Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the FO sector of Escherichia coli ATP synthase

Subunit rotation within the F1 catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded FO sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the single-copy a subunit (stator). Here we report evidence for an energy-dependent rotation at this interface. FOF1 was expressed with a pair of substituted cysteines positioned to allow an intersubunit disulfide crosslink between subunit a and a c subunit [aN214C/cM65C; Jiang, W. & Fillingame, R. H. (1998) Proc. Natl. Acad. Sci. USA 95, 6607–6612]. Membranes were treated with N,N′-dicyclohexyl-[14C]carbodiimide to radiolabel the D61 residue on less than 20% of the c subunits. After oxidation to form an a–c crosslink, the c subunit properly aligned to crosslink to subunit a was found to contain very little 14C label relative to other members of the c ring. However, exposure to MgATP before oxidation significantly increased the radiolabel in the a–c crosslink, indicating that a different c subunit was now aligned with subunit a. This increase was not induced by exposure to MgADP/Pi. Furthermore, preincubation with MgADP and azide to inhibit F1 or with high concentrations of N,N′-dicyclohexylcarbodiimide to label most c subunits prevented the ATP effect. These results provide evidence for an energy-dependent rotation of the c ring relative to subunit a.

Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering

A previously unknown chemical structure, 6-desmethyl-6-ethylerythromycin A (6-ethylErA), was produced through directed genetic manipulation of the erythromycin (Er)-producing organism Saccharopolyspora erythraea. In an attempt to replace the methyl side chain at the C-6 position of the Er polyketide backbone with an ethyl moiety, the methylmalonate-specific acyltransferase (AT) domain of the Er polyketide synthase was replaced with an ethylmalonate-specific AT domain from the polyketide synthase involved in the synthesis of the 16-member macrolide niddamycin. The genetically altered strain was found to produce ErA, however, and not the ethyl-substituted derivative. When the strain was provided with precursors of ethylmalonate, a small quantity of a macrolide with the mass of 6-ethylErA was produced in addition to ErA. Because substrate for the heterologous AT seemed to be limiting, crotonyl-CoA reductase, a primary metabolic enzyme involved in butyryl-CoA production in streptomycetes, was expressed in the strain. The primary macrolide produced by the reengineered strain was 6-ethylErA.

On the reaction mechanism of l-lactate oxidase: Quantitative structure-activity analysis of the reaction with para-substituted l-mandelates

The rate constants for reduction of the flavoenzyme, l-lactate oxidase, and a mutant (in which alanine 95 is replaced by glycine), by a series of para-substituted mandelates, in both the 2-1H- and 2-2H- forms, have been measured by rapid reaction spectrophotometry. In all cases, significant isotope effects (1H/2H = 3–7) on the rate constants of flavin reduction were found, indicating that flavin reduction is a direct measure of α-C-H bond breakage. The rate constants show only a small influence of the electronic characteristics of the substituents, but show a good correlation when combined with some substituent volume parameters. A surprisingly good correlation is found with the molecular mass of the substrate. The results are compatible with any mechanism in which there is little development of charge in the transition state. This could be a transfer of hydride to the flavin N(5) position or a synchronous mechanism in which the α-C-H is formally abstracted as a H+ while the resulting charge is simultaneously neutralized by another event.

Up-regulation of specific tyrosinase mRNAs in mouse melanomas with the c2j gene substituted for the wild-type tyrosinase allele: Utilization in design of syngeneic immunotherapy models

The expression of cell-specialization genes is likely to be changing in tumor cells as their differentiation declines. Functional changes in these genes might yield unusual peptide epitopes with anti-tumor potential and could occur without modification in the DNA sequence of the gene. Melanomas undergo a characteristic decline in melanization that may reflect altered contributions of key melanocytic genes such as tyrosinase. Quantitative reverse transcriptase–PCR of the wild-type (C) tyrosinase gene in transgenic (C57BL/6 strain) mouse melanomas has revealed a shift toward alternative splicing of the pre-mRNA that generated increased levels of the Δ1b and Δ1d mRNA splice variants. The spontaneous c2j albino mutation of tyrosinase (in the C57BL/6 strain) changes the pre-mRNA splicing pattern. In c2j/c2j melanomas, alternative splicing was again increased. However, while some mRNAs (notably Δ1b) present in C/C were obligatorily absent, others (Δ3 and Δ1d) were elevated. In c2j/c2j melanomas, the percentage of total tyrosinase transcripts attributable to Δ3 reached approximately 2-fold the incidence in c2j/c2j or C/C skin melanocytes. The percentage attributable to Δ1d rose to approximately 2-fold the incidence in c2j/c2j skin, and to 10-fold that in C/C skin. These differences provide a basis for unique mouse models in which the melanoma arises in skin grafted from a C/C or c2j/c2j transgenic donor to a transgenic host of the same or opposite tyrosinase genotype. Immunotherapy designs then could be based on augmenting those antigenic peptides that are novel or overrepresented in a tumor relative to the syngeneic host.

Structure and function in rhodopsin: Further elucidation of the role of the intradiscal cysteines, Cys-110, -185, and -187, in rhodopsin folding and function

The disulfide bond between Cys-110 and Cys-187 in the intradiscal domain is required for correct folding in vivo and function of mammalian rhodopsin. Misfolding in rhodopsin, characterized by the loss of ability to bind 11-cis-retinal, has been shown to be caused by an intradiscal disulfide bond different from the above native disulfide bond. Further, naturally occurring single mutations of the intradiscal cysteines (C110F, C110Y, and C187Y) are associated with retinitis pigmentosa (RP). To elucidate further the role of every one of the three intradiscal cysteines, mutants containing single-cysteine replacements by alanine residues and the above three RP mutants have been studied. We find that C110A, C110F, and C110Y all form a disulfide bond between C185 and C187 and cause loss of retinal binding. C185A allows the formation of a C110–C187 disulfide bond, with wild-type-like rhodopsin phenotype. C187A forms a disulfide bond between C110 and C185 and binds retinal, and the pigment formed has markedly altered bleaching behavior. However, the opsin from the RP mutant C187Y forms no rhodopsin chromophore.

Role of cysteines in Plasmodium falciparum circumsporozoite protein: Interactions with heparin can rejuvenate inactive protein mutants

Various pathogenic bacteria, viruses, and protozoan bind to glycosaminoglycan-based receptors on host cells and initiate an infection. Sporozoites of Plasmodium predominantly express circumsporozoite (CS) protein on their surface, which binds to heparan sulfate proteoglycans on liver cell surface that subsequently leads to malaria. Here we show that the interaction of free heparin with this parasite ligand has the potential to be a critical component of invasion. CS protein of P. falciparum contains four cysteines at positions 361, 365, 396, and 401. In this study, all four cysteine residues were mutagenized to alanine both individually and in different combinations. Conversion of cysteine 396 to alanine (protein CS3) led to a 10-fold increase in the binding activity of the protein to HepG2 cells. Replacement of cysteines at positions 361, 365, and 401 either alone or in different combinations led to a near total loss of binding. Surprisingly, activity in these inactive mutants could be effectively restored in the presence of submolar concentrations of heparin. Heparin also up-regulated binding of CS3 at submolar concentrations with respect to the protein but down-regulated binding when present in excess. Given the significantly different concentrations of heparin in different organs of the host and the in vitro results described here one can consider in vivo ramifications of this phenomenon for pathogen targeting of specific organs and for the functional effects of antigenic variation on receptor ligand interaction.

Zinc- and iron-rubredoxins from Clostridium pasteurianum at atomic resolution: a high-precision model of a ZnS4 coordination unit in a protein.

The Zn(Scys)4 unit is present in numerous proteins, where it assumes structural, regulatory, or catalytic roles. The same coordination is found naturally around iron in rubredoxins, several structures of which have been refined at resolutions of, or near to, 1 A. The fold of the small protein rubredoxin around its metal ion is an excellent model for many zinc finger proteins. Zn-substituted rubredoxin and its Fe-containing counterpart were both obtained as the products of the expression in Escherichia coli of the rubredoxin-encoding gene from Clostridium pasteurianum. The structures of both proteins have been refined with an anisotropic model at atomic resolution (1.1 A, R = 8.3% for Fe-rubredoxin, and 1.2 A, R = 9.6% for Zn-rubredoxin) and are very similar. The most significant differences are increased lengths of the M-S bonds in Zn-rubredoxin (average length, 2.345 A) as compared with Fe-rubredoxin (average length, 2.262 A). An increase of the CA-CB-SG-M dihedral angles involving Cys-6 and Cys-39, the first cysteines of each of the Cys-Xaa-Xaa-Cys metal binding motifs, has been observed. Another consequence of the replacement of iron by zinc is that the region around residues 36-46 undergoes larger displacements than the remainder of the polypeptide chain. Despite these changes, the main features of the FeS4 site, namely a local 2-fold symmetry and the characteristic network of N-H...S hydrogen bonds, are conserved in the ZnS4 site. The Zn-substituted rubredoxin provides the first precise structure of a Zn(Scys)4 unit in a protein. The nearly identical fold of rubredoxin around iron or zinc suggests that at least in some of the sites where the metal has mainly a structural role-e.g., zinc fingers-the choice of the relevant metal may be directed by its cellular availability and mobilization processes rather than by its chemical nature.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

Mutagenesis of palmitoylation sites in endothelial nitric oxide synthase identifies a novel motif for dual acylation and subcellular targeting.

The endothelial nitric oxide synthase (ec-NOS) plays a key role in the transduction of signals from the bloodstream to the underlying smooth muscle. ecNOS undergoes a complex series of covalent modifications, including myristoylation and palmitoylation, which appear to play a role in ecNOS membrane association. Mutagenesis of the myristoylation site, which prevents both myristoylation and palmitoylation, blocks ecNOS targeting to cell membranes. Further, as described for some G-protein alpha subunits, both membrane association and palmitoylation of ecNOS are dynamically regulated: in response to agonists, the enzyme undergoes partial redistribution to the cell cytosol concomitant with depalmitoylation. To clarify the role of palmitoylation in determining ecNOS subcellular localization, we have constructed palmitoylation-deficient mutants of ecNOS. Serine was substituted for cysteine at two potential palmitoylation sites (Cys-15 and Cys-26) by site-directed mutagenesis. Immunoprecipitation of ecNOS mutants following cDNA transfection and biosynthetic labeling with [3H]palmitate revealed that mutagenesis of either cysteine residue attenuated palmitoylation, whereas replacement of both residues completely eliminated palmitoylation. Analysis of N-terminal deletion mutations of ecNOS demonstrated that the region containing these two cysteine residues is both necessary and sufficient for enzyme palmitoylation. The cysteines thus identified as the palmitoylation sites for ecNOS are separated by an unusual (Gly-Leu)5 sequence and appear to define a sequence motif for dual acylation. We analyzed the subcellular distribution of ecNOS mutants by differential ultracentrifugation and found that mutagenesis of the ecNOS palmitoylation sites markedly reduced membrane association of the enzyme. These results document that ecNOS palmitoylation is an important determinant for the subcellular distribution of ecNOS and identify a new motif for the reversible palmitoylation of signaling proteins.

Evidence that the pathway of disulfide bond formation in Escherichia coli involves interactions between the cysteines of DsbB and DsbA.

Disulfide bond formation is catalyzed in the periplasm of Escherichia coli. This process involves at least two proteins: DsbA and DsbB. Recent evidence suggests that DsbA, a soluble periplasmic protein directly catalyzes disulfide bond formation in proteins, whereas DsbB, an inner membrane protein, is involved in the reoxidation of DsbA. Here we present direct evidence of an interaction between DsbA and DsbB. (Kishigami et al. [Kishigami, S., Kanaya, E., Kikuchi, M. & Ito, K. (1995) J. Biol. Chem. 270, 17072-17074] have described similar findings.) We isolated a dominant negative mutant of dsbA, dsbAd, where Cys-33 of the DsbA active site is changed to tyrosine. Both DsbAd and DsbA are able to form a mixed disulfide with DsbB, which may be an intermediate in the reoxidation of DsbA. This complex is more stable with DsbAd. The dominance can be suppressed by increasing the production of DsbB. By using mutants of DsbB in which one or two cysteines have been changed to alanine, we show that only Cys-104 is important for complex formation. Therefore, we suggest that in vivo, reduced DsbA forms a complex with DsbB in which Cys-30 of DsbA is disulfide-bonded to Cys-104 of DsbB. Cys-104 is rapidly replaced by Cys-33 of DsbA to generate the oxidized form of this protein.