Conserved noncanonical residue Gly-126 confers instability to the middle part of the tropomyosin molecule

Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle α-Tm either with an Ala residue, which should stabilize the Tm α-helix, or with an Arg residue, which is expected to stabilize both α-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60-70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.     

Glutamate 83 is important for stabilization of domain-domain conformation of Thermus aquaticus glycerol kinase

The gene glpK, encoding glycerol kinase (GlpK) of Thermus aquaticus, has recently been identified. The protein encoded by glpK was found to have an unusually high identity of 81% with the sequence of GlpK from Bacillus subtilis. Three residues (Arg-82, Glu-83, and Asp-244) of T. aquaticus GlpK are conserved in all the known GlpK sequences, including those from various bacteria, yeast and human. The roles that these three residues play in the catalytic mechanism were investigated by using site-directed mutagenesis to produce three mutants: Arg-82-Ala, Glu-83-Ala, and Asp-244-Ala. Replacement of Asp-244 by Ala resulted in a complete loss of activity, thus suggesting that Asp-244 is important for catalysis. Taking k(cat)/K(m) as a simple measure of catalytic efficiency, the mutants Arg-82-Ala and Glu-83-Ala were judged to cause 190- and 37,000-fold decrease, respectively, when compared to the wild-type GlpK. Thus, these three residues play a critical role in the catalytic mechanism. However, only mutant Glu-83-Ala was cleaved by alpha-chymotrypsin, and proteolysis studies showed that the mutant Glu-83-Ala involves a change in the exposure of Tyr-331 at the alpha-chymotrypsin site. This indicates a large domain conformational change, since the residues corresponding to Glu-83 and Tyr-331 in the Escherichia coli GlpK sequence are located in domain IB and domain IIB, respectively. The apparent conformational change caused by replacement of Glu-83 leads us to propose that Glu-83 is an important residue for stabilization of domain conformation.     

Characterization of the putative fusogenic domain in vesicular stomatitis virus glycoprotein G.

The envelope glycoprotein G of vesicular stomatitis virus induces membrane fusion at low pH. Site-directed mutagenesis of specific amino acids within a segment spanning amino acids 123 to 137 of G protein, which is highly conserved in vesiculoviruses and was previously shown by us to be involved in fusogenic activity (Y. Li, C. Drone, E. Sat, and H. P. Ghosh, J. Virol. 67:4070-4077, 1993), was used to determine the role of this region in low-pH-induced membrane fusion. The mutant glycoproteins expressed in COS cells were assayed for acid-pH-induced cell-cell fusion. Substitution of the variant Pro-123 with Leu had no effect on the fusogenic activity, while substitution of conserved Phe-125 and Asp-137 with Tyr and Asn, respectively, shifted the pH optimum of membrane fusion to a more acidic pH value and decreased the fusion efficiency. The deletion of amino acid residues 124 to 127, 131 to 137, or 124 to 137 produced mutants defective in transport. Mutation of the conserved residues Gly-124 and Pro-127 to Ala and to Gly or Leu, respectively, inhibited cell-cell fusion activity by about 90% without affecting transport of the mutant proteins to the cell surface, suggesting that these two residues may be present within the fusion peptide and thus may be directly involved in fusion. This highly conserved domain containing neutral amino acids of G protein may therefore represent the putative fusion domain of vesicular stomatitis virus G protein.     

Contributions of conserved residues at the gating interface of glycine receptors

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.     

Site-specific mutations of conserved C-terminal residues in aminoglycoside 3'-phosphotransferase II: phenotypic and structural analysis of mutant enzymes.

In order to test the biological importance of amino acids in the C-terminal quarter of aminoglycoside 3'-phosphotransferase II enzyme, seven of the highly conserved residues in this region, His-188, Asp-190, Asp-208, Gly-210, Arg-211, Asp-216 and Asp-220, were changed via site-directed mutagenesis. The phenotype of each mutant was compared to wildtype in terms of antibiotic susceptibilities and enzymatic activities. All of the substitutions either abolished or significantly reduced the resistance of the resulting strains to kanamycin, neomycin, butirosin, ribostamycin, paromomycin, gentamicin A, and G-418. Similarly, enzyme activities in crude extracts were substantially reduced for the mutant strains. Affinity of the enzyme for Mg+2-ATP decreased with His-188, Asp-190, Asp-216 and Asp-220 substitutions as revealed by Km measurements. Secondary structure analysis predicted that substitutions at the conserved residues caused severe conformational distortions at the corresponding regions of the protein.     

Conserved Asp-137 Is Important for both Structure and Regulatory Functions of Cardiac α-Tropomyosin (α-TM) in a Novel Transgenic Mouse Model Expressing α-TM-D137L

α-Tropomyosin (α-TM) has a conserved, charged Asp-137 residue located in the hydrophobic core of its coiled-coil structure, which is unusual in that the residue is found at a position typically occupied by a hydrophobic residue. Asp-137 is thought to destabilize the coiled-coil and so impart structural flexibility to the molecule, which is believed to be crucial for its function in the heart. A previous in vitro study indicated that the conversion of Asp-137 to a more typical canonical Leu alters flexibility of TM and affects its in vitro regulatory functions. However, the physiological importance of the residue Asp-137 and altered TM flexibility is unknown. In this study, we further analyzed structural properties of the α-TM-D137L variant and addressed the physiological importance of TM flexibility in cardiac function in studies with a novel transgenic mouse model expressing α-TM-D137L in the heart. Our NMR spectroscopy data indicated that the presence of D137L introduced long range rearrangements in TM structure. Differential scanning calorimetry measurements demonstrated that α-TM-D137L has higher thermal stability compared with α-TM, which correlated with decreased flexibility. Hearts of transgenic mice expressing α-TM-D137L showed systolic and diastolic dysfunction with decreased myofilament Ca²⁺ sensitivity and cardiomyocyte contractility without changes in intracellular Ca²⁺ transients or post-translational modifications of major myofilament proteins. We conclude that conversion of the highly conserved Asp-137 to Leu results in loss of flexibility of TM that is important for its regulatory functions in mouse hearts. Thus, our results provide insight into the link between flexibility of TM and its function in ejecting hearts.     

Mutational analysis of the Pyrococcus furiosus holliday junction resolvase hjc revealed functionally important residues for dimer formation, junction DNA binding, and cleavage activities

The Holliday junction cleavage protein, Hjc resolvase of Pyrococcus furiosus, is the first Holliday junction resolvase to be discovered in Archaea. Although the archaeal resolvase shares certain biochemical properties with other non-archaeal junction resolvases, no amino acid sequence similarity has been identified. To investigate the structure-function relationship of this new Holliday junction resolvase, we constructed a series of mutant hjc genes using site-directed mutagenesis targeted at the residues conserved among the archaeal orthologs. The products of these mutant genes were purified to homogeneity. With analysis of the activity of the mutant proteins to bind and cleave synthetic Holliday junctions, one acidic residue, Glu-9, and two basic residues, Arg-10 and Arg-25, were found to play critical roles in enzyme action. This is in addition to the three conserved residues, Asp-33, Glu-46, and Lys-48, which are also conserved in the motif found in the type II restriction endonuclease family proteins. Two aromatic residues, Phe-68 and Phe-72, are important for the formation of the homodimer probably through hydrophobic interactions. The results of these studies have provided insights into the structure-function relationships of the archaeal Holliday junction resolvase as well as the universality and diversity of the Holliday junction cleavage reaction.     

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 M phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K _m and k _cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K _m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K _m and k _cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.     

Arginine-349 and aspartate-373 of the Na(+)/dicarboxylate cotransporter are conformationally sensitive residues.

The conserved residues, Arg-349 and Asp-373, of the renal Na(+)/dicarboxylate cotransporter (NaDC-1) have been shown in our previous studies to affect substrate affinity and cation binding. In this study, amino acids surrounding Arg-349 and Asp-373 were individually mutated to cysteines and their sensitivity to methanethiosulfonate reagents (MTS) was tested. Only three of the 21 mutants were sensitive to MTS reagents: R349C, S372C, and D373C. The R349C mutant had reduced activity which was restored by chemical modification with MTSEA. The effect of MTSEA was only observed in the presence of sodium, indicating that Arg-349 is conformationally accessible. The succinate transport activity of the S372C mutant was stimulated by both MTSEA and MTSET. The D373C mutant was very sensitive to inhibition by MTSET (K(i) = 0.5 microM) in sodium buffer. The inhibition of D373C by MTSET was prevented by substrate, suggesting that the substrate-induced conformational change occludes the residue. We conclude that the accessibility of Arg-349 and Asp-373 is likely to change with the conformational states of the transport cycle.     

An electron-sharing network involved in the catalytic mechanism is functionally conserved in different glutathione transferase classes

In Anopheles dirus glutathione transferase D3-3, there are electrostatic interactions between the negatively charged glutamyl alpha-carboxylate group of glutathione, the positively charged Arg-66, and the negatively charged Asp-100. This ionic interaction is stabilized by a network of hydrogen bonds from Ser-65, Thr-158, Thr-162, and a conserved water-mediated contact. This alternating ionic bridge interaction between negatively and positively charged residues stabilized by a network of hydrogen bonding we have named an electron-sharing network. We show that the electron-sharing network assists the glutamyl alpha-carboxylate of glutathione to function as a catalytic base accepting the proton from the thiol group forming an anionic glutathione, which is a crucial step in the glutathione transferase (GST) catalysis. Kinetic studies demonstrate that the mutation of electron-sharing network residues results in a decreased ability to lower the pKa of the thiol group of glutathione. Although the residues that contribute to the electron-sharing network are not conserved in the primary sequence, structural characterizations indicate that the presence of the network can be mapped to the same region in all GST classes. A structural diversification but functional conservation suggests a significant role for the electron-sharing network in catalysis as the purpose was maintained during the divergent evolution of GSTs. This network appears to be a functionally conserved motif that contributes to the "base-assisted deprotonation" model suggested to be essential for the glutathione ionization step of the catalytic mechanism.     

Phosphorylation of CREB at Ser-133 induces complex formation with CREB-binding protein via a direct mechanism.

We have characterized a phosphoserine binding domain in the coactivator CREB-binding protein (CBP) which interacts with the protein kinase A-phosphorylated, and hence activated, form of the cyclic AMP-responsive factor CREB. The CREB binding domain, referred to as KIX, is alpha helical and binds to an unstructured kinase-inducible domain in CREB following phosphorylation of CREB at Ser-133. Phospho-Ser-133 forms direct contacts with residues in KIX, and these contacts are further stabilized by hydrophobic residues in the kinase-inducible domain which flank phospho-Ser-133. Like the src homology 2 (SH2) domains which bind phosphotyrosine-containing peptides, phosphoserine 133 appears to coordinate with a single arginine residue (Arg-600) in KIX which is conserved in the CBP-related protein P300. Since mutagenesis of Arg-600 to Gln severely reduces CREB-CBP complex formation, our results demonstrate that, as in the case of tyrosine kinase pathways, signal transduction through serine/threonine kinase pathways may also require protein interaction motifs which are capable of recognizing phosphorylated amino acids.     

Identification of thromboxane synthase amino acid residues involved in heme-propionate binding

Thromboxane A2 synthase (TXAS) is a member of the cytochrome P450 superfamily and catalyzes an isomerization reaction that converts prostaglandin H2 to thromboxane A2. As a step toward understanding the structure/function relationships of TXAS, we mutated amino acid residues predicted to bind the propionate groups of A- and D-pyrrole rings of the heme. These mutations at each of these residues (Asn-110, Trp-133, Arg-137, Arg-413, and Arg-478) resulted in altered heme binding, as evidenced by perturbation of the absorption spectra and EPR. The mutations, although causing no significant changes in the secondary structure of the proteins, induced tertiary structural changes that led to increased susceptibility to trypsin digestion and alteration of the intrinsic protein fluorescence. Moreover, these mutant proteins lost their binding affinity to the substrate analog, had a lower heme content and retained less than 5% of the wild-type catalytic activity. However, mutations at the neighboring amino acid of the aforementioned residues yielded mutant proteins retaining the biochemical and biophysical properties of the wild type TXAS. Aligning the TXAS sequence with the structurally known P450s, we proposed that in TXAS the A-ring propionate of the heme is hydrogen bonded to Asn-110, Arg-413, and Arg-478, whereas D-ring propionate is hydrogen bonded to Trp-133 and Arg-137. Furthermore, both A- and D-ring propionates bulge away from the heme plane and both lie on the proximal face of heme plane, a structure similar to P450terp.     

Role of aspartate-133 and histidine-458 in the mechanism of tryptophan indole-lyase from Proteus vulgaris

Tryptophan indole-lyase (Trpase) from Proteus vulgaris is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Trp to yield indole and ammonium pyruvate. Asp-133 and His-458 are strictly conserved in all sequences of Trpase, and they are located in the proposed substrate-binding region of Trpase. These residues were mutated to alanine to probe their role in substrate binding and catalysis. D133A mutant Trpase has no measurable activity with L-Trp as substrate, but still retains activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase has 1.6% of wild-type Trpase activity with L-Trp, and high activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase does not exhibit the pK(a) of 5.3 seen in the pH dependence of k(cat)/K(m) of L-Trp for wild-type Trpase. Both mutant enzymes are inhibited by L-Ala, L-Met, and L-Phe, with K(i) values similar to those of wild-type Trpase, but oxindolyl-L-alanine and beta-phenyl-DL-serine show much weaker binding to the mutant enzymes, suggesting that Asp-133 and His-458 are involved in the binding of these ligands. D133A and H458A mutant Trpase exhibit absorption and CD spectra in the presence of substrates and inhibitors that are similar to wild-type Trpase, with peaks at about 420 and 500 nm. The rate constants for formation of the 500 nm bands for the mutant enzymes are equal to or greater than those of wild-type Trpase, indicating that Asp-133 and His-458 do not play a role in the formation of quinonoid intermediates. In constrast to wild-type and H458A mutant Trpase, D133A mutant Trpase forms an intermediate from S-ethyl-L-Cys that absorbs at 345 nm, and is likely to be an alpha-aminoacrylate. Crystals of D133A and H458A mutant Trpase bind amino acids with similar affinity as the proteins in solution, except for L-Ala, which binds to D133A mutant Trpase crystals about 20-fold stronger than in solution. These results suggest that Asp-133 and His-458 play an important role in the elimination reaction of L-Trp. Asp-133 likely forms a hydrogen bond directly to the indole NH of the substrate, while His-458 probably is hydrogen bonded to Asp-133.     

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Structure-function analysis of the reactive site in the first Kunitz-type domain of human tissue factor pathway inhibitor-2

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type proteinase inhibitor that regulates a variety of serine proteinases involved in coagulation and fibrinolysis through their non-productive interaction with a P(1) residue (Arg-24) in its first Kunitz-type domain (KD1). Previous kinetic studies revealed that TFPI-2 was a more effective inhibitor of plasmin than several other serine proteinases, but the molecular basis for this specificity was unclear. In this study, we employed molecular modeling and mutagenesis strategies to produce several variants of human TFPI-2 KD1 in an effort to identify interactive site residues other than the P(1) Arg that contribute significantly to its inhibitory activity and specificity. Molecular modeling of KD1 based on the crystal structure of bovine pancreatic trypsin inhibitor revealed that KD1 formed a more energetically favorable complex with plasmin versus trypsin and/or the factor VIIa-tissue factor complex primarily due to strong ionic interactions between Asp-19 (P(6)) and Arg residues in plasmin (Arg-644, Arg-719, and Arg-767), Arg-24 (P(1)) with Asp-735 in plasmin, and Arg-29 (P(5)') with Glu-606 in plasmin. In addition, Leu-26 through Leu-28 (P(2)'-P(4)') in KD1 formed strong van der Waals contact with a hydrophobic cluster in plasmin (Phe-583, Met-585, and Phe-587). Mutagenesis of Asp-19, Tyr-20, Arg-24, Arg-29, and Leu-26 in KD1 resulted in substantial reductions in plasmin inhibitory activity relative to wild-type KD1, but the Asp-19 and Tyr-20 mutations revealed the importance of these residues in the specific inhibition of plasmin. In addition to the reactive site residues in the P(6)-P(5)' region of KD1, mutation of a highly conserved Phe at the P(18)' position revealed the importance of this residue in the inhibition of serine proteinases by KD1. Thus, together with the P(1) residue, the nature of other residues flanking the P(1) residue, particularly at P(6) and P(5)', strongly influences the inhibitory activity and specificity of human TFPI-2.     

Alanine-scanning mutagenesis of the small-subunit beta A-beta B loop of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase: substitution at Arg-71 affects thermal stability and CO2/O2 specificity

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) enzymes from different species differ with respect to carboxylation catalytic efficiency and CO2/O2 specificity, but the structural basis for these differences is not known. Whereas much is known about the chloroplast-encoded large subunit, which contains the alpha/beta-barrel active site, much less is known about the role of the nuclear-encoded small subunit in Rubisco structure and function. In particular, a loop between beta-strands A and B contains 21 or more residues in plants and green algae, but only 10 residues in prokaryotes and nongreen algae. To determine the significance of these additional residues, a mutant of the green alga Chlamydomonas reinhardtii, which lacks both small-subunit genes, was used as a host for transformation with directed-mutant genes. Although previous studies had indicated that the betaA-betaB loop was essential for holoenzyme assembly, Ala substitutions at residues conserved among land plants and algae (Arg-59, Tyr-67, Tyr-68, Asp-69, and Arg-71) failed to block assembly or eliminate function. Only the Arg-71 --> Ala substitution causes a substantial decrease in holoenzyme thermal stability. Tyr-68 --> Ala and Asp-69 --> Ala enzymes have lower K(m)(CO2) values, but these improvements are offset by decreases in carboxylation V(max) values. The Arg-71 --> Ala enzyme has a decreased carboxylation V(max) and increased K(m)(CO2) and K(m)(O2) values, which account for an observed 8% decrease in CO2/O2 specificity. Despite the fact that Arg-71 is more than 20 A from the large-subunit active site, it is apparent that the small-subunit betaA-betaB loop region can influence catalytic efficiency and CO2/O2 specificity.     

Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.     

Key residues responsible for acyl carrier protein and beta-ketoacyl-acyl carrier protein reductase (FabG) interaction

Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes collectively known as type II fatty-acid synthase. Each enzyme interacts with acyl carrier protein (ACP), which shuttles the pathway intermediates between the proteins. The type II enzymes do not possess primary sequence similarity that defines a common ACP-binding site, but rather are hypothesized to possess an electropositive/hydrophobic surface feature that interacts with the electronegative/hydrophobic residues along helix alpha2 of ACP (Zhang, Y.-M., Marrakchi, H., White, S. W., and Rock, C. O. (2003) J. Lipid Res. 44, 1-10). We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on beta-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix alpha2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix alpha2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.     

Escherichia coli H(+)-ATPase: role of the delta subunit in binding Fl to the Fo sector.

The roles of the Escherichia coli H(+)-ATPase (FoFl) delta subunit (177 amino acid residues) was studied by analyzing mutants. The membranes of nonsense (Gln-23----end, Gln-29----end, Gln-74----end) and missense (Gly-150----Asp) mutants had very low ATPase activities, indicating that the delta subunit is essential for the binding of the Fl portion to Fo. The Gln-176----end mutant had essentially the same membrane-bound activity as the wild type, whereas in the Val-174----end mutant most of the ATPase activity was in the cytoplasm. Thus Val-174 (and possibly Leu-175 also) was essential for maintaining the structure of the subunit, whereas the two carboxyl terminal residues Gln-176 and Ser-177 were dispensable. Substitutions were introduced at various residues (Thr-11, Glu-26, Asp-30, Glu-42, Glu-82, Arg-85, Asp-144, Arg-154, Asp-161, Ser-163), including apparently conserved hydrophilic ones. The resulting mutants had essentially the same phenotypes as the wild type, indicating that these residues do not have any significant functional role(s). Analysis of mutations (Gly-150----Asp, Pro, or Ala) indicated that Gly-150 itself was not essential, but that the mutations might affect the structure of the subunit. These results suggest that the overall structure of the delta subunit is necessary, but that individual residues may not have strict functional roles.     

Charged amino acids of the N-terminal domain are involved in coupling binding and gating in alpha7 nicotinic receptors

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of conformational changes resulting in channel opening. Previously, we have shown that the aspartate residue Asp-266 at the M2-M3 linker of the alpha7 nicotinic receptor is involved in connecting binding and gating. High resolution structural data suggest that this region could interact with the so-called loops 2 and 7 of the extracellular N-terminal region. In this case, certain charged amino acids present in these loops could integrate together with Asp-266 and other amino acids, a mechanism involved in channel activation. To test this hypothesis, all charged residues in these loops, Asp-42, Asp-44, Glu-45, Lys-46, Asp-128, Arg-130, and Asp-135, were substituted with other amino acids, and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Glu-45, Lys-46, and Asp-135 exhibited poor or null functional responses to different nicotinic agonists regardless of significant membrane expression, whereas D128A showed a gain of function effect. Because the double reverse charge mutant K46D/D266K did not restore receptor function, a gating mechanism controlled by the pairwise electrostatic interaction between these residues is not likely. Rather, a network of interactions formed by residues Lys-46, Asp-128, Asp-135, Asp-266, and possibly others appears to link agonist binding to channel gating.