxidative stress is of paramount importance for its survival within the host. One of the key enzymes that are involved in protecting the bacterium from reactive oxygen species is the catalase-peroxidase (KatG). However, in strains resistant to the antibiotic isoniazid, KatG is rendered ineffective, which is associated with an increased expression of alkylhydroperoxide reductase subunit C (AhpC). Mycobacterial AhpC possesses a unique helical displacement when compared to its bacterial counterparts. Here, via mutagenesis studies, we demonstrate the importance of this helix for redox modulation of the catalytic activity of AhpC. Along with structural insights from crystallographic data, the impact of critical residues on the structure and flexibility of the helix and on AhpC oligomerization is described.

3D reconstruction and flexibility of the hybrid engine Acetobacterium woodii F-ATP synthase.

The Na+-translocating F1FO ATP synthase from Acetobacterium woodii (AwF-ATP synthase) with a subunit stoichiometry of α3:ß3:γ:δ:ε:a:b2:(c2/3)9:c1 represents an evolutionary path between ATP-synthases and vacuolar ATPases, by containing a heteromeric rotor c-ring, composed of subunits c1, c2 and c3, and an extra loop (γ195-211) within the rotary γ subunit. Here, the recombinant AwF-ATP synthase was subjected to negative stain electron microscopy and single particle analysis. The reference free 2D class averages revealed high flexibility of the enzyme, wherein the F1 and FO domains distinctively bended to adopt multiple conformations. Moreover, both the F1 and FO domains tilted relative to each other to a maximum extent of 28° and 30°, respectively. The first 3D reconstruction of the AwF-ATP synthase was determined which accommodates well the modelled structure of the AwF-ATP synthase as well as the γ195-211-loop. Molecular simulations of the enzyme underlined the bending features and flexibility observed in the electron micrographs, and enabled assessment of the dynamics of the extra γ195-211-loop.

Unique structural and mechanistic properties of mycobacterial F-ATP synthases: Implications for drug design.

The causative agent of Tuberculosis (TB) Mycobacterium tuberculosis (Mtb) encounters unfavourable environmental conditions in the lungs, including nutrient limitation, low oxygen tensions and/or low/high pH values. These harsh conditions in the host triggers Mtb to enter a dormant state in which the pathogen does not replicate and uses host-derived fatty acids instead of carbohydrates as an energy source. Independent to the energy source, the bacterium's energy currency ATP is generated by oxidative phosphorylation, in which the F1FO-ATP synthase uses the proton motive force generated by the electron transport chain. This catalyst is essential in Mtb and inhibition by the diarylquinoline class of drugs like Bedaquilline, TBAJ-587, TBAJ-876 or squaramides demonstrated that this engine is an attractive target in TB drug discovery. A special feature of the mycobacterial F-ATP synthase is its inability to establish a significant proton gradient during ATP hydrolysis, and its latent ATPase activity, to prevent energy waste and to control the membrane potential. Recently, unique epitopes of mycobacterial F1FO-ATP synthase subunits absent in their prokaryotic or mitochondrial counterparts have been identified to contribute to the regulation of the low ATPase activity. Most recent structural insights into individual subunits, the F1 domain or the entire mycobacterial enzyme added to the understanding of mechanisms, regulation and differences of the mycobacterial F1FO-ATP synthase compared to other bacterial and eukaryotic engines. These novel insights provide the basis for the design of new compounds targeting this engine and even novel regimens for multidrug resistant TB.

Structure and subunit arrangement of Mycobacterial F1FO ATP synthase and novel features of the unique mycobacterial subunit δ.

In contrast to other prokaryotes, the Mycobacterial F1FO ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) is essential for growth. The mycobacterial enzyme is also unique as a result of its 111 amino acids extended δ subunit, whose gene is fused to the peripheral stalk subunit b. Recently, the crystallographic structures of the mycobacterial α3:ß3:γ:ε-domain and c subunit ring were resolved. Here, we report the first purification protocol of the intact M. smegmatis F1FO ATP synthase including the F1-domain, the entire membrane-embedded FO sector, and the stator subunits b' and the fused b-δ. This enzyme purification enabled the determination of the first projected 2D- and 3D structure of the intact M. smegmatis F1FO ATP synthase by electron microscopy (EM) and single particle analysis. Expression and purification of the fused mycobacterial b-δ24-446 construct, excluding the membrane-embedded N-terminal amino acids, provided insight into its secondary structure. By combining these data with homology and ab-initio modeling techniques, a model of the mycobacterial peripheral stalk subunits b-δ and b' was generated. Superposition of the 3D M. smegmatis F-ATP synthase EM-structure, the α3:ß3:γ:ε and c-ring, and the derived structural models of the peripheral stalk enabled a clear assignment of all F-ATP synthase subunits, in particular with respect to the unique mycobacterial peripheral stalk subunit b' and the elongated δ fused with subunit b. The arrangement of δ relative to the N-termini of the catalytic α3ß3-headpiece and its potential as a drug target are discussed.

Effect of the additional cysteine 503 of vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F (AhpF) and the mechanism of AhpF and subunit C assembling.

The vancomycin-resistant Enterococcus faecalis alkyl hydroperoxide reductase complex (AhpR) with its subunits AhpC (EfAhpC) and AhpF (EfAhpF) is of paramount importance to restore redox homeostasis. Therefore, knowledge about this defense system is essential to understand its antibiotic-resistance and survival in hosts. Recently, we described the crystallographic structures of EfAhpC, the two-fold thioredoxin-like domain of EfAhpF, the novel phenomenon of swapping of the catalytic domains of EfAhpF as well as the unique linker length, connecting the catalytically active N-and C-terminal domains of EfAhpF. Here, using mutagenesis and enzymatic studies, we reveal the effect of an additional third cysteine (C503) in EfAhpF, which might optimize the functional adaptation of the E. faecalis enzyme under various physiological conditions. The crystal structure and solution NMR data of the engineered C503A mutant of the thioredoxin-like domain of EfAhpF were used to describe alterations in the environment of the additional cysteine residue during modulation of the redox-state. To glean insight into the epitope and mechanism of EfAhpF and -AhpC interaction as well as the electron transfer from the thioredoxin-like domain of EfAhpF to AhpC, NMR-titration experiments were performed, showing a coordinated disappearance of peaks in the thioredoxin-like domain of EfAhpF in the presence of full length EfAhpC, and indicating a stable EfAhpF-AhpC-complex. Combined with docking studies, the interacting residues of EfAhpF were identified and a mechanism of electron transfer of the EfAhpF donor to the electron acceptor EfAhpC is described.

Structure and function of Mycobacterium-specific components of F-ATP synthase subunits α and ε.

The Mycobacterium tuberculosis (Mtb) F1FO-ATP synthase (α3:ß3:γ:δ:ε:a:b:b':c9) is an essential enzyme that supplies energy for both the aerobic growing and the hypoxic dormant stage of the mycobacterial life cycle. Employing the heterologous F-ATP synthase model system αchi3:ß3:γ we showed previously, that transfer of the C-terminal domain (CTD) of Mtb subunit α (Mtα514-549) to a standard F-ATP synthase α subunit suppresses ATPase activity. Here we determined the 3D reconstruction from electron micrographs of the αchi3:ß3:γ complex reconstituted with the Mtb subunit ε (Mtε), which has been shown to crosstalk with the CTD of Mtα. Together with the first solution shape of Mtb subunit α (Mtα), derived from solution X-ray scattering, the structural data visualize the extended C-terminal stretch of the mycobacterial subunit α. In addition, Mtε mutants MtεR62L, MtεE87A, Mtε6-121, and Mtε1-120, reconstituted with αchi3:ß3:γ provided insight into their role in coupling and in trapping inhibiting MgADP. NMR solution studies of MtεE87A gave insights into how this residue contributes to stability and crosstalk between the N-terminal domain (NTD) and the CTD of Mtε. Analyses of the N-terminal mutant Mtε6-121 highlight the differences of the NTD of mycobacterial subunit ε to the well described Geobacillus stearothermophilus or Escherichia coli counterparts. These data are discussed in context of a crosstalk between the very N-terminal amino acids of Mtε and the loop region of one c subunit of the c-ring turbine for coupling of proton-translocation and ATP synthesis activity.

Molecular mechanism of the Escherichia coli AhpC in the function of a chaperone under heat-shock conditions.

Peroxiredoxins (Prxs) are ubiquitous antioxidants utilizing a reactive cysteine for peroxide reduction and acting as a molecular chaperone under various stress conditions. Besides other stimulating factors, oxidative- and heat stress conditions trigger their ATP-independent chaperoning function. So far, many studies were intended to reveal the chaperoning mechanisms of the so-called sensitive Prxs of eukaryotes, which are susceptible to inactivation by over-oxidation of its reactive cysteine during H2O2 reduction. In contrast, the chaperone mechanisms of bacterial Prxs, which are mostly robust against inactivation by over-oxidation, are not well understood. Herein, comprehensive biochemical and biophysical studies demonstrate that the Escherichia coli alkyl hydroperoxide reductase subunit C (EcAhpC) acquires chaperone activity under heat stress. Interestingly, their chaperoning activity is independent of its redox-states but is regulated in a temperature-dependent manner. Data are presented, showing that oxidized EcAhpC, which forms dimers at 25 °C, self-assembled into high molecular weight (HMW) oligomers at higher temperatures and supressed aggregation of client proteins at heat-shock conditions. In addition, we unravelled the essential role of the C-terminal tail of EcAhpC on heat-induced HMW oligomer formation and chaperoning activity. Our findings suggest a novel molecular mechanism for bacterial Prxs to function as chaperone at heat-shock conditions.

Active site CP-loop dynamics modulate substrate binding, catalysis, oligomerization, stability, over-oxidation and recycling of 2-Cys Peroxiredoxins.

Peroxiredoxins (Prxs) catalyse the rapid reduction of hydrogen peroxide, organic hydroperoxide and peroxynitrite, using a fully conserved peroxidatic cysteine (CP) located in a conserved sequence Pxxx(T/S)xxCP motif known as CP-loop. In addition, Prxs are involved in cellular signaling pathways and regulate several redox-dependent process related disease. The effective catalysis of Prxs is associated with alterations in the CP-loop between reduced, Fully Folded (FF), and oxidized, Locally Unfolded (LU) conformations, which are linked to dramatic changes in the oligomeric structure. Despite many studies, little is known about the precise structural and dynamic roles of the CP-loop on Prxs functions. Herein, the comprehensive biochemical and biophysical studies on Escherichia coli alkyl hydroperoxide reductase subunit C (EcAhpC) and the CP-loop mutants, EcAhpC-F45A and EcAhpC-F45P reveal that the reduced form of the CP-loop adopts conformational dynamics, which is essential for effective peroxide reduction. Furthermore, the point mutants alter the structure and dynamics of the reduced form of the CP-loop and, thereby, affect substrate binding, catalysis, oligomerization, stability and overoxidiation. In the oxidized form, due to restricted CP-loop dynamics, the EcAhpC-F45P mutant favours a decamer formation, which enhances the effective recycling by physiological reductases compared to wild-type EcAhpC. In addition, the study reveals that residue F45 increases the specificity of Prxs-reductase interactions. Based on these studies, we propose an evolution of the CP-loop with confined sequence conservation within Prxs subfamilies that might optimize the functional adaptation of Prxs into various physiological roles.

Essential role of the flexible linker on the conformational equilibrium of bacterial peroxiredoxin reductase for effective regeneration of peroxiredoxin.

Reactive oxygen species (ROS) can damage DNA, proteins, and lipids, so cells have antioxidant systems that regulate ROS. In many bacteria, a dedicated peroxiredoxin reductase, alkyl hydroperoxide reductase subunit F (AhpF), catalyzes the rapid reduction of the redox-active disulfide center of the antioxidant protein peroxiredoxin (AhpC) to detoxify ROS such as hydrogen peroxide, organic hydroperoxide, and peroxynitrite. AhpF is a flexible multidomain protein that enables a series of electron transfers among the redox centers by accepting reducing equivalents from NADH. A flexible linker connecting the N-terminal domain (NTD) and C-terminal domain (CTD) of AhpF suggests that the enzyme adopts a large-scale domain motion that alternates between the closed and open states to shuttle electrons from the CTD via the NTD to AhpC. Here, we conducted comprehensive mutational, biochemical, and biophysical analyses to gain insights into the role of the flexible linker and the residues critical for the domain motions of Escherichia coli AhpF (EcAhpF) during electron transfer. Small-angle X-ray scattering studies of linker mutants revealed that a group of charged residues, 200EKR202, is crucial for the swiveling motion of the NTD. Moreover, NADH binding significantly affected EcAhpF flexibility and the movement of the NTD relative to the CTD. The mutants also exhibited a decrease in H2O2 reduction by the AhpF-AhpC ensemble. We propose that a concerted movement involving the NTD, C-terminal NADH, and FAD domains, and the flexible linker between them is essential for optimal intra-domain cross-talk and for efficient electron transfer to the redox partner AhpC required for peroxidation.

Transition steps in peroxide reduction and a molecular switch for peroxide robustness of prokaryotic peroxiredoxins.

In addition to their antioxidant function, the eukaryotic peroxiredoxins (Prxs) facilitate peroxide-mediated signaling by undergoing controlled inactivation by peroxide-driven over-oxidation. In general, the bacterial enzyme lacks this controlled inactivation mechanism, making it more resistant to high H2O2 concentrations. During peroxide reduction, the active site alternates between reduced, fully folded (FF), and oxidized, locally unfolded (LU) conformations. Here we present novel insights into the divergence of bacterial and human Prxs in robustness and sensitivity to inactivation, respectively. Structural details provide new insights into sub-steps during the catalysis of peroxide reduction, enabling the transition from an FF to a LU conformation. Complementary to mutational and enzymatic results, these data unravel the essential role of the C-terminal tail of bacterial Prxs to act as a molecular switch, mediating the transition from an FF to a LU state. In addition, we propose that the C-terminal tail has influence on the propensity of the disulphide bond formation, indicating that as a consequence on the robustness and sensitivity to over-oxidation. Finally, a physical linkage between the catalytic site, the C-terminal tail and the oligomer interface is described.

Low resolution solution structure of an enzymatic active AhpC10:AhpF2 ensemble of the Escherichia coli Alkyl hydroperoxide Reductase.

The ability of bacteria to combat oxidative stress is imperative for their survival. The Alkyl hydroperoxide Reductase (AhpR) system, composed of the AhpC and AhpF proteins, is one of the dominant antioxidant defense systems required for scavenging hydrogen peroxide and organic peroxide. Therefore, it is necessary to understand the mechanism of the AhpR ensemble formation. In previous studies, we were able to elucidate conformational flexibility of Escherichia coli AhpF during the catalytic cycle and its binding site, the N-terminal domain (NTD), to AhpC. We proposed the novel binding and release mechanism of EcAhpC-AhpF, which is mediated by the well defined redox-state linked conformational changes associated with the C-terminal tail and active site regions of EcAhpC. Here, we have proceeded further to elucidate the solution structure of E. coli AhpC and the stable ensemble formation with EcAhpF using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. The EcAhpC-AhpF complex structure with a stoichiometry of AhpC10:AhpF2 reveals that dimeric EcAhpF in its extended conformation enables the NTD disulphide centers to come in close proximity to the redox-active disulphide centers of EcAhpC, and provides an efficient electron transfer. Furthermore, the significance of the C-terminal tail of EcAhpC in ensemble formation is elucidated. SAXS data-based modeling revealed the flexible C-terminal tail of EcAhpC in solution, and its exposed nature, making it possible to contact the NTD of EcAhpF for stable complex formation.

NMR studies reveal a novel grab and release mechanism for efficient catalysis of the bacterial 2-Cys peroxiredoxin machinery.

In bacteria, an ensemble of alkyl hydroperoxide reductase subunits C (AhpC) and F (AhpF) is responsible for scavenging H2O2. AhpC donates electrons for the reduction of H2O2, which are provided after NADH oxidation by AhpF. The latter contains an N-terminal domain (NTD), catalyzing the electron transfer from NADH via a FAD of the C-terminal domain (CTD) into AhpC. The NADH-bound Escherichia coli AhpF structure revealed that NADH binding brings the substrate to the re-face of the FAD, making the Cys-Cys center of the CTD accessible to the NTD disulfide center for electron transfer (Kamariah et al. (2015) Biochim Biophys Acta 1847, 1139-1152). So far insight into the epitope and mechanism of AhpF and AhpC interaction as well as the electron transfer from the NTD to AhpC have been lacking. Here using NMR spectroscopy, we glean insight into the interaction of the NTD of AhpF with AhpC from E. coli. A coordinated disappearance of EcAhpF NTD peaks was observed in the presence of full length EcAhpC, indicating a long-lived AhpC-AhpF complex. C-terminal truncated EcAhpC resulted in a more dynamic interaction, revealing specific residue chemical shift perturbation and hence the binding epitope of the complex. Combined with docking studies, we have suggested that the C terminus of AhpC binds to the backside groove of the NTD. In addition, AhpC-AhpF formation is abolished under reducing conditions. We propose for the first time a binding mechanism in which the C terminus of AhpC wraps around the NTD, slowing the dissociation rate for an efficient electron transfer process, and a release mechanism mediated by the conformational change of the C terminus of AhpC upon reduction.

Crystallographic and solution studies of NAD(+)- and NADH-bound alkylhydroperoxide reductase subunit F (AhpF) from Escherichia coli provide insight into sequential enzymatic steps.

Redox homeostasis is significant for the survival of pro- and eukaryotic cells and is crucial for defense against reactive oxygen species like superoxide and hydrogen peroxide. In Escherichia coli, the reduction of peroxides occurs via the redox active disulfide center of the alkyl hydroperoxide reductase C subunit (AhpC), whose reduced state becomes restored by AhpF. The 57kDa EcAhpF contains an N-terminal domain (NTD), which catalyzes the electron transfer from NADH via an FAD of the C-terminal domain into EcAhpC. The NTD is connected to the C-terminal domain via a linker. Here, the first crystal structure of E. coli AhpF bound with NADH and NAD(+) has been determined at 2.5Å and 2.4Å resolution, respectively. The NADH-bound form of EcAhpF reveals that the NADH-binding domain is required to alter its conformation to bring a bound NADH to the re-face of the isoalloxazine ring of the flavin, and thereby render the NADH-domain dithiol center accessible to the NTD disulfide center for electron transfer. The NAD(+)-bound form of EcAhpF shows conformational differences for the nicotinamide end moieties and its interacting residue M467, which is proposed to represent an intermediate product-release conformation. In addition, the structural alterations in EcAhpF due to NADH- and NAD(+)-binding in solution are shown by small angle X-ray scattering studies. The EcAhpF is revealed to adopt many intermediate conformations in solution to facilitate the electron transfer from the substrate NADH to the C-terminal domain, and subsequently to the NTD of EcAhpF for the final step of AhpC reduction.