Luminal Binding Site Research Articles

The discovery and structural basis of two distinct state-dependent inhibitors of BamA

BamA is the central component of the essential β-barrel assembly machine (BAM), a conserved multi-subunit complex that dynamically inserts and folds β-barrel proteins into the outer membrane of Gram-negative bacteria. Despite recent advances in our mechanistic and structural understanding of BamA, there are few potent and selective tool molecules that can bind to and modulate BamA activity. Here, we explored in vitro selection methods and different BamA/BAM protein formulations to discover peptide macrocycles that kill Escherichia coli by targeting extreme conformational states of BamA. Our studies show that Peptide Targeting BamA-1 (PTB1) targets an extracellular divalent cation-dependent binding site and locks BamA into a closed lateral gate conformation. By contrast, PTB2 targets a luminal binding site and traps BamA into an open lateral gate conformation. Our results will inform future antibiotic discovery efforts targeting BamA and provide a template to prospectively discover modulators of other dynamic integral membrane proteins.

Eu3+ detects two functionally distinct luminal Ca2+ binding sites in ryanodine receptors

Ryanodine receptors (RyRs) are Ca2+ release channels, gated by Ca2+ in the cytosol and the sarcoplasmic reticulum lumen. Their regulation is impaired in certain cardiac and muscle diseases. Although a lot of data is available on the luminal Ca2+ regulation of RyR, its interpretation is complicated by the possibility that the divalent ions used to probe the luminal binding sites may contaminate the cytoplasmic sites by crossing the channel pore. In this study, we used Eu3+, an impermeable agonist of Ca2+ binding sites, as a probe to avoid this complication and to gain more specific information about the function of the luminal Ca2+ sensor. Single-channel currents were measured from skeletal muscle and cardiac RyRs (RyR1 and RyR2) using the lipid bilayer technique. We show that RyR2 is activated by the luminal addition of Ca2+, whereas RyR1 is inhibited. These results were qualitatively reproducible using Eu3+. The luminal regulation of RyR1 carrying a mutation associated with malignant hyperthermia was not different from that of the wild-type.RyR1 inhibition by Eu3+ was extremely voltage dependent, whereas RyR2 activation did not depend on the membrane potential. These results suggest that the RyR1 inhibition site is in the membrane’s electric field (channel pore), whereas the RyR2 activation site is outside. Using in silico analysis and previous results, we predicted putative Ca2+ binding site sequences. We propose that RyR2 bears an activation site, which is missing in RyR1, but both isoforms share the same inhibitory Ca2+ binding site near the channel gate.

CLIP-170S is a microtubule+TIP variant that confers resistance to taxanes by impairing drug-target engagement.

Taxanes are widely used cancer chemotherapeutics. However, intrinsic resistance limits their efficacy without any actionable resistance mechanism. We have discovered a microtubule (MT) plus-end-binding CLIP-170 protein variant, hereafter CLIP-170S, which we found enriched in taxane-resistant cell lines and patient samples. CLIP-170S lacks the first Cap-Gly motif, forms longer comets, and impairs taxane access to its MT luminal binding site. CLIP-170S knockdown reversed taxane resistance in cells and xenografts, whereas its re-expression led to resistance, suggesting causation. Using a computational approach in conjunction with the connectivity map, we unexpectedly discovered that Imatinib was predicted to reverse CLIP-170S-mediated taxane resistance. Indeed, Imatinib treatment selectively depleted CLIP-170S, thus completely reversing taxane resistance. Other RTK inhibitors also depleted CLIP-170S, suggesting a class effect. Herein, we identify CLIP-170S as a clinically prevalent variant that confers taxane resistance, whereas the discovery of Imatinib as a CLIP-170S inhibitor provides novel therapeutic opportunities for future trials.

Structural basis of ER-associated protein degradation mediated by the Hrd1 ubiquitin ligase complex.

Misfolded luminal endoplasmic reticulum (ER) proteins undergo ER-associated degradation (ERAD-L): They are retrotranslocated into the cytosol, polyubiquitinated, and degraded by the proteasome. ERAD-L is mediated by the Hrd1 complex (composed of Hrd1, Hrd3, Der1, Usa1, and Yos9), but the mechanism of retrotranslocation remains mysterious. Here, we report a structure of the active Hrd1 complex, as determined by cryo-electron microscopy analysis of two subcomplexes. Hrd3 and Yos9 jointly create a luminal binding site that recognizes glycosylated substrates. Hrd1 and the rhomboid-like Der1 protein form two "half-channels" with cytosolic and luminal cavities, respectively, and lateral gates facing one another in a thinned membrane region. These structures, along with crosslinking and molecular dynamics simulation results, suggest how a polypeptide loop of an ERAD-L substrate moves through the ER membrane.

Cardiac ryanodine receptor: Selectivity for alkaline earth metal cations points to the EF-hand nature of luminal binding sites

A growing body of evidence suggests that the regulation of cardiac ryanodine receptor (RYR2) by luminal Ca2+ is mediated by luminal binding sites located on the RYR2 channel itself and/or its auxiliary protein, calsequestrin. The localization and structure of RYR2-resident binding sites are not known because of the lack of a high-resolution structure of RYR2 luminal regions. To obtain the first structural insight, we probed the RYR2 luminal face stripped of calsequestrin by alkaline earth metal divalents (M2+: Mg2+, Ca2+, Sr2+ or Ba2+). We show that the RYR2 response to caffeine at the single-channel level is significantly modified by the nature of luminal M2+. Moreover, we performed competition experiments by varying the concentration of luminal M2+ (Mg2+, Sr2+ or Ba2+) from 8mM to 53mM and investigated its ability to compete with 1mM luminal Ca2+. We demonstrate that all tested M2+ bind to exactly the same RYR2 luminal binding sites. Their affinities decrease in the order: Ca2+>Sr2+>Mg2+~Ba2+, showing a strong correlation with the M2+ affinity of the EF-hand motif. This indicates that the RYR2 luminal binding regions and the EF-hand motif likely share some structural similarities because the structure ties directly to the function.

Endothelial cells respond to hyperglycemia by increasing the LPL transporter GPIHBP1.

In diabetes, when glucose uptake and oxidation are impaired, the heart is compelled to use fatty acid (FA) almost exclusively for ATP. The vascular content of lipoprotein lipase (LPL), the rate-limiting enzyme that determines circulating triglyceride clearance, is largely responsible for this FA delivery and increases following diabetes. Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein [GPIHBP1; a protein expressed abundantly in the heart in endothelial cells (EC)] collects LPL from the interstitial space and transfers it across ECs onto the luminal binding sites of these cells, where the enzyme is functional. We tested whether ECs respond to hyperglycemia by increasing GPIHBP1. Streptozotocin diabetes increased cardiac LPL activity and GPIHBP1 gene and protein expression. The increased LPL and GPIHBP1 were located at the capillary lumen. In vitro, passaging EC caused a loss of GPIHBP1, which could be induced on exposure to increasing concentrations of glucose. The high-glucose-induced GPIHBP1 increased LPL shuttling across EC monolayers. GPIHBP1 expression was linked to the EC content of heparanase. Moreover, active heparanase increased GPIHBP1 gene and protein expression. Both ECs and myocyte heparan sulfate proteoglycan-bound platelet-derived growth factor (PDGF) released by heparanase caused augmentation of GPIHBP1. Overall, our data suggest that this protein "ensemble" (heparanase-PDGF-GPIHBP1) cooperates in the diabetic heart to regulate FA delivery and utilization by the cardiomyocytes. Interrupting this axis may be a novel therapeutic strategy to restore metabolic equilibrium, curb lipotoxicity, and help prevent or delay heart dysfunction that is characteristic of diabetes.

Identification and Characterization of an Intermediate Taxol Binding Site Within Microtubule Nanopores and a Mechanism for Tubulin Isotype Binding Selectivity

Tubulin, the primary subunit of microtubules, is remarkable for the variety of small molecules to which it binds. Many of these are very useful or promising agents in cancer chemotherapy. One of the most useful of these is paclitaxel. The tubulin molecule is itself an alpha/beta heterodimer, both alpha- and beta-tubulin monomers existing as multiple isotypes. Despite the success of paclitaxel as an anticancer drug, resistance often occurs in cancer cells and has been associated with variations in tubulin isotype expression, most notably with the increased expression of betaIII-tubulin. Paclitaxel is thought to reach its binding site on beta-tubulin by diffusion through nanopores in the microtubule wall. It has been suggested that a transitional step in this process may be the binding of paclitaxel to an intermediate site within a nanopore, from which it moves directly to its binding site in the microtubule interior facing the lumen. To test this hypothesis, we have computationally docked paclitaxel within a microtubule nanopore and simulated its passage to the intermediate binding site. Targeted molecular dynamics was then used to test the hypothesis that paclitaxel utilizes the H6/H7 loop as a hinge to move directly from this intermediate binding site to its final position in the luminal binding site. We observed that this motion appears to be stabilized by the formation of a hydrogen bond involving serine 275 in beta-tubulin isotypes I, IIa, IIb, IVa, IVb, V, VII, and VIII. Interestingly, this residue is replaced by alanine in the betaIII and VI isotypes. This observation raises the possibility that the observed isotype difference in paclitaxel binding may be a kinetic effect arising from the isotype difference at this residue. We are now able to suggest derivatives of paclitaxel that may reverse the isotype-specificity or lead to an alternate stabilizing hydrogen-bond interaction with tubulin, thus increasing the rate of passage to the luminal binding site and hopefully offering a therapeutic advantage in paclitaxel resistant cases.

Linking Calsequestrin to Lumenal Control of SR Ca 2+ Release

See related article, pages 617–626 The heart, beating constantly over the course of the human lifetime, operates continuously through the process of excitation-contraction coupling. Initiated by a depolarizing influx of Na, a small Ca2+ flux across the sarcolemma causes a large release of Ca2+ from the sarcoplasmic reticulum (SR). Release of this Ca2+ takes place via the ryanodine receptor (RyR), a Ca2+ channel within the SR membrane which is gated by cytoplasmic Ca2+ ([Ca2+]i).1 This gating takes place to a small but significant extent even at the relatively low [Ca2+]i found during diastole within the myocyte (SR Ca2+ leak). Among the striking features of this SR Ca2+ release is its steep nonlinear dependence on the total concentration of Ca2+ found in the lumen of the SR ([Ca2+]SRT). The degree of release at diastolic [Ca2+]i is very low when the [Ca2+]SRT is approximately 50% of the level usually found in an isolated cardiac myocyte.2 However, it increases dramatically as [Ca2+]SRT increases toward its normal level. Of special interest is the effect of SR [Ca2+] on the SR Ca2+ leak through the RyR. In the cardiac ventricular myocyte, this leak takes place just under the sarcolemmal membrane, the location of many proteins whose effects are regulated by Ca2+. The magnitude of SR Ca2+ leak may alter the activity of these proteins. Among the candidate proteins which may be affected by the subsarocolemmal Ca2+ is the sodium-calcium exchanger (NCX). This may be particularly relevant in cells from hearts undergoing chronic failure because the myocytes from these heart have a higher leak rate than normal.2 These …

Amino Acid Residues Gln4020 and Lys4021 of the Ryanodine Receptor Type 1 Are Required for Activation by 4-Chloro-m-cresol

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

Electrogenic Partial Reactions of the Gastric H,K-ATPase

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K(+) and H(+) ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E(1) and P-E(2), are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and </=4.5 in the E(1) conformation; 6.7 and </=2 in the P-E(2) conformation. The equilibrium dissociation constants for K(+) binding on the cytoplasmic side were 11 and 16 mM. The respective equilibrium dissociation constants on the luminal side were obtained via K(+) concentration dependence of the enzyme activity and determined to be 0.11 mM for both luminal binding sites.

The inhibition of the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase by macrocyclic lactones and cyclosporin A.

The pharmacology of macrocyclic lactones is varied, with many beneficial effects in treating disease processes. FK-506, rapamycin and ascomycin have been utilized as immunosuppressant agents. Ivermectin is typically used to treat parasitic worm infections in mammals. Another immunosuppressant, cyclosporin A, is a cyclic oligotide that has similar immunosuppressant properties to those exerted by macrocyclic lactones. Here we report on the inhibition by these compounds of sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase (SERCA) Ca(2+) pumps. Ivermectin, cyclosporin A and rapamycin all inhibited the skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1). In addition, although ivermectin inhibited brain microsomal endoplasmic reticulum (type 2b) Ca(2+)-ATPase, cyclosporin A and rapamycin did not. As cyclosporin A also did not inhibit cardiac Ca(2+)-ATPase activity, this would suggest that it could be an isoform-specific inhibitor. Ivermectin was shown to be the most potent Ca(2+)-ATPase inhibitor of the macrocyclic lactones (IC(50)=7 microM). It appears to show a 'competitive' inhibition with respect to high concentrations of ATP by increasing the regulatory binding site K(m) but without affecting the catalytic site K(m). In addition, ivermectin stabilizes the ATPase in an E1 conformational state, and inhibits Ca(2+) release from the enzyme during turnover. This would suggest that ivermectin inhibits Ca(2+) release from the luminal binding sites of the phosphoenzyme intermediate, a step that is known to be accelerated by high [ATP].

Opioid peptides encrypted in intact milk protein sequences

Opioid agonistic and antagonistic peptides which are inactive within the sequence of the precursor milk proteins can be released and thus activated by enzymatic proteolysis, for example during gastrointestinal digestion or during food processing. Activated opioid peptides are potential modulators of various regulatory processes in the body. Opioid peptides can interact with subepithelial opioid receptors or specific luminal binding sites in the intestinal tract. Furthermore, they may be absorbed and then reach endogenous opioid receptors.

Luminal and non-luminal non-competitive inhibitor binding sites on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor presents two very well differentiated domains for ligand binding that account for different cholinergic properties. In the hydrophilic extracellular region of the alpha subunit exist the binding sites for agonists such as the neurotransmitter acetylcholine, which upon binding trigger the channel opening, and for competitive antagonists such as d-tubocurarine, which compete for the former inhibiting its pharmacological action. For non-competitive inhibitors, a population of low-affinity binding sites have been found at the lipid-protein interface of the nicotinic acetylcholine receptor. In addition, at the M2 transmembrane domain, several high-affinity binding sites have been found for non-competitive inhibitors such as chlorpromazine, triphenylmethylphosphonium, the local anaesthetic QX-222 and the hydrophobic probe trifluoromethyl-iodophenyldiazirine. They are known as luminal binding sites. Although the local anaesthetic meproadifen seems to be located between the hydrophobic domains M2-M3, this locus is considered to form part of the channel mouth, thus this site can also be called a luminal binding site. In contraposition, experimental evidences support the hypothesis of the existence of other high-affinity binding sites for non-competitive inhibitors located not at the channel lumen, but at non-luminal binding domains. Among them, we can quote the binding site for quinacrine, which is located at the lipid-protein interface of the alpha M1 domain, and the binding site for ethidium, which is believed to interact with the wall of the vestibule very far away from both the lumen channel and the lipid membrane surface. The aim of this review is to discuss these recent findings relative to both structurally and functionally relevant aspects of non-competitive inhibitors of the nicotinic acetylcholine receptor. We will put special emphasis on the description of the localization of molecules with non-competitive antagonist properties that bind with high-affinity to luminal and non-luminal domains. The information described herein was principally obtained by means of methods such as photolabelling and site-directed mutagenesis in combination with patch-clamp. Our laboratory has contributed with data obtained by using biophysical approaches such as paramagnetic electron spin resonance and quantitative fluorescence spectroscopy.

Activation and binding volumes of the sarcoplasmic reticulum transport enzyme activated by calcium or strontium

The effect of pressure on the hydrolysis of dinitrophenyl phosphate (DnpP) and p-nitrophenyl phosphate (NpP) by the sarcoplasmatic reticulum transport enzyme in permeabilized and native closed vesicles activated by calcium or strontium, respectively, in aqueous and Me2SO-containing media has been studied. At atmospheric pressure, the enzyme in permeabilized vesicles, saturated with respect to substrates and activating ions, hydrolyzes DnpP ten times faster than NpP; for both substrates, calcium activation exceeds that by strontium only a little (20%). In aqueous media the enzyme displays, under all activating conditions, an almost identical curvilinear relationship between the logarithm of enzyme activity and pressure. The data were analysed on the basis of a simplified reaction scheme, in which two unidirectionally proceeding substrate-driven pressure-dependent reactions (k2, k4) cyclically transfer high-affinity into low-affinity binding sites which are assumed to be in equilibrium with either calcium or strontium. The fitting procedure yielded two sets of positive activation volumes delta V2* = 90-110 ml/mol and delta V4* = 15-25 ml/mol. Substrate specificity, as well as the effect of temperature, are exclusively localized in the pressure-independent rate constants k'2 and k'4. Considerable different pressure/activity relations characterized by a single activation volume of 20 ml/mol were obtained for the strongly suppressed substrate hydrolysis of native closed vesicles. At atmospheric pressure DnpP hydrolysis of open vesicles is inhibited by Me2SO, while NpP hydrolysis is considerably activated, irrespective of its activation by calcium or strontium. In the presence of 22.5% Me2SO, the activation volumes are reduced by 50-70 ml/mol. The rate constants of DnpP and NpP hydrolysis are either augmented or reduced by rising Me2SO concentrations, depending on the corresponding supporting substrate. Me2SO has only a slight effect on the pressure dependence of substrate hydrolysis by native vesicles. The small activation volume observed for the activity of native vesicles could be assigned on account of the simplified reaction scheme of the slow reaction step k4, by which the enzyme is transferred from its low-affinity into its high-affinity binding state. Volume changes connected with the binding of calcium or strontium to the luminal binding site of the enzyme were deduced from the observed activation volume and the computed volume change of the slow reaction step (delta V4*).