Amphiphilic Α-helices Research Articles

Scorpion Venom Antimicrobial Peptide Derivative BmKn2-T5 Inhibits Enterovirus 71 in the Early Stages of the Viral Life Cycle In Vitro.

Enterovirus 71 (EV71), a typical representative of unenveloped RNA viruses, is the main pathogenic factor responsible for hand, foot, and mouth disease (HFMD) in infants. This disease seriously threatens the health and lives of humans worldwide, especially in the Asia-Pacific region. Numerous animal antimicrobial peptides have been found with protective functions against viruses, bacteria, fungi, parasites, and other pathogens, but there are few studies on the use of scorpion-derived antimicrobial peptides against unenveloped viruses. Here, we investigated the antiviral activities of scorpion venom antimicrobial peptide BmKn2 and five derivatives, finding that BmKn2 and its derivative BmKn2-T5 exhibit a significant inhibitory effect on EV71. Although both peptides exhibit characteristics typical of amphiphilic α-helices in terms of their secondary structure, BmKn2-T5 displayed lower cellular cytotoxicity than BmKn2. BmKn2-T5 was further found to inhibit EV71 in a dose-dependent manner in vitro. Moreover, time-of-drug-addition experiments showed that BmKn2-T5 mainly restricts EV71, but not its virion or replication, at the early stages of the viral cycle. Interestingly, BmKn2-T5 was also found to suppress the replication of the enveloped viruses DENV, ZIKV, and HSV-1 in the early stages of the viral cycle, which suggests they may share a common early infection step with EV71. Together, the results of our study identified that the scorpion-derived antimicrobial peptide BmKn2-T5 showed valuable antiviral properties against EV71 in vitro, but also against other enveloped viruses, making it a potential new candidate therapeutic molecule.

Machine Learning Guided Discovery of Non-Hemolytic Membrane Disruptive Anticancer Peptides.

Most antimicrobial peptides (AMPs) and anticancer peptides (ACPs) fold into membrane disruptive cationic amphiphilic α‐helices, many of which are however also unpredictably hemolytic and toxic. Here we exploited the ability of recurrent neural networks (RNN) to distinguish active from inactive and non‐hemolytic from hemolytic AMPs and ACPs to discover new non‐hemolytic ACPs. Our discovery pipeline involved: 1) sequence generation using either a generative RNN or a genetic algorithm, 2) RNN classification for activity and hemolysis, 3) selection for sequence novelty, helicity and amphiphilicity, and 4) synthesis and testing. Experimental evaluation of thirty‐three peptides resulted in eleven active ACPs, four of which were non‐hemolytic, with properties resembling those of the natural ACP lasioglossin III. These experiments show the first example of direct machine learning guided discovery of non‐hemolytic ACPs.

Structure and dynamics of the SARS-CoV-2 envelope protein monomer.

Coronaviruses, especially severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), present an ongoing threat to human wellbeing. Consequently, elucidation of molecular determinants of their function and interaction with the host is an important task. Whereas some of the coronaviral proteins are extensively characterized, others remain understudied. Here, we use molecular dynamics simulations to analyze the structure and dynamics of the SARS-CoV-2 envelope (E) protein (a viroporin) in the monomeric form. The protein consists of the hydrophobic α-helical transmembrane domain (TMD) and amphiphilic α-helices H2 and H3, connected by flexible linkers. We show that TMD has a preferable orientation in the membrane, while H2 and H3 reside at the membrane surface. Orientation of H2 is strongly influenced by palmitoylation of cysteines Cys40, Cys43, and Cys44. Glycosylation of Asn66 affects the orientation of H3. We also observe that the monomeric E protein both generates and senses the membrane curvature, preferably localizing with the C-terminus at the convex regions of the membrane; the protein in the pentameric form displays these properties as well. Localization to curved regions may be favorable for assembly of the E protein oligomers, whereas induction of curvature may facilitate the budding of the viral particles. The presented results may be helpful for a better understanding of the function of the coronaviral E protein and viroporins in general, and for overcoming the ongoing SARS-CoV-2 pandemic.

Membrane Interactions of Latarcins: Antimicrobial Peptides from Spider Venom.

A group of seven peptides from spider venom with diverse sequences constitute the latarcin family. They have been described as membrane-active antibiotics, but their lipid interactions have not yet been addressed. Using circular dichroism and solid-state 15N-NMR, we systematically characterized and compared the conformation and helix alignment of all seven peptides in their membrane-bound state. These structural results could be correlated with activity assays (antimicrobial, hemolysis, fluorescence vesicle leakage). Functional synergy was not observed amongst any of the latarcins. In the presence of lipids, all peptides fold into amphiphilic α-helices as expected, the helices being either surface-bound or tilted in the bilayer. The most tilted peptide, Ltc2a, possesses a novel kind of amphiphilic profile with a coiled-coil-like hydrophobic strip and is the most aggressive of all. It indiscriminately permeabilizes natural membranes (antimicrobial, hemolysis) as well as artificial lipid bilayers through the segregation of anionic lipids and possibly enhanced motional averaging. Ltc1, Ltc3a, Ltc4a, and Ltc5a are efficient and selective in killing bacteria but without causing significant bilayer disturbance. They act rather slowly or may even translocate towards intracellular targets, suggesting more subtle lipid interactions. Ltc6a and Ltc7, finally, do not show much antimicrobial action but can nonetheless perturb model bilayers.

Characterisation and comparative analysis of hydrophobin isolated from Pleurotus floridanus (PfH)

Hydrophobins are a class of small cysteine rich surface active proteins produced exclusively by filamentous fungi. It forms a nano layer in the cell-water interface, thereby protecting the emerging fungal hyphae from surrounding water. Even though hydrophobins have similar functions in fungi, they share less sequence similarity. In the current study, we made a comparative study of the hydrophobin produced by the mushroom Pleurotus floridanus (PfH). Mushroom P. floridanus was cultured in PD broth. The hydrophobin was purified by foam fractionation and characterized in terms of molecular weight, solubility and glycosylation. In the RP-HPLC analysis, the hydrophobin eluted at a retention time of 45.56 min. The molecular weight of the PfH was found to be 13.52 kDa by MALDI-TOF MS and the LC-MS/MS showed no similar sequence in MASCOT database. The hydrophobin gene of P. floridanus was amplified using custom-designed primers and the BLAST analysis showed 80% sequence similarity with the Vmh2-1 gene of Pleurotus ostreatus. The sequence was translated into protein using ExPASy, secondary and tertiary structure predictions were carried out using Jpred4 and Phyre2. The tertiary structure showed 91.5% similarity with the HYD1 hydrophobin of Schizophyllum commune. A comparative study of PfH with Vmh2-1 and HYD1 was performed using bioinformatics tools. Hydrophobic cluster analysis revealed that three of these proteins have uniformity in terms of amphiphilic and non-amphiphilic α-helices, whereas PfH has a unique proline clustering. Physicochemical analysis by ProtParam revealed that PfH shares similar properties with HYD1 and Vmh2-1, which can be correlated with its function.

Sustained photodynamic effect of single chirality-enriched single-walled carbon nanotubes

Semiconducting single-walled carbon nanotubes (s-SWNTs) are capable of fluorescence emission as well as photothermal and photodynamic actions, resulting from their near-infrared (NIR) absorptions corresponding to their S11 and S22 transitions. Here, we show that one chiral s-SWNTs, (6,4)-SWNTs, photogenerates all three of the major reactive oxygen species, i.e., singlet oxygen (1O2), superoxide anion (O2•–), and hydroxyl radical (•OH), in a sustainable manner. Its efficiency for •OH generation is dramatically higher than those of the other two chiral s-SWNTs tested, as well as a clinical NIR dye. This sustained and relatively high photodynamic effect in (6,4)-SWNTs is due to their extraordinary high photostability and relatively high generation efficiency of their triplet excited state. In the colloidal stabilization of (6,4)-SWNTs under physiological conditions, a serum protein consisting of a tandem repeat of amphiphilic α-helices is found to be useful for dispersion. The protein-coated (6,4)-SWNTs are capable of effectively ablating cancer cells and disintegrating amyloid beta peptide aggregates through sustained photodynamic action.

A small mycobacteriophage-derived peptide and its improved isomer restrict mycobacterial infection via dual mycobactericidal-immunoregulatory activities

Mycobacteriophages express various peptides/proteins to infect Mycobacterium tuberculosis (M. tb). Particular attention has been paid to mycobacteriophage-derived endolysin proteins. We herein characterized a small mycobacteriophage-derived peptide designated AK15 with potent anti-M. tb activity. AK15 adopted cationic amphiphilic α-helical structure, and on the basis of this structure, we designed six isomers with increased hydrophobic moment by rearranging amino acid residues of the helix. We found that one of these isomers, AK15-6, exhibits enhanced anti-mycobacterial efficiency. Both AK15 and AK15-6 directly inhibited M. tb by trehalose 6,6'-dimycolate (TDM) binding and membrane disruption. They both exhibited bactericidal activity, cell selectivity, and synergistic effects with rifampicin, and neither induced drug resistance to M. tb They efficiently attenuated mycobacterial load in the lungs of M. tb-infected mice. We observed that lysine, arginine, tryptophan, and an α-helix are key structural requirements for their direct anti-mycobacterial action. Of note, they also exhibited immunomodulatory effects, including inhibition of proinflammatory response in TDM-stimulated or M. tb-infected murine bone marrow-derived macrophages (BMDMs) and M.tb-infected mice and induction of only a modest level of cytokine (tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6)) production in murine BMDMs and a T-cell cytokine (interferin-γ (IFN-γ) and TNF-α) response in murine lung and spleen. In summary, characterization of a small mycobacteriophage-derived peptide and its improved isomer revealed that both efficiently restrain M. tb infection via dual mycobactericidal-immunoregulatory activities. Our work provides clues for identifying small mycobacteriophage-derived anti-mycobacterial peptides and improving those that have cationic amphiphilic α-helices.

BANFF: bending of bilayer membranes by amphiphilic α-helices is necessary for form and function of organelles 1.

By binding to and inserting into the lipid bilayer, amphiphilic α-helices of proteins are involved in the curvature of biological membranes in all organisms. In particular, they are involved in establishing the complex membrane architecture of intracellular organelles like the endoplasmatic reticulum, Golgi apparatus, mitochondria, and chloroplasts. Thus, amphiphilic α-helices are essential for maintenance of cellular metabolism and fitness of organisms. Here we focus on the structure and function of membrane-intrinsic proteins, which are involved in membrane curvature by amphiphilic α-helices, in mitochondria and chloroplasts of the eukaryotic model organisms yeast and Arabidopsis thaliana. Further, we propose a model for transport of fatty acids and lipid compounds across the envelope of chloroplasts in which amphiphilic α-helices might play a role.

Yeast Surface Display of Antheraea pernyi Lysozyme Revealed α-Helical Antibacterial Peptides in Its N-Terminal Domain.

The present study investigated a novel lysozyme ApLyz from the Chinese oak silkmoth, Antheraea pernyi, for its active expression with N- or C-terminus fused to the yeast cell surface, and the antimicrobial activities of the corresponding expressed lysozymes were evaluated. The bactericidal activity of C-terminal fusion of ApLyz surpassed that of the N-terminal fusion, which revealed the implication of an N-terminal stretch of ApLyz in the bactericidal function based on the structural mobility of this region. Two N-terminal peptides of ApLyz (residues 1-15 and 1-32), which primarily consist of amphiphilic α-helices, exerted similar bactericidal efficacy and had a strong preference for the Gram-negative strains. Further investigation revealed that the N-terminal peptides are membrane-targeting peptides causing cell permeabilization and also possess nonmembrane disturbing bactericidal mechanism. Overall, in addition to the key findings of novel bactericidal peptides from silkmoth lysozyme, this work laid the foundation for future improvement of ApLyz by protein engineering.

Prolines in the α-helix confer the structural flexibility and functional integrity of importin-β.

The karyopherin family of nuclear transport receptors is composed of a long array of amphiphilic α-helices and undergoes flexible conformational changes to pass through the hydrophobic crowding barrier of the nuclear pore. Here, we focused on the characteristic enrichment of prolines in the middle of the outer α-helices of importin-β. When these prolines were substituted with alanine, nuclear transport activity was reduced drastically in vivo and in vitro, and caused a severe defect in mitotic progression. These mutations did not alter the overall folding of the helical repeat or affect its interaction with cargo or the regulatory factor Ran. However, in vitro and in silico analyses revealed that the mutant lost structural flexibility and could not undergo rapid conformational changes when transferring from a hydrophilic to hydrophobic environment or vice versa. These findings reveal the essential roles of prolines in ensuring the structural flexibility and functional integrity of karyopherins.

Structure of purotoxin-2 from wolf spider: modular design and membrane-assisted mode of action in arachnid toxins

Traditionally, arachnid venoms are known to contain two particularly important groups of peptide toxins. One is disulfide-rich neurotoxins with a predominance of β-structure that specifically target protein receptors in neurons or muscle cells. The other is linear cationic cytotoxins that form amphiphilic α-helices and exhibit rather non-specific membrane-damaging activity. In the present paper, we describe the first 3D structure of a modular arachnid toxin, purotoxin-2 (PT2) from the wolf spider Alopecosa marikovskyi (Lycosidae), studied by NMR spectroscopy. PT2 is composed of an N-terminal inhibitor cystine knot (ICK, or knottin) β-structural domain and a C-terminal linear cationic domain. In aqueous solution, the C-terminal fragment is hyper-flexible, whereas the knottin domain is very rigid. In membrane-mimicking environment, the C-terminal domain assumes a stable amphipathic α-helix. This helix effectively tethers the toxin to membranes and serves as a membrane-access and membrane-anchoring device. Sequence analysis reveals that the knottin + α-helix architecture is quite widespread among arachnid toxins, and PT2 is therefore the founding member of a large family of polypeptides with similar structure motifs. Toxins from this family target different membrane receptors such as P2X in the case of PT2 and calcium channels, but their mechanism of action through membrane access may be strikingly similar.

Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana

A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm.

Physicochemical and biological characterizations of Pxt peptides from amphibian (Xenopus tropicalis) skin.

Pxt peptides (Pxt-1 through Pxt-12) have been isolated from amphibian, Xenopus tropicalis Pxt-related peptides (Pxt-2, Pxt-5, Pxt-12, reverse Pxt-2, reverse Pxt-5 and reverse Pxt-12) with significant foaming properties were further characterized. In the physicochemical experiments, all Pxt-related peptides formed significant amphiphilic α-helices in 50% 2,2,2-trifluoroethanol by circular dichroism measurements. Among Pxt-related peptides, both Pxt-5 and reverse Pxt-5 were the most effective in reducing their surface tensions. Moreover, Pxt-2, Pxt-5 and reverse Pxt-5 produced constant surface tensions above their critical association concentrations, suggesting the micelle-like assemblies. In the biological experiments, Pxt-5 possessed the most potent hemolytic activity, while reverse Pxt-5 exhibited the most remarkable gene expression of interleukin 8 and heme oxygenase 1 and the most potent cytotoxicity in HaCaT cells. In contrast, Pxt-12 and reverse Pxt-12 were much weaker in antimicrobial assays for Gram-negative bacteria, Gram-positive bacteria and yeasts, as well as in hemolytic, cell viability and cytotoxicity assays in HaCaT cells. All Pxt-related peptides exhibited about 20-50% of the total cellular histamine release at 10(-5) M, as well as mastoparan and melittin in mast cells. Real-time polymerase chain reaction analysis confirmed the gene expressions of Pxt-5 in testis and Pxt-12 in muscle, in addition to skin, while Pxt-2 was only in skin.

Organization of the multiaminoacyl-tRNA synthetase complex and the cotranslational protein folding.

Aminoacyl-tRNA synthetases (ARSs) play an essential role in the protein synthesis by catalyzing an attachment of their cognate amino acids to tRNAs. Unlike their prokaryotic counterparts, ARSs in higher eukaryotes form a multiaminoacyl-tRNA synthetase complex (MARS), consisting of the subset of ARS polypeptides and three auxiliary proteins. The intriguing feature of MARS complex is the presence of only nine out of twenty ARSs, specific for Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro, regardless of the organism, cell, or tissue types. Although existence of MARSs complex in higher eukaryotes has been already known for more than four decades, its functional significance remains elusive. We found that seven of the nine corresponding amino acids (Arg, Gln, Glu, Ile, Leu, Lys, and Met) together with Ala form a predictor of the protein α-helicity. Remarkably, all amino acids (besides Ala) in the predictor have the highest possible number of side-chain rotamers. Therefore, compositional bias of a typical α-helix can contribute to the helix's stability by increasing the entropy of the folded state. It also appears that position-specific α-helical propensity, specifically periodic alternation of charged and hydrophobic residues in the helices, may well be provided by the structural organization of the complex. Considering characteristics of MARS complex from the perspective of the α-helicity, we hypothesize that specific composition and structure of the complex represents a functional mechanism for coordination of translation with the fast and correct folding of amphiphilic α-helices.

Structure analysis and conformational transitions of the cell penetrating peptide transportan 10 in the membrane-bound state.

Structure analysis of the cell-penetrating peptide transportan 10 (TP10) revealed an exemplary range of different conformations in the membrane-bound state. The bipartite peptide (derived N-terminally from galanin and C-terminally from mastoparan) was found to exhibit prominent characteristics of (i) amphiphilic α-helices, (ii) intrinsically disordered peptides, as well as (iii) β-pleated amyloid fibrils, and these conformational states become interconverted as a function of concentration. We used a complementary approach of solid-state 19F-NMR and circular dichroism in oriented membrane samples to characterize the structural and dynamical behaviour of TP10 in its monomeric and aggregated forms. Nine different positions in the peptide were selectively substituted with either the L - or D -enantiomer of 3-(trifluoromethyl)-bicyclopent-[1.1.1]-1-ylglycine (CF3 -Bpg) as a reporter group for 19F-NMR. Using the L -epimeric analogs, a comprehensive three-dimensional structure analysis was carried out in lipid bilayers at low peptide concentration, where TP10 is monomeric. While the N-terminal region is flexible and intrinsically unstructured within the plane of the lipid bilayer, the C-terminal α-helix is embedded in the membrane with an oblique tilt angle of ∼55° and in accordance with its amphiphilic profile. Incorporation of the sterically obstructive D -CF3 -Bpg reporter group into the helical region leads to a local unfolding of the membrane-bound peptide. At high concentration, these helix-destabilizing C-terminal substitutions promote aggregation into immobile β-sheets, which resemble amyloid fibrils. On the other hand, the obstructive D -CF3 -Bpg substitutions can be accommodated in the flexible N-terminus of TP10 where they do not promote aggregation at high concentration. The cross-talk between the two regions of TP10 thus exerts a delicate balance on its conformational switch, as the presence of the α-helix counteracts the tendency of the unfolded N-terminus to self-assemble into β-pleated fibrils.

Structure of a dominant-negative helix-loop-helix transcriptional regulator suggests mechanisms of autoinhibition

Helix-loop-helix (HLH) family transcription factors regulate numerous developmental and homeostatic processes. Dominant-negative HLH (dnHLH) proteins lack DNA-binding ability and capture basic HLH (bHLH) transcription factors to inhibit cellular differentiation and enhance cell proliferation and motility, thus participating in patho-physiological processes. We report the first structure of a free-standing human dnHLH protein, HHM (Human homologue of murine maternal Id-like molecule). HHM adopts a V-shaped conformation, with N-terminal and C-terminal five-helix bundles connected by the HLH region. In striking contrast to the common HLH, the HLH region in HHM is extended, with its hydrophobic dimerization interfaces embedded in the N- and C-terminal helix bundles. Biochemical and physicochemical analyses revealed that HHM exists in slow equilibrium between this V-shaped form and the partially unfolded, relaxed form. The latter form is readily available for interactions with its target bHLH transcription factors. Mutations disrupting the interactions in the V-shaped form compromised the target transcription factor specificity and accelerated myogenic cell differentiation. Therefore, the V-shaped form of HHM may represent an autoinhibited state, and the dynamic conformational equilibrium may control the target specificity.

Short Stories - on Antimicrobial Peptides that are Too Short to Span the Membrane

Solid-state 19F-NMR structure analysis of membrane-bound peptides is routinely feasible now in macroscopically oriented samples. For many long amphiphilic α-helices, their membrane-induced folding, re-alignment and insertion into the lipid bilayer has been characterized as a series of consecutive steps, ultimately leading to pore-formation across the membrane. More recently, however, we have examined various short peptides that are not long enough to span the lipid bilayer, and whose biological action cannot be explained in terms of classical models of oligomeric pore formation.

Amphiphilic α-Helical Potential: A Putative Folding Motif Adding Few Constraints to Protein Evolution

Evidence from a number of studies indicates that protein folding is dictated not only by factors stabilizing the native state, but also by potentially independent factors that create folding pathways. How natural selection might cope simultaneously with two independent factors was addressed in this study within the framework of the "Lim-model" of protein folding, which postulates that the early stages of folding of all globular proteins, regardless of their native structure, are directed at least in part by potential to form amphiphilic α-helices. For this purpose, the amphiphilic α-helical potential in randomly ordered amino acid sequences and the conservation in phylogeny of amphiphilic α-helical potential within various proteins were assessed. These analyses revealed that amphiphilic α-helical potential is a common occurrence in random sequences, and that the presence of amphiphilic α-helical potential is present but not conserved in phylogeny within a given protein. The results suggest that the rapid formation of molten globules and the variable behavior of those globules depending on the protein may be a fundamental property of polymers of naturally occurring amino acids more so than a trait that must be derived or maintained by natural selection. Further, the results point toward the utility of randomly occurring process in protein function and evolution, and suggest that the formation of efficient pathways that determine early processes in protein folding, unlike the formation of stable, native protein structure, does not present a substantial hurdle during the evolution of amino acid sequences.

Novel Amphiphilic α-Helix Mimetics Based on a Bis-benzamide Scaffold

To mimic amphiphilic alpha-helices, a new scaffold was designed based on a bis-benzamide that places four side-chain functional groups found at the i, i+2, i+5, and i+7 positions of a helix. Its two hydrogen bonds fix the conformation and provide accurate bifacial arrangement of the four substituents, simultaneously representing two opposing helical sides. An efficient synthetic route was achieved for the construction of bis-benzamides, and their superior alpha-helix mimicry was confirmed by X-ray crystallography.

Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.