Localization Of Binding Sites Research Articles

Anesthetic Binding Sites within the Transmembrane Domain of the α7 Nicotinic Acetylcholine Receptor

General anesthetics inhibit the α7 neuronal nicotinic acetylcholine receptor (nAChR) with different sensitivities, but the location of anesthetic binding sites remains unclear. Here we used high-resolution solution NMR to study the interaction of general anesthetics ketamine and halothane with the transmembrane domain (TMD) of the human α7 nAChR. The TMD was expressed in E. coli and purified into detergent LDAO micelles. The specific residues involved in anesthetic binding were identified by anesthetics-induced chemical shift changes in 1H-15N HSQC NMR spectra. Ketamine binds to an intrasubunit site involving residues of all four helices at the intracellular end of the TMD. Two halothane interaction sites were detected. The first site was located near the intracellular end of the TMD, similar to the ketamine site, but involved only residues in TM1 and TM2. The second halothane site was formed by residues in the middle of TM1 and TM3. This site was not observed for ketamine binding, probably because ketamine was too large to penetrate deep into the TMD. Interestingly, unlike the previous finding in the α4β2 nAChR, anesthetic binding to the extracellular end of the TMD was not observed in the α7 nAChR. This difference may be responsible for their distinct sensitivity to halothane. In addition, halothane modulated motion less in α7 than in α4β2 on the micro- to milli-second (μs-ms) timescale, as demonstrated by changes in peak intensity, line width, and peak splitting for some residues. This work was supported by NIH grants: R01GM066358, R01GM056257, and R37GM049202.

Building multi-component crystals from cations and co-crystals: the use of chaperones

Ternary crystalline complexes consisting of both salts and ionic co-crystals have been created through the crystallisation of the binary co-crystal 3,5-dinitrobenzoic acid–4-(dimethylamino)benzoic acid with group 1 or ammonium cations. The size and charge density of the cation can be used to adjust the protonation level and local geometry of the acid pair. The selectivity and coordination geometry of the chaperone cation may be further adjusted by the inclusion of a crown ether to reduce the number and location of potential binding sites.

Exploring the ATP-binding site of P2X receptors

P2X receptors are ATP-gated non-selective cation channels involved in many different physiological processes, such as synaptic transmission, inflammation, and neuropathic pain. They form homo- or heterotrimeric complexes and contain three ATP-binding sites in their extracellular domain. The recent determination of X-ray structures of a P2X receptor solved in two states, a resting closed state and an ATP-bound, open-channel state, has provided unprecedented information not only regarding the three-dimensional shape of the receptor, but also on putative conformational changes that couple ATP binding to channel opening. These data provide a structural template for interpreting the huge amount of functional, mutagenesis, and biochemical data collected during more than fifteen years. In particular, the interfacial location of the ATP binding site and ATP orientation have been successfully confirmed by these structural studies. It appears that ATP binds to inter-subunit cavities shaped like open jaws, whose tightening induces the opening of the ion channel. These structural data thus represent a firm basis for understanding the activation mechanism of P2X receptors.

Competing hydrogen-bond and halogen-bond donors in crystal engineering

In order to study the structure-directing competition between hydrogen- and halogen-bond donors we have synthesized two ligands, 3,3′-azobipyridine and 4,4′-azobipyridine, and co-crystallized them with a series of bi-functional donor molecules comprising an activated halogen-bond donor (I or Br) as well as a hydrogen-bond donor (acid, phenol or oxime) on the same backbone. Based on the subsequent single-crystal analysis, 5 of 6 co-crystals of 3,3′-azobipyridine are assembled using hydrogen bonds as the primary driving force accompanied by weaker secondary (C–X⋯O) interactions. However, in 5 out of the 6 co-crystals of 4,4′-azobipyridine, both hydrogen bonds (O–H⋯N) and halogen bonds (C–X⋯N) are present as structure-directing interactions leading to 1-D chains. Since the charges on the acceptor sites in 3,3′- and 4,4′-azobipyridine are very similar, the observed difference in binding behaviour highlights the importance of binding-site location on the acceptor molecules (anti-parallel in 3,3′-azobipyridine and co-linear in 4,4′-azobipyridine) as a direct influence over the structural balance between hydrogen- and halogen-bond donors.

Pitx1 broadly associates with limb enhancers and is enriched on hindlimb cis-regulatory elements

Extensive functional analyses have demonstrated that the pituitary homeodomain transcription factor Pitx1 plays a critical role in specifying hindlimb morphology in vertebrates. However, much less is known regarding the target genes and cis-regulatory elements through which Pitx1 acts. Earlier studies suggested that the hindlimb transcription factors Tbx4, HoxC10, and HoxC11 might be transcriptional targets of Pitx1, but definitive evidence for direct regulatory interactions has been lacking. Using ChIP-Seq on embryonic mouse hindlimbs, we have pinpointed the genome-wide location of Pitx1 binding sites during mouse hindlimb development and identified potential gene targets for Pitx1. We determined that Pitx1 binding is significantly enriched near genes involved in limb morphogenesis, including Tbx4, HoxC10, and HoxC11. Notably, Pitx1 is bound to the previously identified HLEA and HLEB hindlimb enhancers of the Tbx4 gene and to a newly identified Tbx2 hindlimb enhancer. Moreover, Pitx1 binding is significantly enriched on hindlimb relative to forelimb-specific cis-regulatory features that are differentially marked by H3K27ac. However, our analysis revealed that Pitx1 also strongly associates with many functionally verified limb enhancers that exhibit similar levels of activity in the embryonic mesenchyme of forelimbs and hindlimbs. We speculate that Pitx1 influences hindlimb morphology both through the activation of hindlimb-specific enhancers as well as through the hindlimb-specific modulation of enhancers that are active in both sets of limbs.

Insights into the Glycosaminoglycan-Mediated Cytotoxic Mechanism of Eosinophil Cationic Protein Revealed by NMR

Protein-glycosaminoglycan interactions are essential in many biological processes and human diseases, yet how their recognition occurs is poorly understood. Eosinophil cationic protein (ECP) is a cytotoxic ribonuclease that interacts with glycosaminoglycans at the cell surface; this promotes the destabilization of the cellular membrane and triggers ECP's toxic activity. To understand this membrane destabilization event and the differences in the toxicity of ECP and its homologues, the high resolution solution structure of the complex between full length folded ECP and a heparin-derived trisaccharide (O-iPr-α-D-GlcNS6S-α(1-4)-L-IdoA2S-α(1-4)-D-GlcNS6S) has been solved by NMR methods and molecular dynamics simulations. The bound protein retains the tertiary structure of the free protein. The (2)S(0) conformation of the IdoA ring is preferably recognized by the protein. We have identified the precise location of the heparin binding site, dissected the specific interactions responsible for molecular recognition, and defined the structural requirements for this interaction. The structure reveals the contribution of Arg7, Gln14, and His15 in helix α1, Gln40 in strand β1, His64 in loop 4, and His128 in strand β6 in the recognition event and corroborates the previously reported participation of residues Arg34-Asn39. The participation of the catalytic triad (His15, Lys38, His128) in recognizing the heparin mimetic reveals, at atomic resolution, the mechanism of heparin's inhibition of ECP's ribonucleolytic activity. We have integrated all the available data to propose a molecular model for the membrane interaction process. The solved NMR complex provides the structural model necessary to design inhibitors to block ECP's toxicity implicated in eosinophil pathologies.

Binding of Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase to the Cytoplasmic Tails of Plasmodium falciparum Merozoite Duffy Binding-Like and Reticulocyte Homology Ligands

ABSTRACTInvasion of erythrocytes by Plasmodium falciparum requires a connection between the cytoplasmic tail of the parasite’s ligands for its erythrocyte receptors and the actin-myosin motor of the parasite. For the thromobospondin-related anonymous protein (TRAP) ligand on Plasmodium sporozoites, aldolase forms this connection and requires tryptophan and negatively charged amino acids in the ligand’s cytoplasmic tail. Because of the importance of the Duffy binding-like (DBL) and the reticulocyte homology (RH) ligand families in erythrocyte binding and merozoite invasion, we characterized the ability of their cytoplasmic tails to bind aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), both of which bind actin. We tested the binding of the cytoplasmic peptides of the two ligand families to aldolase and GAPDH. Only the cytoplasmic peptides of some RH ligands showed strong binding to aldolase, and the binding depended on the presence of an aromatic amino acid (phenylalanine or tyrosine), rather than tryptophan, in the context of negatively charged amino acids. The binding was confirmed by surface plasmon resonance analysis and was found to represent affinity similar to that seen with TRAP. An X-ray crystal structure of aldolase at 2.5 Å in the presence of RH2b peptide suggested that the binding site location was near the TRAP-binding site. GAPDH bound to some of the cytoplasmic tails of certain RH and DBL ligands in an aromatic amino acid-dependent manner. Thus, the connection between Plasmodium merozoite ligands and erythrocyte receptors and the actin motor can be achieved through the activity of either aldolase or GAPDH by mechanisms that do not require tryptophan but, rather, other aromatic amino acids.

Protein-DNA docking with a coarse-grained force field

BackgroundProtein-DNA interactions are important for many cellular processes, however structural knowledge for a large fraction of known and putative complexes is still lacking. Computational docking methods aim at the prediction of complex architecture given detailed structures of its constituents. They are becoming an increasingly important tool in the field of macromolecular assemblies, complementing particularly demanding protein-nucleic acids X ray crystallography and providing means for the refinement and integration of low resolution data coming from rapidly advancing methods such as cryoelectron microscopy.ResultsWe present a new coarse-grained force field suitable for protein-DNA docking. The force field is an extension of previously developed parameter sets for protein-RNA and protein-protein interactions. The docking is based on potential energy minimization in translational and orientational degrees of freedom of the binding partners. It allows for fast and efficient systematic search for native-like complex geometry without any prior knowledge regarding binding site location.ConclusionsWe find that the force field gives very good results for bound docking. The quality of predictions in the case of unbound docking varies, depending on the level of structural deviation from bound geometries. We analyze the role of specific protein-DNA interactions on force field performance, both with respect to complex structure prediction, and the reproduction of experimental binding affinities. We find that such direct, specific interactions only partially contribute to protein-DNA recognition, indicating an important role of shape complementarity and sequence-dependent DNA internal energy, in line with the concept of indirect protein-DNA readout mechanism.

Multiple instance learning of Calmodulin binding sites

Motivation: Calmodulin (CaM) is a ubiquitously conserved protein that acts as a calcium sensor, and interacts with a large number of proteins. Detection of CaM binding proteins and their interaction sites experimentally requires a significant effort, so accurate methods for their prediction are important.Results: We present a novel algorithm (MI-1 SVM) for binding site prediction and evaluate its performance on a set of CaM-binding proteins extracted from the Calmodulin Target Database. Our approach directly models the problem of binding site prediction as a large-margin classification problem, and is able to take into account uncertainty in binding site location. We show that the proposed algorithm performs better than the standard SVM formulation, and illustrate its ability to recover known CaM binding motifs. A highly accurate cascaded classification approach using the proposed binding site prediction method to predict CaM binding proteins in Arabidopsis thaliana is also presented.Availability: Matlab code for training MI-1 SVM and the cascaded classification approach is available on request.Contact: fayyazafsar@gmail.com or asa@cs.colostate.edu

Complementary strand relocation may play vital roles in RecA-based homology recognition

RecA-family proteins mediate homologous recombination and recombinational DNA repair through homology search and strand exchange. Initially, the protein forms a filament with the incoming single-stranded DNA (ssDNA) bound in site I. The RecA–ssDNA filament then binds double-stranded DNA (dsDNA) in site II. Non-homologous dsDNA rapidly unbinds, whereas homologous dsDNA undergoes strand exchange yielding heteroduplex dsDNA in site I and the leftover outgoing strand in site II. We show that applying force to the ends of the complementary strand significantly retards strand exchange, whereas applying the same force to the outgoing strand does not. We also show that crystallographically determined binding site locations require an intermediate structure in addition to the initial and final structures. Furthermore, we demonstrate that the characteristic dsDNA extension rates due to strand exchange and free RecA binding are the same, suggesting that relocation of the complementary strand from its position in the intermediate structure to its position in the final structure limits both rates. Finally, we propose that homology recognition is governed by transitions to and from the intermediate structure, where the transitions depend on differential extension in the dsDNA. This differential extension drives strand exchange forward for homologs and increases the free energy penalty for strand exchange of non-homologs.

Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization

Laulimalide is a natural product that has strong taxoid-like properties but binds to a distinct site on β-tubulin in the microtubule (MT) lattice. At elevated concentrations, it generates MTs that are resistant to depolymerization, and it induces a conformational state indistinguishable from taxoid-treated MTs. In this study, we describe the effect of low-dose laulimalide on various stages of the cell cycle and compare these effects to docetaxel as a representative of taxoid stabilizers. No evidence of MT bundling in interphase was observed with laulimalide, in spite of the fact that MTs are stabilized at low dose. Cells treated with laulimalide enter mitosis but arrest at prometaphase by generating multiple asters that coalesce into supernumerary poles and interfere with the integrity of the metaphase plate. Cells with a preformed bipolar spindle exist under heightened tension under laulimalide treatment, and chromosomes rapidly shear from the plate, even though the bipolar spindle is well-preserved. Docetaxel generates a similar phenotype for HeLa cells entering mitosis, but when treated at metaphase, cells undergo chromosomal fragmentation and demonstrate reduced centromere dynamics, as expected for a taxoid. Our results suggest that laulimalide represents a new class of molecular probe for investigating MT-mediated events, such as kinetochore-MT interactions, which may reflect the location of the ligand binding site within the interprotofilament groove.

LeuO is a global regulator of gene expression in Salmonella enterica serovar Typhimurium

We report the first investigation of the binding of the Salmonella enterica LeuO LysR-type transcription regulator to its genomic targets in vivo. Chromatin-immunoprecipitation-on-chip identified 178 LeuO binding sites on the chromosome of S. enterica serovar Typhimurium strain SL1344. These sites were distributed across both the core and the horizontally acquired genome, and included housekeeping genes and genes known to contribute to virulence. Sixty-eight LeuO targets were co-bound by the global repressor protein, H-NS. Thus, while LeuO may function as an H-NS antagonist, these functions are unlikely to involve displacement of H-NS. RNA polymerase bound 173 of the 178 LeuO targets, consistent with LeuO being a transcription regulator. Thus, LeuO targets two classes of genes, those that are bound by H-NS and those that are not bound by H-NS. LeuO binding site analysis revealed a logo conforming to the TN(11) A motif common to LysR-type transcription factors. It differed in some details from a motif that we composed for Escherichia coli LeuO binding sites; 1263 and 1094 LeuO binding site locations were predicted in the S. Typhimurium SL1344 and E. coli MG1655 genomes respectively. Despite differences in motif composition, many LeuO target genes were common to both species. Thus, LeuO is likely to be a more important global regulator than previously suspected.

Membrane permeable local anesthetics modulate NaV1.5 mechanosensitivity

Voltage-gated sodium selective ion channel NaV1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. NaV1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon NaV1.5 to modulate activity by multiple mechanisms. This study examined whether NaV1.5 mechanosensitivity is modulated by local anesthetics. NaV1.5 channels wereexpressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V1/2a) and inactivation (V1/2i) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V1/2a but not V1/2i. Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V1/2a. Lidocaine inhibited mechanosensitivity in NaV1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A NaV1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of NaV1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions.

Local Anesthetic-like Inhibition of Voltage-gated Na+Channels by the Partial μ-opioid Receptor Agonist Buprenorphine

Opioids induce analgesia mainly by inhibiting synaptic transmission via G protein-coupled opioid receptors. In addition to analgesia, buprenorphine induces a pronounced antihyperalgesia and is an effective adjuvant to local anesthetics. These properties only partially apply to other opioids, and thus targets other than opioid receptors are likely to be employed. Here we asked if buprenorphine inhibits voltage-gated Na(+) channels. Na(+) currents were examined by whole cell patch clamp recordings on different recombinant Na(+) channel α-subunits. The effect of buprenorphine on unmyelinated mouse C-fibers was examined with the skin-nerve preparation. Data are presented as mean ± SEM. Buprenorphine induced a concentration-dependent tonic (IC(50) 33 ± 2 μM) and use-dependent block of endogenous Na(+) channels in ND7/23 cells. This block was state-dependent and displayed slow on and off characteristics. The effect of buprenorphine was reduced on local anesthetic insensitive Nav1.4-mutant constructs and was more pronounced on the inactivation-deficient Nav1.4-WCW mutant. Neuronal (Nav1.3, Nav1.7, and Nav1.8), cardiac (Nav1.5), and skeletal muscle (Nav1.4) α-subunits displayed small differences in tonic block, but similar degrees of use-dependent block. According to our patch clamp data, buprenorphine blocked electrically evoked action potentials in C-fiber nerve terminals. Buprenorphine was more potent than other opioids, including morphine (IC(50) 378 ± 20 μM), fentanyl (IC(50) 95 ± 5 μM), sufentanil (IC(50) 111 ± 6 μM), remifenatil (IC(50) 612 ± 17 μM), and tramadol (IC(50) 194 ± 9 μM). Buprenorphine is a potent local anesthetic and blocks voltage-gated Na(+) channels via the local anesthetic binding site. This property is likely to be relevant when buprenorphine is used for pain treatment and for local anesthesia.

Inferring gene regulatory logic from high-throughput measurements of thousands of systematically designed promoters

Despite much research, our understanding of the rules by which cis-regulatory sequences are translated into expression levels is still lacking. We devised a method for obtaining parallel and highly accurate expression measurements of thousands of fully designed promoters, and applied it to measure the effect of systematic changes to location, number, orientation, affinity and organization of transcription factor (TF) binding sites and of nucleosome disfavoring sequences. Our analyses reveal a clear relationship between expression and binding site number, and TF-specific dependencies of expression on the distance between sites and gene starts including a striking ~10bp periodic relationship. We also demonstrate the utility of our approach for measuring TF sequence specificities and sensitivity of TF sites to surrounding sequence context, and for profiling the activity of most yeast transcription factors. Our method is readily applicable for studying both the cis and trans effects of genotype on transcriptional, post-transcriptional, and translational control.

MicroRNA-29 Stabilizes IFN-γ mRNA by inhibiting GW182 (174.5)

Abstract Tight regulation of interferon-gamma (IFN-γ) expression is critical for an optimal immune response. We have extensively studied the mechanisms responsible for the post-transcriptional regulation of IFN-γ expression. AU-rich element (ARE)-mediated decay (AMD) via the tristetraprolin (TTP) complex is responsible for robust degradation of the IFN-γ mRNA. We found that microRNA-29 (miR-29) targets the IFN-γ 3’UTR and, unlike the conventional inhibitory miRNAs, stabilizes the mRNA. Resolution of the secondary structure showed that the ARE and miR-29 binding site are in close proximity and a series of experiments manipulating the RNA structure revealed that this proximity enables miR-29 to antagonize TTP degradation. Interestingly, miR-29 is unable to degrade the mRNA even in the absence of TTP. Recently we have found that GW182, a major degradation protein in the miRISC, is unable to be recruited to the mRNA as a result of the location of the miR-29 binding site in the secondary structure. The ability of miR-29 to stabilize the IFN-γ mRNA demonstrates a novel molecular mechanism of miRNA regulation.

High-Resolution Single-Molecule Recognition Imaging of the Molecular Details of Ricin–Aptamer Interaction

We studied the molecular details of DNA aptamer-ricin interactions. The toxic protein ricin molecules were immobilized on a Au(111) surface using a N-hydroxysuccinimide (NHS) ester to specifically react with lysine residues located on the ricin B chains. A single ricin molecule was visualized in situ using the AFM tip modified with an antiricin aptamer. Computer simulation was used to illustrate the protein and aptamer structures, the single-molecule ricin images on a Au(111) surface, and the binding conformations of ricin-aptamer and ricin-antibody complexes. The various ricin conformations on a Au(111) surface were caused by the different lysine residues reacting with the NHS ester. It was also observed that most of the binding sites for aptamer and antibody on the A chains of ricin molecules were not interfered by the immobilization reaction. The different locations of the ricin binding sites to aptamer and antibody were also distinguished by AFM recognition images and interpreted by simulations.

Conserved Motifs and Prediction of Regulatory Modules inCaenorhabditis elegans

Transcriptional regulation, a primary mechanism for controlling the development of multicellular organisms, is carried out by transcription factors (TFs) that recognize and bind to their cognate binding sites. In Caenorhabditis elegans, our knowledge of which genes are regulated by which TFs, through binding to specific sites, is still very limited. To expand our knowledge about the C. elegans regulatory network, we performed a comprehensive analysis of the C. elegans, Caenorhabditis briggsae, and Caenorhabditis remanei genomes to identify regulatory elements that are conserved in all genomes. Our analysis identified 4959 elements that are significantly conserved across the genomes and that each occur multiple times within each genome, both hallmarks of functional regulatory sites. Our motifs show significant matches to known core promoter elements, TF binding sites, splice sites, and poly-A signals as well as many putative regulatory sites. Many of the motifs are significantly correlated with various types of experimental data, including gene expression patterns, tissue-specific expression patterns, and binding site location analysis as well as enrichment in specific functional classes of genes. Many can also be significantly associated with specific TFs. Combinations of motif occurrences allow us to predict the location of cis-regulatory modules and we show that many of them significantly overlap experimentally determined enhancers. We provide access to the predicted binding sites, their associated motifs, and the predicted cis-regulatory modules across the whole genome through a web-accessible database and as tracks for genome browsers.

Characterization of α2B-adrenoceptor ligand binding in the presence of Muscarinic toxin α and delineation of structural features of receptor binding selectivity

Muscarinic toxin α (MTα), a peptide isolated from the venom of the African black mamba, was recently found to selectively antagonize the human α2B-adrenoceptor. To gain more information about the binding of this peptide toxin, we studied the properties of the [3H]UK14,304 agonist and the [3H]MK-912 antagonist binding to the α2B-adrenoceptor in the presence of MTα. In equilibrium binding experiments, MTα decreased the binding of the orthosteric ligands, but failed to completely displace these. This effect of MTα was due to noncompetitive inhibition of Bmax without change in radioligand affinity. On the contrary, cellular signaling via the α2B-adrenoceptor could be titrated to zero despite the incomplete receptor blockade. To locate binding sites for MTα on the receptor protein, we generated chimeric receptors of α2B- and α2A- or α2C-adrenoceptors. Data based on these constructs revealed the extracellular loop two (ECL2) as the structural entity that enables MTα binding. Cumulative exchange of parts of ECL2 of α2B for α2A-adrenoceptor sequence resulted in a gradual decrease in the affinity for MTα, indicating that MTα binds to the α2B-adrenoceptor through multiple sites dispersed over the whole ECL2. Together the results suggest that binding of MTα to the α2B-adrenoceptor occludes orthosteric ligand access to the binding pocket. Putative homomeric receptor complexes as factors underlying the apparent noncompetitivity are also discussed.

Fenpyroximate Binds to the Interface between PSST and 49 kDa Subunits in Mitochondrial NADH-Ubiquinone Oxidoreductase

Using a photoaffinity labeling technique, Nakamaru-Ogiso et al. demonstrated that fenpyroximate, a strong inhibitor of bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I), binds to the ND5 subunit [Nakamaru-Ogiso, E., et al. (2003) Biochemistry 42, 746-754]. Considering that the main body of the ND5 subunit composed of transmembrane helixes 1-15 is located at the distal end of the membrane domain [Efremov, R. G., et al. (2010) Nature 465, 441-445], however, their result may be questionable. Because establishing the number and location of inhibitors and/or quinone binding sites in the membrane domain is necessary to elucidate the function of the enzyme, it is critical to clarify whether there is an additional inhibitor and/or quinone binding site besides the interface between the hydrophilic and membrane domains. We therefore performed photoaffinity labeling experiments using two newly synthesized fenpyroximate derivatives [[(125)I]-4-azidophenyl fenpyroximate ([(125)I]APF) and [(125)I]-3-azido-5-iodobenzyl fenpyroximate ([(125)I]AIF)] possessing a photoreactive azido group at and far from the pharmacophoric core moiety, respectively. Doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that [(125)I]APF and [(125)I]AIF bind to the PSST and 49 kDa subunits, respectively. Careful examination of the fragmentation patterns of the labeled PSST and 49 kDa subunits generated by limited proteolysis indicated that the residues labeled by [(125)I]APF and [(125)I]AIF are located in the Ser43-Arg66 (PSST) and Asp160-Arg174 (49 kDa) regions, respectively, which face the supposed quinone-binding pocket formed at the interface of the PSST, 49 kDa, and ND1 subunits. We conclude that fenpyroximate does not bind to the distal end of the membrane domain but rather resides at the interface between the two domains in a manner such that the pharmacophoric pyrazole ring and side chain of the inhibitor orient toward the PSST and 49 kDa subunits, respectively. This study answers a critical question relating to complex I.