Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

Withdrawal of IL-7 induces Bax translocation from cytosol to mitochondria through a rise in intracellular pH

IL-7 functions as a trophic factor during T lymphocyte development by a mechanism that is partly based on the induction of Bcl-2, which protects cells from apoptosis. Here we report a mechanism by which cytokine withdrawal activates the prodeath protein Bax. On loss of IL-7 in a dependent cell line, Bax protein translocated from the cytosol to the mitochondria, where it integrated into the mitochondrial membrane. This translocation was attributable to a conformational change in the Bax protein itself. We show that a rise in intracellular pH preceded mitochondrial translocation and triggered the change in Bax conformation. Intracellular pH in the IL-7-dependent cells rose steadily to peak over pH 7.8 by 6 hr after cytokine withdrawal, paralleling the time point of Bax translocation (a similar alkalinization and Bax translocation was also observed after IL-3 withdrawal from a dependent cell line). The conformation of Bax was directly altered by pH of 7.8 or higher and was demonstrated by increased protease sensitivity, exposure of N terminus epitopes, and exposure of a hydrophobic domain in the C terminus. Eliminating charged amino acids at the C or N termini of Bax induced a conformational change similar to that induced by raising pH, implicating these residues in the pH effect. Therefore, we have shown that by either cytokine withdrawal, experimental manipulation of pH, or site-directed mutagenesis, Bax protein changes conformation, exposing membrane-seeking domains, thereby inducing mitochondrial translocation and initiating the cascade of events leading to apoptotic death.

Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position

Acyl-acyl carrier protein (ACP) desaturases introduce double bonds at specific positions in fatty acids of defined chain lengths and are one of the major determinants of the monounsaturated fatty acid composition of vegetable oils. Mutagenesis studies were conducted to determine the structural basis for the substrate and double bond positional specificities displayed by acyl-ACP desaturases. By replacement of specific amino acid residues in a Δ6-palmitoyl (16:0)-ACP desaturase with their equivalents from a Δ9-stearoyl (18:0)-ACP desaturase, mutant enzymes were identified that have altered fatty acid chain-length specificities or that can insert double bonds into either the Δ6 or Δ9 positions of 16:0- and 18:0-ACP. Most notably, by replacement of five amino acids (A181T/A200F/S205N/L206T/G207A), the Δ6-16:0-ACP desaturase was converted into an enzyme that functions principally as a Δ9-18:0-ACP desaturase. Many of the determinants of fatty acid chain-length specificity in these mutants are found in residues that line the substrate binding channel as revealed by x-ray crystallography of the Δ9-18:0-ACP desaturase. The crystallographic model of the active site is also consistent with the diverged activities associated with naturally occurring variant acyl-ACP desaturases. In addition, on the basis of the active-site model, a Δ9-18:0-ACP desaturase was converted into an enzyme with substrate preference for 16:0-ACP by replacement of two residues (L118F/P179I). These results demonstrate the ability to rationally modify acyl-ACP desaturase activities through site-directed mutagenesis and represent a first step toward the design of acyl-ACP desaturases for the production of novel monounsaturated fatty acids in transgenic oilseed crops.

A σ32 mutant with a single amino acid change in the highly conserved region 2.2 exhibits reduced core RNA polymerase affinity

σ32, the product of the rpoH gene in Escherichia coli, provides promoter specificity by interacting with core RNAP. Amino acid sequence alignment of σ32 with other sigma factors in the σ70 family has revealed regions of sequence homology. We have investigated the function of the most highly conserved region, 2.2, using purified products of various rpoH alleles. Core RNAP binding analysis by glycerol gradient sedimentation has revealed reduced core RNAP affinity for one of the mutant σ32 proteins, Q80R. This reduced core interaction is exacerbated in the presence of σ70, which competes with σ32 for binding of core RNAP. When a different but more conserved amino acid was introduced at this position by site-directed mutagenesis (Q80N), this mutant sigma factor still displayed a significant reduction in its core RNAP affinity. Based on these results, we conclude that at least one specific amino acid in region 2.2 is involved in core RNAP interaction.

Activity-based protein profiling: The serine hydrolases

With the postgenome era rapidly approaching, new strategies for the functional analysis of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chemical synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue extracts, we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Additionally, we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.

Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an α-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R2 = 0.97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.

Structure and function in rhodopsin: Topology of the C-terminal polypeptide chain in relation to the cytoplasmic loops*

Cysteine mutagenesis and site-directed spin labeling in the C-terminal region of rhodopsin have been used to probe the local structure and proximity of that region to the cytoplasmic loops. Each of the native amino acids in the sequence T335–T340 was replaced with Cys, one at a time. The sulfhydryl groups of all mutants reacted rapidly with the sulfhydryl reagent 4,4′-dithiodipyridine, which indicated a high degree of solvent accessibility. Furthermore, to probe the proximity relationships, a series of double Cys mutants was constructed. One Cys in all sets was at position 338 and the other was at a position in the sequence S240–V250 in the EF interhelical loop, at position 65 in the AB interhelical loop, or at position 140 in the CD interhelical loop. In the dark state, no significant disulfide formation was observed between C338 and C65 or C140 under the conditions used, whereas a relatively rapid disulfide formation was observed between C338 and C242 or C245. Spin labels in the double Cys mutants showed the strongest magnetic interactions between the nitroxides attached to C338 and C245 or C246. Light activation of the double mutant T242C/S338C resulted in slower disulfide formation, whereas interactions between nitroxides at C338 and C245 or C246 decreased. These results suggest the proximity of the C-terminal residue C338 to residues located on the outer face of a cytoplasmic helical extension of the F helix with an apparent increase of distance upon photoactivation.

Novel mechanism for carbamoyl-phosphate synthetase: A nucleotide switch for functionally equivalent domains

Carbamoyl-phosphate synthetases (CPSs) utilize two molecules of ATP at two internally duplicated domains, B and C. Domains B and C have recently been shown to be structurally [Thoden, J. B., Holden, H. M., Wesenberg, G., Raushel, F. M. & Rayment, I. (1997) Biochemistry 36, 6305–6316] and functionally [Guy, H. I. & Evans, D. R. (1996) J. Biol. Chem. 271, 13762–13769] equivalent. We have carried out a site-directed mutagenic analysis that is consistent with ATP binding to a palmate motif rather than to a Walker A/B motif in domains B and C. To accommodate our present findings, as well as the other recent findings of structural and functional equivalence, we are proposing a novel mechanism for CPS. In this mechanism utilization of ATP bound to domain C is coupled to carbamoyl-phosphate synthesis at domain B via a nucleotide switch, with the energy of ATP hydrolysis at domain C allowing domain B to cycle between two alternative conformations.

ExoY, an adenylate cyclase secreted by the Pseudomonas aeruginosa type III system

The exoenzyme S regulon is a set of coordinately regulated virulence genes of Pseudomonas aeruginosa. Proteins encoded by the regulon include a type III secretion and translocation apparatus, regulators of gene expression, and effector proteins. The effector proteins include two enzymes with ADP-ribosyltransferase activity (ExoS and ExoT) and an acute cytotoxin (ExoU). In this study, we identified ExoY as a fourth effector protein of the regulon. ExoY is homologous to the extracellular adenylate cyclases of Bordetella pertussis (CyaA) and Bacillus anthracis (EF). The homology among the three adenylate cyclases is limited to two short regions, one of which possesses an ATP-binding motif. In assays for adenylate cyclase activity, recombinant ExoY (rExoY) catalyzed the formation of cAMP with a specific activity similar to the basal activity of CyaA. In contrast to CyaA and EF, rExoY activity was not stimulated or activated by calmodulin. A 500-fold stimulation of activity was detected following the addition of a cytosolic extract from Chinese hamster ovary (CHO) cells. These results indicate that a eukaryotic factor, distinct from calmodulin, enhances rExoY catalysis. Site-directed mutagenesis of residues within the putative active site of ExoY abolished adenylate cyclase activity. Infection of CHO cells with ExoY-producing strains of P. aeruginosa resulted in the intracellular accumulation of cAMP. cAMP accumulation within CHO cells depended on an intact type III translocation apparatus, demonstrating that ExoY is directly translocated into the eukaryotic cytosol.

Chemical complementation identifies a proton acceptor for redox-active tyrosine D in photosystem II

Through the use of site-directed mutagenesis and chemical rescue, we have identified the proton acceptor for redox-active tyrosine D in photosystem II (PSII). Effects of chemical rescue on the tyrosyl radical were monitored by EPR spectroscopy. We also have acquired the Fourier–transform infrared (FT-IR) spectrum associated with the oxidation of tyrosine D and concomitant protonation of the acceptor. Mutant and isotopically labeled PSII samples are used to assign vibrational lines in the 3,600–3,100 cm−1 region to N-H modes of His-189 in the D2 polypeptide. When His-189 in D2 is changed to a leucine (HL189D2) in PSII, dramatic alterations of both EPR and FT-IR spectra are observed. When imidazole is introduced into HL189D2 samples, results from both EPR and FT-IR spectroscopy argue that imidazole is functionally reconstituted into an accessible pocket and that imidazole acts as a chemical mimic for His-189. Small perturbations of EPR and FT-IR spectra are consistent with access to this pocket in wild-type PSII, as well. Structures of the analogous site in bacterial reaction centers suggest that an accessible pocket, large enough to contain imidazole, is bordered by tyrosine D and His-189 in the D2 polypeptide. These data provide evidence that His-189 in the D2 polypeptide of PSII acts as a proton acceptor for redox-active tyrosine D and that proton transfer to the imidazole ring facilitates the efficient oxidation/reduction of tyrosine D.

Association of INAD with NORPA is essential for controlled activation and deactivation of Drosophila phototransduction in vivo

Visual transduction in Drosophila is a G protein-coupled phospholipase C-mediated process that leads to depolarization via activation of the transient receptor potential (TRP) calcium channel. Inactivation-no-afterpotential D (INAD) is an adaptor protein containing PDZ domains known to interact with TRP. Immunoprecipitation studies indicate that INAD also binds to eye-specific protein kinase C and the phospholipase C, no-receptor-potential A (NORPA). By overlay assay and site-directed mutagenesis we have defined the essential elements of the NORPA–INAD association and identified three critical residues in the C-terminal tail of NORPA that are required for the interaction. These residues, Phe-Cys-Ala, constitute a novel binding motif distinct from the sequences recognized by the PDZ domain in INAD. To evaluate the functional significance of the INAD–NORPA association in vivo, we generated transgenic flies expressing a modified NORPA, NORPAC1094S, that lacks the INAD interaction. The transgenic animals display a unique electroretinogram phenotype characterized by slow activation and prolonged deactivation. Double mutant analysis suggests a possible inaccessibility of eye-specific protein kinase C to NORPAC1094S, undermining the observed defective deactivation, and that delayed activation may similarly result from NORPAC1094S being unable to localize in close proximity to the TRP channel. We conclude that INAD acts as a scaffold protein that facilitates NORPA–TRP interactions required for gating of the TRP channel in photoreceptor cells.

Recognition Signal for C-Mannosylation of Trp-7 in RNase 2 Consists of Sequence Trp-x-x-Trp

C2-α-Mannosyltryptophan was discovered in human RNase 2, an enzyme that occurs in eosinophils and is involved in host defense. It represents a novel way of attaching carbohydrate to a protein in addition to the well-known N- and O-glycosylations. The reaction is specific, as in RNase 2 Trp-7, but never Trp-10, which is modified. In this article, we address which structural features provide the specificity of the reaction. Expression of chimeras of RNase 2 and nonglycosylated RNase 4 and deletion mutants in HEK293 cells identified residues 1–13 to be sufficient for C-mannosylation. Site-directed mutagenesis revealed the sequence Trp-x-x-Trp, in which the first Trp becomes mannosylated, as the specificity determinant. The Trp residue at position +3 can be replaced by Phe, which reduces the efficiency of the reaction threefold. Interpretation of the data in the context of the three-dimensional structure of RNase 2 strongly suggests that the primary, rather than the tertiary, structure forms the determinant. The sequence motif occurs in 336 mammalian proteins currently present in protein databases. Two of these proteins were analyzed protein chemically, which showed partial C-glycosylation of recombinant human interleukin 12. The frequent occurrence of the protein recognition motif suggests that C-glycosides could be part of the structure of more proteins than assumed so far.

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Phosphorylation of the α-subunit of Na+,K+-ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na+,K+-ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na+,K+-ATPase α-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the α-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat α-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86Rb uptake in opossum kidney cells expressing mutant rat α1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na+,K+-ATPase α-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

Molecular basis for a link between complement and the vascular complications of diabetes

Activated terminal complement proteins C5b to C9 form the membrane attack complex (MAC) pore. Insertion of the MAC into endothelial cell membranes causes the release of growth factors that stimulate tissue growth and proliferation. The complement regulatory membrane protein CD59 restricts MAC formation. Because increased cell proliferation characterizes the major chronic vascular complications of human diabetes and because increased glucose levels in diabetes cause protein glycation and impairment of protein function, we investigated whether glycation could inhibit CD59. Glycation-inactivation of CD59 would cause increased MAC deposition and MAC-stimulated cell proliferation. Here, we report that (i) human CD59 is glycated in vivo, (ii) glycated human CD59 loses its MAC-inhibitory function, and (iii) inactivation of CD59 increases MAC-induced growth factor release from endothelial cells. We demonstrate by site-directed mutagenesis that residues K41 and H44 form a preferential glycation motif in human CD59. The presence of this glycation motif in human CD59, but not in CD59 of other species, may help explain the distinct propensity of humans to develop vascular proliferative complications of diabetes.

Protein engineering of Bacillus thuringiensis δ-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis δ-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) ≈4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering δ-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.