Contribution of the Disulfide Bond of the A Subunit to the Action of Escherichia coli Heat-Labile Enterotoxin

Escherichia coli heat-labile enterotoxin (LT) consists of an A subunit and five B subunits. These subunits oligomerize into an assembled holotoxin within the periplasm. Structural analysis of LT has revealed that the A subunit interacts with the B subunit through its carboxy terminus. This indicates that the carboxy-terminal portion of the protein is required for assembly of holotoxin in the periplasm. However, it is not known whether other regions of the A subunit contribute to the assembly. The A subunit constituting the holotoxin contains a disulfide bond between Cys-187 and Cys-199. It has been observed in many proteins that the intramolecular disulfide bond is deeply involved in the function and tertiary structure of the protein. We speculated that the disulfide bond of the A subunit contributes to the assembly in the periplasm, although the bond is not a structural element of the carboxy-terminal portion of the A subunit. We replaced these cysteine residues of the A subunit by oligonucleotide-directed site-specific mutagenesis and analyzed the LTs produced by cells containing the mutant LT genes. The amount of the mutant holotoxin produced was small compared with that of the wild-type strain, indicating that the disulfide bond of the A subunit contributes to the structure which functions as the site of nucleation in the assembly. A reconstitution experiment in vitro supported the notion. Subsequently, we found that the mutant A subunit constituting holotoxin is easily degraded by trypsin and that in cells incubated with mutant LTs, the lag until the intracellular cyclic AMP begins to accumulate is longer than in cells incubated with native LTs. These results might be useful for the analysis of the interaction of LT with target cells at the molecular level.     

Analysis of Shiga toxin subunit association by using hybrid A polypeptides and site-specific mutagenesis.

Shiga toxin (STX), a bacterial toxin produced by Shigella dysenteriae type 1, is a hexamer composed of five receptor-binding B subunits which encircle an alpha-helix at the carboxyl terminus of the enzymatic A polypeptide. Hybrid toxins constructed by fusing the A polypeptide sequences of STX and Shiga-like toxin type II were used to confirm that the carboxyl terminus of the A subunits governs association with the B pentamers. The alpha-helix of the 293-amino-acid STX A subunit contains nine residues (serine 279 to methionine 287) which penetrate the nonpolar pore of the B-subunit pentamer. Site-directed mutagenesis was used to establish the involvement of two residues bordering this alpha-helix, aspartic acid 278 and arginine 288, in coupling the C terminus of StxA to the B pentamer. Amino acid substitutions at StxB residues arginine 33 and tryptophan 34, which are on the membrane-contacting surface of the pentamer, reduced cytotoxicity without affecting holotoxin formation. Although these B-subunit mutations did not involve receptor-binding residues, they may have induced an electrostatic repulsion between the holotoxin and the mammalian cell membrane or disrupted cytoplasmic translocation.     

Functional diversity of heat-labile toxins (LT) produced by enterotoxigenic Escherichia coli: differential enzymatic and immunological activities of LT1 (hLT) AND LT4 (pLT)

Heat-labile toxins (LTs) have ADP-ribosylation activity and induce the secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains in different mammalian hosts. LTs also act as adjuvants following delivery via mucosal, parenteral, or transcutaneous routes. Previously we have shown that LT produced by human-derived ETEC strains encompass a group of 16 polymorphic variants, including the reference toxin (LT1 or hLT) produced by the H10407 strain and one variant that is found mainly among bacterial strains isolated from pigs (LT4 or pLT). Herein, we show that LT4 (with six polymorphic sites in the A (K4R, K213E, and N238D) and B (S4T, A46E, and E102K) subunits) displays differential in vitro toxicity and in vivo adjuvant activities compared with LT1. One in vitro generated LT mutant (LTK4R), in which the lysine at position 4 of the A subunit was replaced by arginine, showed most of the LT4 features with an ～10-fold reduction of the cytotonic effects, ADP-ribosylation activity, and accumulation of intracellular cAMP in Y1 cells. Molecular dynamic studies of the A subunit showed that the K4R replacement reduces the N-terminal region flexibility and decreases the catalytic site crevice. Noticeably, LT4 showed a stronger Th1-biased adjuvant activity with regard to LT1, particularly concerning activation of cytotoxic CD8(+) T lymphocytes when delivered via the intranasal route. Our results further emphasize the relevance of LT polymorphism among human-derived ETEC strains that may impact both the pathogenicity of the bacterial strain and the use of these toxins as potential vaccine adjuvants.     

Identification of three amino acid residues in the B subunit of Shiga toxin and Shiga-like toxin type II that are essential for holotoxin activity.

Shiga toxin of Shigella dysenteriae type I and Shiga-like toxins I and II (SLT-I and SLT-II, respectively) of enterohemorrhagic Escherichia coli are functionally similar protein cytotoxins. These toxin molecules have a bipartite molecular structure which consists of an enzymatically active A subunit that inhibits protein synthesis in eukaryotic cells and an oligomeric B subunit that binds to globotriaosylceramide glycolipid receptors on eukaryotic cells. Regionally directed chemical mutagenesis of the B subunit of SLT-II was used to identify amino acids in the B subunit that are critical for SLT-II holotoxin cytotoxic activity. Three noncytotoxic mutants were isolated, and their mutations were mapped. The substitutions of arginine with cysteine at codon 32, alanine with threonine at codon 42, and glycine with aspartic acid at codon 59 in the 70-amino-acid mature SLT-II B polypeptide resulted in the complete abolition of cytotoxicity. The analogous arginine, alanine, and glycine residues were conserved at codons 33, 43, and 60 in the 69-amino-acid mature B polypeptide of Shiga toxin. Comparable mutations induced in the B-subunit gene of Shiga toxin by oligonucleotide-directed, site-specific mutagenesis resulted in drastically decreased cytotoxicity (10(3)- to 10(6)-fold) as compared with that of wild-type Shiga toxin. The mutant SLT-II and Shiga toxin B subunits were characterized for stability, receptor binding, immunoreactivity, and ability to be assembled into holotoxin.     

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for G_M1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, G_Sα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.     

A switch 3 point mutation in the alpha subunit of transducin yields a unique dominant-negative inhibitor

The rhodopsin/transducin-coupled vertebrate vision system has served as a paradigm for G protein-coupled signaling. We have taken advantage of this system to identify new types of constitutively active, transducin-alpha (alphaT) subunits. Here we have described a novel dominant-negative mutation, made in the background of a chimera consisting of alphaT and the alpha subunit of G(i1) (designated alphaT*), which involves the substitution of a conserved arginine residue in the conformationally sensitive Switch 3 region. Changing Arg-238 to either lysine or alanine had little or no effect on the ability of alphaT* to undergo rhodopsin-stimulated GDP-GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an alphaT* subunit (alphaT*(R238E)) that was incapable of undergoing rhodopsin-dependent nucleotide exchange and was unable to bind or stimulate the target/effector enzyme (cyclic GMP phosphodiesterase). Moreover, unlike the GDP-bound forms of alphaT*, alphaT*(R238A) and alphaT*(R238K), the alphaT*(R238E) mutant did not respond to aluminum fluoride (AlF4(-)), as read out by changes in Trp-207 fluorescence. However, surprisingly, we found that alphaT*(R238E) effectively blocked rhodopsin-catalyzed GDP-GTP exchange on alphaT*, as well as rhodopsin-stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the alphaT*(R238E) mutant exists in a nucleotide-free state. Nucleotide-free forms of G alpha subunits were typically very sensitive to proteolytic degradation, but alphaT*(R238E) exhibited a resistance to trypsin-proteolysis similar to that observed with activated forms of alphaT*. Overall, these findings indicated that by mutating a single residue in Switch 3, it is possible to generate a unique type of dominant-negative G alpha subunit that can effectively block signaling by G protein-coupled receptors.     

Identification of the Shiga toxin A-subunit residues required for holotoxin assembly.

Recent X-ray crystallographic analyses have demonstrated that the receptor-binding (B) subunits of Shiga toxin (STX) are arranged as a doughnut-shaped pentamer. The C terminus of the enzymatic (A) subunit presumably penetrates the nonpolar pore of the STX B pentamer, and the holotoxin is stabilized by noncovalent interactions between the polypeptides. We identified a stretch of nine nonpolar amino acids near the C terminus of StxA which were required for subunit association by using site-directed mutagenesis to introduce progressive C-terminal deletions in the polypeptide and assessing holotoxin formation by a receptor analog enzyme-linked immunosorbent assay, immunoprecipitation, and a cytotoxicity assay. Tryptophan and aspartic acid residues which form the N-terminal boundary, as well as two arginine residues which form the C-terminal boundary of the nine-amino-acid sequence, were implicated as the stabilizers of subunit association. Our model proposes that residues 279 to 287 of the 293-amino-acid STX A subunit penetrate the pore while the tryptophan, aspartic acid, and 2 arginine residues interact with other charged or aromatic amino acids outside the pore on the planar surfaces of the STX B pentamer.     

A single amino acid substitution in B subunit of Escherichia coli enterotoxin affects its oligomer formation

We isolated a mutant strain of enterotoxigenic Escherichia coli by nitrosoguanidine mutagenesis, which produces an immunologically altered B subunit of heat-labile enterotoxin. This mutant B subunit was detected as a monomer on sodium dodecyl sulfate-polyacrylamide gel electrophoresis even without prior heating, suggesting a problem in oligomer formation. Furthermore, this mutant B subunit could not form holotoxin with the native A subunit, and the affinity to GM1-ganglioside receptor was 10-fold lower than that of the native B subunit. The amino acid sequence analysis of this mutant B subunit revealed only one amino acid substitution compared with the native B subunit, at the 64th position from the N terminus (valine instead of alanine). These data suggest that the alanine at position 64 from the N terminus is important for the native B subunit to form an oligomer structure and express its functions.     

Critical molecular regions for elicitation of the sweetness of the sweet-tasting protein, thaumatin I

Thaumatin is an intensely sweet-tasting protein. To identify the critical amino acid residue(s) responsible for elicitation of the sweetness of thaumatin, we prepared mutant thaumatin proteins, using Pichia pastoris, in which alanine residues were substituted for lysine or arginine residues, and the sweetness of each mutant protein was evaluated by sensory analysis in humans. Four lysine residues (K49, K67, K106 and K163) and three arginine residues (R76, R79 and R82) played significant roles in thaumatin sweetness. Of these residues, K67 and R82 were particularly important for eliciting the sweetness. We also prepared two further mutant thaumatin I proteins: one in which an arginine residue was substituted for a lysine residue, R82K, and one in which a lysine residue was substituted for an arginine residue, K67R. The threshold value for sweetness was higher for R82K than for thaumatin I, indicating that not only the positive charge but also the structure of the side chain of the arginine residue at position 82 influences the sweetness of thaumatin, whereas only the positive charge of the K67 side chain affects sweetness.     

Two conserved arginines in the extracellular N-terminal domain of the GABA(A) receptor alpha(5) subunit are crucial for receptor function

The gamma-aminobutyric acid (GABA) binding pocket within the GABA(A) receptor complex has been suggested to contain arginine residues. The aim of this study was to test this hypothesis by mutating arginine residues potentially contributing to the formation of a GABA binding pocket. Thus, six arginines conserved in human GABA(A) receptor alpha subunits (arginine 34, 70, 77, 123, 135, and 224) as well as two nonconserved arginines (79 and 190), all located in the extracellular N-terminal segment of the alpha(5) subunit, were substituted by lysines. The individual alpha(5) subunit mutants were coexpressed with human beta(2) and gamma(2s) GABA(A) receptor subunits in Chinese hamster ovary cells by transient transfection. Electrophysiological whole-cell patch-clamp recordings show that, of the eight arginine residues tested, the two arginines at positions 70 and 123 appear to be essential for the GABA-gated chloride current because the EC(50) values of the two mutant constructs increase >100-fold compared with the wild-type alpha(5),beta(2), gamma(2s) GABA(A) receptor. However, diazepam and allopregnanolone modulation and pentobarbital stimulation properties are unaffected by the introduction of lysines at positions 70 and 123. A double mutant carrying lysine substitutions at positions 70 and 123 is virtually insensitive to GABA, suggesting alterations of one or more GABA binding sites.     

Heat-labile enterotoxin in Escherichia coli. Kinetics of association of subunits into periplasmic holotoxin

We have investigated the assembly of the heat-labile enterotoxin (LT) subunits after their processing and segregation into the periplasmic space as mature LT A and LT B polypeptides. LT B starts associating into oligomers during or immediately after translocation through the cytoplasmic membrane. Binding to LT A occurs immediately after oligomerization. Over 80% of the LT B subunits have oligomerized, and over 50% have associated with LT A into holotoxin within 1 min after synthesis. The fate of newly synthesized LT A is totally different. There is an extensive overproduction of LT A relative to LT B and after membrane translocation it becomes part of a periplasmic pool of free LT A. It is then bound by LT B oligomers or degraded at such a rate that the free periplasmic LT A disappears from the pool with a half-time of 20-25 min. About half of the LT A is incorporated into holotoxin, while the other half is degraded. We conclude that LT subunits are translocated and processed in a ratio of about 2 A to 5 B. Since free LT A is either degraded slowly or bound to newly synthesized LT B oligomers, the net result is a steady state of 1.4 to 1.7 A subunits to 5 B subunits in the periplasm. About 60% of this LT A is bound by LT B to form periplasmic holotoxin with a subunit ratio of about 1 A to 5 B. The remaining 40% of periplasmic LT A occurs free.     

Three subunits contribute amino acids to the active site of tetrameric adenylosuccinate lyase: Lys268 and Glu275 are required

Tetrameric adenylosuccinate lyase (ASL) of Bacillus subtilis catalyzes the cleavage of adenylosuccinate to form AMP and fumarate. We previously reported that two distinct subunits contribute residues to each active site, including the His68 and His89 from one and His141 from a second subunit [Brosius, J. L., and Colman, R. F. (2000) Biochemistry 39, 13336-13343]. Glu(275) is 2.8 A from His141 in the ASL crystal structure, and Lys268 is also in the active site region; Glu275 and Lys268 come from a third, distinct subunit. Using site-directed mutagenesis, we have replaced Lys268 by Arg, Gln, Glu, and Ala, with specific activities of the purified mutant enzymes being 0.055, 0.00069, 0.00028, and 0.0, respectively, compared to 1.56 units/mg for wild-type (WT) enzyme. Glu275 was substituted by Gln, Asp, Ala, and Arg; none of these homogeneous mutant enzymes has detectable activity. Circular dichroism and light scattering reveal that neither the secondary structure nor the oligomeric state of the Lys268 mutant enzymes has been perturbed. Native gel electrophoresis and circular dichroism indicate that the Glu275 mutant enzymes are tetramers, but their conformation is altered slightly. For K268R, the K(m)s for all substrates are similar to WT enzyme. Binding studies using [2-3H]-adenylosuccinate reveal that none of the Glu275 mutant enzymes, nor inactive K268A, can bind substrate. We propose that Lys268 participates in binding substrate and that Glu275 is essential for catalysis because of its interaction with His141. Incubation of H89Q with K268Q or E275Q leads to restoration of up to 16% WT activity, while incubation of H141Q with K268Q or E275Q results in 6% WT activity. These complementation studies provide the first functional evidence that a third subunit contributes residues to each intersubunit active site of ASL. Thus, adenylosuccinate lyase has four active sites per enzyme tetramer, each of which is formed from regions of three subunits.     

Adenine nucleotides promote dissociation of pertussis toxin subunits

Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity.     

Role of conserved proline residues in stabilizing tryptophan synthase alpha subunit: analysis by mutants with alanine or glycine

To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant alpha subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six Pro residues (positions 28, 57, 62, 96, 132, and 207) that are conserved in 10 microorganisms, were constructed by means of site-directed mutagenesis. The far-ultraviolet (UV) CD spectra of five mutant alpha subunits with Ala in place of Pro were identical to the spectrum of the wild-type protein, the exception being the mutant at position 207 (P207A). CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha subunit. The negative CD values of the Gly mutants examined (P28G, P96G, and P132G) were also decreased. Calorimetric measurements showed that the two mutants at position 28 (P28G and P28A) gave two peaks in the excess heat capacity curve, whereas the wild type and other Pro mutants had only a single peak. The stability of each mutant protein relative to that of the wild type was about the same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A, which are substituted at less mobile positions. The changes of denaturation entropy (delta delta dS) at the denaturation temperature of the wild-type protein (54.1 degrees C at pH 9.0) were positive for P57A, P62A, and P132A, but negative for P96A, P207A, and P132G.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermostability of manganese- and iron-superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue.

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active-site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site-directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild-type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild-type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.     

Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae.

Cholera toxin and heat-labile enterotoxin (LT) are structurally similar oligomeric proteins which are capable of being efficiently secreted from Vibrio cholerae. Here we report that these proteins transiently enter the periplasm of V. cholerae as they traverse the cell envelope to reach the extracellular milieu. Pulse-chase experiments on V. cholerae TRH7000 harboring an LT-encoding plasmid revealed that radiolabeled LT A and B subunits entered the periplasm rapidly, followed by their slow efflux (half-time, 13 min) into the medium. LT B-subunit efflux from the periplasm was calculated to be at a rate of ca. 170 monomers per min per cell (which is equivalent to 34 assembled LT holotoxin molecules per min per cell). These values were estimated to be sufficient to account for the increase in extracellular enterotoxin concentration during exponential cell growth. Thus, all enterotoxin subunits which are secreted into the medium can be assumed to be channelled via the periplasm. These findings led to an improved model of the pathway of toxin secretion by V. cholerae.     

New insights into the role of conserved, essential residues in the GTP binding/GTP hydrolytic cycle of large G proteins

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.     

Genetic identification of residues involved in association of alpha and beta G-protein subunits.

The GPA1, STE4, and STE18 genes of Saccharomyces cerevisiae encode the alpha, beta, and gamma subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and delta L138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signaling pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be defective in association with the Gpa1 protein. A mutation at position E307 of the Gpa1 protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant alpha subunit to physically associate with a specific mutant G beta subunit. The mutation in the Gpa1 protein is adjacent to the hinge, or switch, region that is required for the conformational change which triggers subunit dissociation, but the mutation does not affect the interaction of the alpha subunit with the wild-type beta subunit. Yeast cells constructed to contain only the mutant alpha and beta subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified G alpha subunit to suppress the Ste4 mutations is allele specific, it is likely that the residues defined by this analysis play a direct role in G-protein subunit association.     

Cooperativity in nicotinamide adenine dinucleotide binding induced by mutations of arginine 475 located at the subunit interface in the human liver mitochondrial class 2 aldehyde dehydrogenase

The low-activity Oriental variant of human mitochondrial aldehyde dehydrogenase possesses a lysine rather than a glutamate at residue 487 in the 500 amino acid homotetrameric enzyme. The glutamate at position 487 formed two salt bonds, one to an arginine at position 264 in the same subunit and the other to arginine 475 in a different subunit [Steinmetz, C. G., Xie, P.-G.,Weiner, H., and Hurley, T. D. (1997) Structure 5, 2487-2505]. Mutating arginine 264 to glutamine produced a recombinantly expressed enzyme with nativelike properties; in contrast, mutating arginine 475 to glutamine produced an enzyme that exhibited positive cooperativity in NAD binding. The K(M) for NAD increased 23-fold with a Hill coefficient of 1.8. The binding of both NAD and NADH was affected by the mutation at position 475. Restoring the salt bonds between residues 487 and either or both 264 and 475 did not restore nativelike properties to the Oriental variant. Further, the R475Q mutant was thermally less stable than the native enzyme, Oriental variant, or other mutants. The presence of NAD restored nativelike stability to the mutant. It is concluded that movement of arginine 475 disrupted salt bonds between it and residues other than the one at 487, which caused the apo-R475Q mutant to have properties typical of an enzyme that exhibits positive cooperativity in substrate binding. Breaking the salt bond between glutamate 487 in the Oriental variant and the two arginine residues cannot be the only reason that this enzyme has altered catalytic properties.     

Dominant inheritance of isolated hypermethioninemia is associated with a mutation in the human methionine adenosyltransferase 1A gene.

Methionine adenosyltransferase (MAT) I/III deficiency, characterized by isolated persistent hypermethioninemia, is caused by mutations in the MAT1A gene encoding MAT(alpha)1, the subunit of major hepatic enzymes MAT I ([alpha1]4) and III([alpha1]2). We have characterized 10 MAT1A mutations in MAT I/III-deficient individuals and shown that the associated hypermethioninemic phenotype was inherited as an autosomal recessive trait. However, dominant inheritance of hypermethioninemia, also hypothesized to be caused by MAT I/III deficiency, has been reported in two families. Here we show that the only mutation uncovered in one of these families, G, is a G-->A transition at nt 791 in exon VII of one MAT1A allele that converts an arginine at position 264 to a histidine (R264H). This single allelic R264H mutation was subsequently identified in two hypermethioninemic individuals in an additional family, C. Family C members were also found to inherit hypermethioninemia in a dominant fashion, and the available affected members analyzed carried the single allelic R264H mutation. Substitution of R-264 with histidine (R264H, the naturally occurring mutant), leucine (R264L), aspartic acid (R264D), or glutamic acid (R264E) greatly reduced MAT activity and severely impaired the ability of the MAT(alpha)1 subunits to form homodimers essential for optimal catalytic activity. On the other hand, when lysine was substituted for R-264 (R264K), the mutant alpha1 subunit was able to form dimers that retain significant MAT activity, suggesting that amino acid 264 is involved in intersubunit salt-bridge formation. Cotransfection studies show that R264/R264H MAT(alpha)1 heterodimers are enzymatically inactive, thus providing an explanation for the R264H-mediated dominant inheritance of hypermethioninemia.