Peroxynitrite-mediated nitration of tyrosine residues in Escherichia coli glutamine synthetase mimics adenylylation: relevance to signal transduction.

Treatment of Escherichia coli glutamine synthetase (GS) with peroxynitrite leads to nitration of some tyrosine residues and conversion of some methionine residues to methionine sulfoxide (MSOX) residues. Nitration, but not MSOX formation, is stimulated by Fe-EDTA. In the absence of Fe-EDTA, nitration of only one tyrosine residue per subunit of unadenylylated GS leads to changes in divalent cation requirement, pH-activity profile, affinity for ADP, and susceptibility to feedback inhibition by end products (tryptophan, AMP, CTP), whereas nitration of one tyrosine residue per subunit in the adenylylated GS leads to complete loss of catalytic activity. In the presence of Fe-EDTA, nitration is a more random process: nitration of five to six tyrosine residues per subunit is needed to convert unadenylylated GS to the adenylylated configuration. These results and the fact that nitration of tyrosine residues is an irreversible process serve notice that the regulatory function of proteins that undergo phosphorylation or adenylylation in signal transduction cascades might be seriously compromised by peroxynitrite-promoted nitration.

Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins.

An enzyme that reduces methionine sulfoxide [Met(O)] residues in proteins [peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli] was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced. The mammalian homologue of E. coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da. This protein has 61% identity with the E. coli MsrA throughout a region encompassing a 199-amino acid overlap. The protein has been overexpressed in E. coli and purified to homogeneity. The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide. Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney. This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.

Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by site-directed mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of the [Fe4S4]3+ core.

Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K+ channel selectively modulates channel gating.

Voltage-gated channel proteins sense a change in the transmembrane electric field and respond with a conformational change that allows ions to diffuse across the pore-forming structure. Site-specific mutagenesis combined with electrophysiological analysis of expressed mutants in amphibian oocytes has previously established the S4 transmembrane segment as an element of the voltage sensor. Here, we show that mutations of conserved negatively charged residues in S2 and S3 of a brain K+ channel, thought of as countercharges for the positively charged residues in S4, selectively modulate channel gating without modifying the permeation properties. Mutations of Glu235 in S2 that neutralize or reverse charge increase the probability of channel opening and the apparent gating valence. In contrast, replacements of Glu272 by Arg or Thr268 by Asp in S3 decrease the open probability and the apparent gating valence. Residue Glu225 in S2 tolerated replacement only by acidic residues, whereas Asp258 in S3 was intolerant to any attempted change. These results imply that S2 and S3 are unlikely to be involved in channel lining, yet, together with S4, may be additional components of the voltage-sensing structure.

Residues in the pathway through a membrane transporter.

The structure of solute transporters is understood largely from analysis of their amino acid sequences, and more direct information is greatly needed. Here we report work that applies cysteine scanning mutagenesis to describe structure-function relations in UhpT, a bacterial membrane transporter. By using an impermeant SH-reactive agent to probe single-cysteine variants, we show that UhpT transmembrane segment 7 spans the membrane as an alpha-helix and that the central portion of this helix is exposed to both membrane surfaces, forming part of the translocation pathway through this transporter.

Inhibition of AP-1 binding and transcription by gold and selenium involving conserved cysteine residues in Jun and Fos.

Gold(I) salts and selenite, which have diverse therapeutic and biological effects, are noted for their reactivity with thiols. Since the binding of Jun-Jun and Jun-Fos dimers to the AP-1 DNA binding site is regulated in vitro by a redox process involving conserved cysteine residues, we hypothesized that some of the biological actions of gold and selenium are mediated via these residues. In electrophoretic mobility-shift analyses, AP-1 DNA binding was inhibited by gold(I) thiolates and selenite, with 50% inhibition occurring at approximately 5 microM and 1 microM, respectively. Thiomalic acid had no effect in the absence of gold(I), and other metal ions inhibited at higher concentrations, in a rank order correlating with their thiol binding affinities. Cysteine-to-serine mutants demonstrated that these effects of gold(I) and selenite require Cys272 and Cys154 in the DNA-binding domains of Jun and Fos, respectively. Gold(I) thiolates and selenite did not inhibit nonspecific protein binding to the AP-1 site and were at least an order of magnitude less potent as inhibitors of sequence-specific binding to the AP-2, TFIID, or NF1 sites compared with the AP-1 site. In addition, 10 microM gold(I) or 10 microM selenite inhibited expression of an AP-1-dependent reporter gene, but not an AP-2-dependent reporter gene. These data suggest a mechanism regulating transcription factor activity by inorganic ions which may contribute to the known antiarthritic action of gold and cancer chemoprevention by selenium.

On the distribution of amino acid residues in transmembrane alpha-helix bundles.

The periodic distribution of residues in the sequence of 469 putative transmembrane alpha-helices from eukaryotic plasma membrane polytopic proteins has been analyzed with correlation matrices. The method does not involve any a priori assumption about the secondary structure of the segments or about the physicochemical properties of individual amino acid residues. Maximal correlation is observed at 3.6 residues per period, characteristic of alpha-helices. A scale extracted from the data describes the propensity of the various residues to lie on the same or on opposite helix faces. The most polar face of transmembrane helices, presumably that buried in the protein core, shows a strong enrichment in aromatic residues, while residues likely to face the fatty acyl chains of lipids are largely aliphatic.

Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

Residue replacements were made at five positions (Arg-73, Asp-76, Tyr-87, Asp-106, and Asp-201) in the Halobacterium salinarium phototaxis receptor sensory rhodopsin I (SR-I) by site-specific mutagenesis. The sites were chosen for their correspondence in position to residues of functional importance in the homologous light-driven proton pump bacteriorhodopsin found in the same organism. This work identifies a residue in SR-I shown to be of vital importance to its attractant signaling function: Asp-201. The effect of the substitution with the isosteric asparagine is to convert the normally attractant signal of orange light stimulation to a repellent signal. In contrast, similar neutral substitution of the four other ionizable residues near the photoactive site allows essentially normal attractant and repellent phototaxis signaling. Wild-type two-photon repellent signaling by the receptor is intact in the Asp-201 mutant, genetically separating the wild-type attractant and repellent signal generation processes. A possible explanation and implications of the inverted signaling are discussed. Results of neutral residue substitution for Asp-76 confirm our previous evidence that proton transfer reactions involving this residue are not important to phototaxis but that Asp-76 functions as the Schiff base proton acceptor in proton translocation by transducer-free SR-I.

Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.

The interacting domains of three MutL heterodimers in man: hMLH1 interacts with 36 homologous amino acid residues within hMLH3, hPMS1 and hPMS2

In human cells, hMLH1, hMLH3, hPMS1 and hPMS2 are four recognised and distinctive homologues of MutL, an essential component of the bacterial DNA mismatch repair (MMR) system. The hMLH1 protein forms three different heterodimers with one of the other MutL homologues. As a first step towards functional analysis of these molecules, we determined the interacting domains of each heterodimer and tried to understand their common features. Using a yeast two-hybrid assay, we show that these MutL homologues can form heterodimers by interacting with the same amino acid residues of hMLH1, residues 492–742. In contrast, three hMLH1 partners, hMLH3, hPMS1 and hPMS2 contain the 36 homologous amino acid residues that interact strongly with hMLH1. Contrary to the previous studies, these homologous residues reside at the N-terminal regions of three subdomains conserved in MutL homologues in many species. Interestingly, these residues in hPMS2 and hMLH3 may form coiled-coil structures as predicted by the MULTICOIL program. Furthermore, we show that there is competition for the interacting domain in hMLH1 among the three other MutL homologues. Therefore, the quantitative balance of these three MutL heterodimers may be important in their functions.

Multidrug resistance proteins QacA and QacB from Staphylococcus aureus: membrane topology and identification of residues involved in substrate specificity.

The closely related multidrug efflux pumps QacA and QacB, from the bacterial pathogen Staphylococcus aureus, both confer resistance to various toxic organic cations but differ in that QacB mediates lower levels of resistance to divalent cations. Cloning and nucleotide sequencing of the qacB gene revealed that qacB differs from qacA by only seven nucleotide substitutions. Random hydroxylamine mutagenesis of qacB was undertaken, selecting for variants that conferred increased resistance to divalent cations. Both QacA and the QacB mutants capable of conferring resistance to divalent cations contain an acidic residue at either amino acid 322 or 323, whereas QacB contains uncharged residues in these positions. Site-directed mutagenesis of qacA confirmed the importance of an acidic residue within this region of QacA in conferring resistance to divalent cations. Membrane topological analysis using alkaline phosphatase and beta-galactosidase fusions indicated that the QacA protein contains 14 transmembrane segments. Thus, QacA represents the first membrane transport protein shown to contain 14 transmembrane segments, and confirms that the major facilitator superfamily contains a family of proteins with 14 transmembrane segments.

The putative actin-binding role of hydrophobic residues Trp546 and Phe547 in chicken gizzard heavy meromyosin.

In the course of myosin-catalyzed ATP hydrolysis, certain amino acid residues in myosin interact with counterparts in actin to produce the relational changes that underlie muscle contraction; some of these interactions are ionic, but the stronger interactions are hydrophobic. In an effort to identify myosin residues participating in hydrophobic interactions, myosin (from smooth muscle) fragments with mutations at suspected sites were engineered and compared with wild-type fragments. It was found that the ATPase of doubly mutated (Trp546Ser and Phe547His) fragments was minimally activated by actin and did not decorate actin well to form the regular arrowhead pattern characteristic of myosin binding to actin filaments. Thus, we suggest that Trp546 and Phe547 are important participants in the hydrophobic actin-myosin interaction.