Microsomal cytochrome P-450: substrate binding, membrane interactions, and topology

The microsomal monoxygenase system is of paramount importance for the metabolism of endogenous substrates and xenobiotics. It is capable of detoxifying many compounds, but also activates procarcinogens to carcinogens. Cytochrome P-450 is the terminal enzyme of the monoxygenase system. In this article we briefly review current knowledge of the nature of its active site, its interaction with the membrane, and its topology in the membrane. In contrast to previous proposals there is now strong evidence that cytochrome P-450 spans the membrane with only one short segment. Analysis of tryptophan fluorescence gives further evidence that most of the protein's mass protrudes from the membrane into the cytosolic space.     

Gene structure, expression analysis, and membrane topology of RDH4

The murine retinol dehydrogenase RDH4 oxidizes several cis-isomers of retinol into their corresponding aldehydes. We have determined the structure of the murine gene, investigated the temporal and spatial expression of the enzyme, and analyzed the membrane topology of the enzyme. The gene has four translated exons, and several alternatively spliced exons in the 5'-untranslated region were identified. Immunohistochemical analysis showed expression of RDH4 in developing and adult mouse eye, particularly in the retinal pigment epithelium. In nonocular adult tissues, including liver, kidney, lung, and skin, RDH4 expression was widespread. The results suggest that RDH4 may have a dual and tissue-specific role in oxidation of 9-cis- and 11-cis-isomers of retinol into 9-cis-retinal and 11-cis-retinal, respectively. Furthermore, the lumenal orientation of the enzyme domain in the ER suggests that oxidation of both cis-isomers of retinol occurs in the ER.     

Membrane topology prediction by hydropathy profile alignment: membrane topology of the Na(+)-glutamate transporter GltS

Structural classification of families of membrane proteins by bioinformatics techniques has become a critical aspect of membrane protein research. We have proposed hydropathy profile alignment to identify structural homology between families of membrane proteins. Here, we demonstrate experimentally that two families of secondary transporters, the ESS and 2HCT families, indeed share similar folds. Members of the two families show highly similar hydropathy profiles but cannot be shown to be homologous by sequence similarity. A structural model was predicted for the ESS family transporters based upon an existing model of the 2HCT family transporters. In the model, the transporters fold into two domains containing five transmembrane segments and a reentrant or pore-loop each. The two pore-loops enter the membrane embedded part of the proteins from opposite sides of the membrane. The model was verified by accessibility studies of cysteine residues in single-Cys mutants of the Na+-glutamate transporter GltS of Escherichia coli, a member of the ESS family. Cysteine residues positioned in predicted periplasmic loops were accessible from the periplasm by a bulky, membrane-impermeable thiol reagent, while cysteine residues in cytoplasmic loops were not. Furthermore, two cysteine residues in the predicted pore-loop entering the membrane from the cytoplasmic side were shown to be accessible for small, membrane-impermeable thiol reagents from the periplasm, as was demonstrated before for the Na+-citrate transporter CitS of Klebsiella pneumoniae, a member of the 2HCT family. The data strongly suggests that GltS of the ESS family and CitS of the 2HCT family share the same fold as was predicted by comparing the averaged hydropathy profiles of the two families.     

Analysis of the pH dependence of folate binding and transport by rat kidney brush border membrane vesicles

Folate reabsorption by the mammalian kidney occurs following a tight binding reaction with the renal brush border membrane. Previous studies have shown that transport of folic acid (PteGlu) by rat kidney brush border membrane vesicles occurs maximally at pH 5.6 via a saturable system that is associated with a binding component. The present studies have shown that the pH dependency of transport was due to the development of the transmembrane pH gradient (7.3 in/5.6 out), not to the acidic pH per se. The pH gradient-mediated transport was stimulated by an inwardly directed ionic gradient, either of NaCl or choline chloride. These gradients also stimulated the membrane binding of PteGlu suggesting that NaCl and choline chloride may have increased PteGlu transport by altering binding to the brush border membrane. Renal brush border membrane vesicular transport of PteGlu was not affected by induction of a relatively positive intravesicular space. Transport was inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene, an anion exchange inhibitor. The results suggest that rat kidney brush border membrane transport of PteGlu is initiated by association with a specific membrane protein, followed by transfer of folate across the membrane. The overall activity is influenced by a transmembrane pH gradient.     

Functional and structural membrane topology of rat liver microsomal glutathione transferase

The membrane topology of rat liver microsomal glutathione transferase was investigated by comparing the tryptic cleavage products from intact and permeabilized microsomes. It was shown that lysine-4 of microsomal glutathione transferase is accessible at the luminal surface of the endoplasmic reticulum, whereas lysine-41 faces the cytosol. These positions are separated by a hydrophobic stretch of 25 amino acids (positions 11–35) which comprises the likely membrane-spanning region. Reaction of cysteine-49 of the microsomal glutathione transferase with the charged sulfhydryl reagent DTNB (2,2′-dithiobis(5-nitrobenzoic acid))) in intact microsomes further supports the cytosolic localization of this portion of the polypeptide chain. The role of two other potential membrane-spanning/associated segments in the C-terminal half of the polypeptide chain was examined by investigating the association of the protein to the membrane after trypsin cleavage at lysine-41. Activity measurements and Western blot analysis after washing with high concentrations of salt, as well as after phase separation in Triton X-114, indicate that this portion of the protein also binds to the membrane. It is also shown that cleavage of the purified protein at Lys-41 and subsequent separation of the fragments obtained yields a functional C-terminal polypeptide with the expected length for the product encompassing positions 42–154. The location of the active site of microsomal glutathione transferase was investigated using radiolabelled glutathione together with a second substrate. Since isolated rat liver microsomes do not take up glutathione or release the glutathione conjugate into the lumen, it can be concluded that the active site of rat liver microsomal glutathione transferase faces the cytosolic side of the endoplasmic reticulum.     

pH dependence of the effect of adenosine triphosphate and ethylenediaminetetraacetate on sodium and magnesium binding by cellular membrane fragments

The pH dependence of previously reported effects of adenosine triphosphate (ATP) and ethylenediaminetetraacetate (EDTA) on cation binding by rat liver microsomes was studied by an equilibration and washing procedure. Equilibration of microsomes in media containing 95 mM NaCl and 4 mM MgCl₂ with pH varied from 4 to 8 resulted in an increase in bound cations from zero below pH 4 to 0.90 mmoles Mg and 0.34 mmoles Na/g N at pH 8; the ratio of bound Na/bound Mg increased from 0.15 at pH 5 to 0.38 at pH 8. Addition of 5 mM EDTA to the equilibration media produced striking changes in cation binding such that bound Na/bound Mg increased from 0.30 at pH 5 to 3.90 at pH 7 and decreased to 3.55 at pH 8. In the presence of added 10 mM ATP, bound Na/bound Mg increased from 0.10 at pH 5 to a maximum of 0.80 at pH 7. The observed changes could generally be correlated with known mass law relationships, although the system containing added ATP was complicated considerably by the hydrolysis of ATP. Results demonstrate that environmental pH is an important factor in determining the effect of ATP and EDTA on the cation binding pattern of cellular membranes. Because hydrogen ion is a product of ATP hydrolysis as well as of other metabolic reactions, the described interactions may be of particular significance in the molecular mechanisms of ATP effects on cation binding and transport in living cells.     

The pH dependence of binding of alpha,alpha'-dibromo-p-xylenesulfonic acid to lysozyme.

The chemical modification of lysozyme (I) has been accomplished with alpha, alpha'-dibromo-p-xylenesulfonic acid (DBX) at five different pH values. I was alkylated by DBX at room temperature (28 degrees C) with decrease in enzyme activity. The rate of inactivation depended upon the pH at which alkylation was carried out. The highest rate was seen at alkaline pH values; the lowest at more acidic pH values. Amino acid analyses showed that-two lysines and two tryptophan residues had been modified at pH 9; two lysines, one tryptophan and one methionine had reacted at pH 8. A histidine residue was bound at pH 6.5 together with a tryptophan residue. At the lower pH values (2.7, 4.5, 6.5), alkylation occurred with a single tryptophan residue each. Fluorescence and CD data both ruled out the participation of tryptophans 62 or 108. Labeling experiments showed that two residues of DBX-35S were bound per molecule of I at both pH9 and pH8; one residue of DBX was bound per molecule of I at the other pH values. Sedimentation coefficients were characteristic of native lysozyme. The stoichiometry of binding and residue modification indicated that intra-molecular cross links were established. The pH dependence of the cross-linking provides means to measure several allowed intra-molecular distances. The results presented here are consistent with the existence of side chain motion in lysozyme.     

HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation,membrane binding,and electrostatics,and implications for HIV-host cell fusion

The HIV gp41 protein catalyzes fusion between HIV and target cell membranes. The fusion states of the gp41 ectodomain include early coiled-coil (CC) structure and final six-helix bundle (SHB) structure. The ectodomain has an additional N-terminal apolar fusion peptide (FP) sequence which binds to target cell membranes and plays a critical role in fusion. One approach to understanding gp41 function is study of vesicle fusion induced by constructs that encompass various regions of gp41. There are apparent conflicting literature reports of either rapid or no fusion of negatively charged vesicles by SHB constructs. These reports motivated the present study, which particularly focused on effects of pH because the earlier high and no fusion results were at pH 3.0 and 7.2, respectively. Constructs include “Hairpin,” which has SHB structure but lacks the FP, “FP-Hairpin” with FP + SHB, and “N70,” which contains the FP and part of the CC but does not have SHB structure. Aqueous solubility, membrane binding, and vesicle fusion function were measured at a series of pHs and much of the pH dependences of these properties were explained by protein charge. At pH 3.5, all constructs were positively charged, bound negatively charged vesicles, and induced rapid fusion. At pH 7.0, N70 remained positively charged and induced rapid fusion, whereas Hairpin and FP-Hairpin were negatively charged and induced no fusion. Because viral entry occurs near pH 7 rather than pH 3, our results are consistent with fusogenic function of early CC gp41 and with fusion arrest by final SHB gp41.     

Mapping the membrane topology and extracellular ligand binding domains of the retinol binding protein receptor

STRA6 is a multitransmembrane domain protein not homologous to any other proteins with known function. It functions as the high-affinity receptor for plasma retinol binding protein (RBP) and mediates cellular uptake of vitamin A from the vitamin A-RBP complex. Consistent with the diverse roles of vitamin A and the wide tissue expression pattern of STRA6, mutations in STRA6 are associated with severe pathological phenotypes in humans. The structural basis for STRA6's biochemical function is unknown. Although computer programs predict 11 transmembrane domains for STRA6, its topology has never been studied experimentally. Elucidating the transmembrane topology of STRA6 is critical for understanding its structure and function. By inserting an epitope tag into all possible extracellular and intracellular domains of STRA6, we systematically analyzed the accessibility of each tag on the surface of live cells, the accessibility of each tag in permeabilized cells, and the effect of each tag on RBP binding and STRA6-mediated vitamin A uptake from the vitamin A-RBP complex. In addition, we used a new lysine accessibility technique combining cell-surface biotinylation and tandem-affinity purification to study a region of the protein not revealed by the epitope tagging method. These studies not only revealed STRA6's extracellular, transmembrane, and intracellular domains but also implicated extracellular regions of STRA6 in RBP binding.     

pH dependence of carbon monoxide binding to ferrous horseradish peroxidase

The kinetic parameters of the reaction of horseradish peroxidase with CO have been determined at pH values between 10 and 3. At pH 7.0 the CO binding equilibrium constant L was measured using submicromolar concentrations of horseradish peroxidase; the value obtained corresponds to the ratio of the association and dissociation kinetic constants as expected for a simple binding mechanism to a monomeric hemeprotein. The CO association rate constant is pH-independent below pH 7, whereas in going from pH 7 to pH 11 a 2-fold increase can be detected, as previously reported (Kertesz, D., Antonini, E., Brunori, M., Wyman, J., and Zito, R. (1965) Biochemistry 4, 2672-2676). On the other hand, CO dissociation displays a peculiar pH rate profile characterized by a progressive decrease from pH 10 to pH 5 and by a very marked increase as the pH is further lowered to pH congruent to 3. Furthermore, the rate of CO dissociation is markedly enhanced in peroxidase reconstituted with protoheme dimethyl ester, suggesting a role of the propionates in the regulation of this process.     

Reaction mechanism of chalcone isomerase. pH dependence, diffusion control, and product binding differences.

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of bicyclic chalcones into tricyclic (S)-flavanones. The activity of CHI is essential for the biosynthesis of flavanone precursors of floral pigments and phenylpropanoid plant defense compounds. We have examined the spontaneous and CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, 2',4'-dihydroxychalcone, and 4,2'-dihydroxychalcone into the corresponding flavanones. The pH dependence of flavanone formation indicates that both the non-enzymatic and enzymatic reactions first require the bulk phase ionization of the substrate 2'-hydroxyl group and subsequently on the reactivity of the newly formed 2'-oxyanion during C-ring formation. Solvent viscosity experiments demonstrate that at pH 7.5 the CHI-catalyzed cyclization reactions of 4,2',4',6'-tetrahydroxychalcone, 4,2',4'-trihydroxychalcone, and 2',4'-dihydroxychalcone are approximately 90% diffusion-controlled, whereas cyclization of 4,2'-dihydroxychalcone is limited by a chemical step that likely reflects the higher pK(a) of the 2'-hydroxyl group. At pH 6.0, the reactions with 4,2',4',6'-tetrahydroxychalcone and 4,2',4'-trihydroxychalcone are approximately 50% diffusion-limited, whereas the reactions of both dihydroxychalcones are limited by chemical steps. Comparisons of the 2.1-2.3 A resolution crystal structures of CHI complexed with the products 7,4'-dihydroxyflavanone, 7-hydroxyflavanone, and 4'-hydroxyflavanone show that the 7-hydroxyflavanones all share a common binding mode, whereas 4'-hydroxyflavanone binds in an altered orientation at the active site. Our functional and structural studies support the proposal that CHI accelerates the stereochemically defined intramolecular cyclization of chalcones into biologically active (2S)-flavanones by selectively binding an ionized chalcone in a conformation conducive to ring closure in a diffusion-controlled reaction.     

RNA binding characteristics and overall topology of the vaccinia poly(A) polymerase-processivity factor-primer complex.

The vaccinia virus-encoded heterodimer responsible for poly(A) tail elongation comprises a polyadenylylation catalytic subunit (VP55) and associated processivity factor (VP39). We show that monomeric VP39's affinity for RNA homopolymers follows the hierarchy poly(I) >poly(U) >poly(G) >poly(A) >poly(C), that the heterodimer interacts stably with 40-45 nucleotide nucleic acid segments, and that its homopolymer preference for polyadenylylation priming is comparable to the VP39 affinity hierarchy (above). For oligonucleotide ligands possessing the previously-identified (rU)2-(N)25-rU heterodimer-binding motif, the heterodimer's affinity and base-type preference are mediated via both the (rU)2and rU portions, with the greater contribution coming from (rU)2. VP39's R107 sidechain contributes to specificity at the downstream rU. Substitution of each ribouridylate of the motif with either ribothymidine or 4-thiodeoxythymidine indicated that the downstream rU interacts with both heterodimer subunits, whereas the upstream (rU)2interacts only with VP55. A 'crosslinking SELEX' approach indicated VP39-base proximity around position -10 of a 4-thioribouridine/deoxycytidine ligand pool. Upon incubating the heterodimer with a panel of identical-sequence oligonucleotides derivatized with azidophenacyl bromide at various phosphate positions, those derivatized at positions -11 to -21 photocrosslinked to both subunits in a coordinated manner. This region may therefore pass through a 'cleft' or enclosed 'channel' at the subunit interface.     

pH dependence of photosystem II photoinhibition: relationship with structural transition of oxygen-evolving complex at the pH of thylakoid lumen

Photosynthesis Research - Ca-depleted photosystem II membranes (PSII[-Ca]) do not contain PsbP and PsbQ proteins protecting the Mn4CaO5 cluster of the PSII oxygen-evolving complex (OEC). Therefore,...     

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface pK_a, antibody pI, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface pK_a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab₂ antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.     

Spontaneous, reversible protein cross-linking in the human erythrocyte membrane. Temperature and pH dependence.

Changes in pH significantly affect the morphology and physical properties of red cell membranes. We have explored the molecular basis for these phenomena by characterizing the pattern of protein disulfide cross-linkages formed spontaneously in ghost exposed to acid pH or elevated temperature (37 degrees C). Protein aggregation was analyzed by two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulfate. incubation of ghosts at pH 4.0 to 5.5 (0-4 degrees C) yielded (i) complexes of spectrin and band 3, (ii) complexes of actin and band 3, (iii) band 3 complexes, i.e. dimer and trimer, and (iv) heterogeneous aggregates involving spectrin, band 3, band 4.2, and actin in varying proportions. Aggregation was maximal near the isoelectric points of the major membrane proteins, and appeared to reflect (i) the aggregation of intramembrane particles including band 3 and (ii) more intimate contact between spectrin-actin meshwork and band 3.     

Studies on membrane topology, N-glycosylation and functionality of SARS-CoV membrane protein

Background

Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis.

Results

Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein.

Conclusion

Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.     

Isocitrate lyase from Pseudomonas indigofera: pH dependence of catalysis and binding of substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomonas indigofera was dependent on two dissociable groups (pK_a's of 6.9 and 8.6). The pH dependence of the pK_i for succinate, a product of isocitrate cleavage, implied that a dissociable group (pK_a of 6.0) on the enzyme functions in binding succinate. The pK_i's for maleate and itaconate (succinate analogs) were similarly pH dependent. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the pK_i's of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the pK_m for isocitrate and the pK_i for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme (pK_a of 5.8). The pH dependence of the pK_i for homoisocitrate was similar. In addition the K_i for succinate and K_m for isocitrate were dependent upon Mg²⁺ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofera, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (pK_a of 5.8) and one on phosphoenolpyruvate (pK_a of 6.35), contributed to the pH dependence observed with phosphoenolpyruvate.