ATP-binding site of human brain hexokinase as studied by molecular modeling and site-directed mutagenesis

The interaction of ATP with the active site of hexokinase is unknown since the crystal structure of the hexokinase-ATP complex is unavailable. It was found that the ATP binding site of brain hexokinase is homologous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned in the catalytic domain of human brain hexokinase, which was modeled from the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is though to be involved in binding the Mg2+ ion of the MgATP2- complex, was mutated to Lys and Glu. The kcat values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 was proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The kcat value of the Thr680Val mutant decreased 2000-fold, whereas the kcat value of the Thr680Ser decreased only 2.5-fold, implying the importance of the hydroxyl group. The Km and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relative to the wild-type enzyme. The Ki values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosphate (Pi) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results from the site-directed mutagenesis demonstrate that the presumed interactions of investigated residues with ATP are important for the stabilization of the transition state.     

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.     

Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding

To provide new insights into ligand/A1 adenosine receptor (A1 AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane (TM) domains of the human A1 AR. Based on multiple sequence analysis of all known ARs, both acidic (glutamic acid and aspartic acid) and polar hydroxy (serine and threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, and Ser6 and Ser23 (TM1) were identified in all A1 ARs. To test the role of these residues, each was individually mutated to alanine. When Ala6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, affinities for [3H]2-chloro-N6-cyclopentyladenosine (CCPA) and [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen for the wild-type receptor. After conversion of Glu16 to Ala16, the affinity for [3H]CCPA and other agonists fell 10-100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. After conversion of Asp55 to Ala55, the affinity for [3H]CCPA and other agonists increased < or = 100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. Studies of the Ala55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [3H]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A1 AR immunoreactivity was present on stable cell lines; however, functional binding sites could not be detected. When Ser94 was converted to Thr94, the affinity for some xanthine antagonists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play important roles in agonist/A1 AR interactions and show that Asp55 is responsible for allosteric regulation of ligand/A1 AR binding by sodium. We also identify Ser94 as an important site for ligand binding.     

Structural studies of receptor binding by cholera toxin mutants

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.     

A study of subunit interface residues of fructose-1,6-bisphosphatase by site-directed mutagenesis: effects on AMP and Mg2+ affinities

The structural transformation of fructose-1,6-bisphosphatase upon binding of the allosteric regulator AMP dramatically changes the interactions across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Met18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose-1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spectrometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in kcat with Met18Arg and Asn9Asp, respectively. The K(m) for fructose 1,6-bisphosphate increased about 2-approximately 4-fold relative to that of the wild-type enzyme in the four mutants. A 50-fold lower Ka value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18Ile with no alteration of the Ki for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fold) without a change in Mg2+ affinity. Increases of 6- and 2-fold in the Ki values for AMP were found with Asn9Asp and Ser87Ala, respectively. There was no difference in the cooperativity for AMP inhibition between the wild-type and the mutant forms of fructose-1,6-bisphosphatase. This study demonstrates that the mutation of residues in the C1-C4 (C2-C3) interface of fructose-1,6-bisphosphatase can significantly affect the affinity for Mg2+, which is presumably bound 30 A away. Moreover the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the corresponding allosteric states of the enzyme. The kinetics and structural modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.     

Allosteric regulation of type I hexokinase: A site-directed mutational study indicating location of the functional glucose 6-phosphate binding site in the N-terminal half of the enzyme

The Type I isozyme of mammalian hexokinase has evolved by a gene duplication-fusion mechanism, with resulting internal duplication of sequence and ligand binding sites. However, 1:1 binding stoichiometry indicates that only one of these is available for binding the product inhibitor, Glc-6-P; the location of that site, in the N- or C-terminal half, remains under debate. Recent structural studies (Aleshin et al., Structure 6, 39-50, 1998; Mulichak et al., Nature Struct. Biol. 5, 555-560, 1998) implicated Asp 84 or its analog in the C-terminal half, Asp 532, in binding of Glc-6-P. Zeng et al. (Biochemistry 35, 13157-13164, 1996) demonstrated that mutation of Asp 532 to Lys or Glu did not affect inhibition by the Glc-6-P analog, 1,5-anhydroglucitol-6-P. These same mutations, as well as mutation to Ala, at the Asp 84 position are now shown to result in increased Ki for 1,5-anhydroglucitol-6-P. The ability of Pi to antagonize inhibition by the Glc-6-P analog is severely diminished or abolished by these mutations, suggesting that antagonism is dependent on precise positioning of the inhibitory hexose 6-phosphate. The structure of the enzyme complexed with Glc and Pi has been determined, and shows that Pi occupies the same site as the 6-phosphate group in the complex with Glc-6-P. Thus, antagonism between these ligands results from competition for a common anion binding site in the N-terminal half.     

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.     

Role of beta87 Thr in the beta6 Val acceptor site during deoxy Hb S polymerization

Three new Hb S variants containing beta87 Leu, Trp, or Asp instead of Thr were expressed in yeast in order to further define the role of the beta87 position in stability and polymerization of deoxy Hb S. Previous studies showed that hydrophobicity at beta85 Phe and beta88 Leu is critical for stabilization of hemoglobin. Results with the three Hb S beta87 variants, however, showed minimal differences in stability, suggesting that beta87 amino acid hydrophobicity is not critical for stabilization of hemoglobin. Polymerization properties of the variants in the deoxy form, however, were affected by the beta87 amino acid. Polymerization of Hb S beta87 Thr --> Leu and Hb S beta87 Thr --> Trp was preceded by a delay time like Hb S, while Hb S beta87 Thr --> Asp did not show a delay time. In addition, changes in time required for half polymer formation (T1/2) as a function of hemoglobin concentration for Hb S beta87 Thr --> Asp were similar to that for beta87 Thr --> Gln. Hb S beta87 Thr --> Leu polymerized at a lower hemoglobin concentration than Hb S while beta87 Thr --> Trp and Hb S beta87 Thr --> Asp required much higher hemoglobin concentrations for polymer formation. Critical concentration required for deoxy Hb S beta87 Thr --> Asp polymerization was 6- and 2.3-fold greater than that for Hb S beta85 Phe --> Glu and Hb S beta88 Leu --> Glu, respectively. These results suggest that even though beta87 Thr is not a direct interaction site for beta6 Val in deoxy Hb S polymers, it does play a critical role in formation of the hydrophobic acceptor pocket which then promotes protein-protein interactions facilitating formation of stable nuclei and polymers of deoxy Hb S.     

Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46

Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were purified by affinity chromatography and investigated as compared to the wild-type enzyme. The specific activity and substrate affinity of Thr42 and Thr46 mutants were not significantly affected. On the contrary, Asn41 mutants showed a residual negligible activity (about 0.05-0.15% as compared to wild-type enzyme), though maintaining an unchanged binding capability of both substrate and inorganic phosphate, an enzyme competitive inhibitor. According to the 1H nuclear magnetic resonance spectroscopy and circular dichroism results, all mutants elicited well-constrained native-like secondary and tertiary structures. Thermodynamic parameters, as calculated from circular dichroism data, demonstrated a significantly decreased stability of the Thr42 mutant under increasing temperatures and urea concentrations. The reported results strongly support a direct participation of Asn41 to the enzyme catalytic mechanism, indicating that Asn41 mutants may well represent a useful tool for the investigation of the enzyme physiological function by the negative dominant approach.     

Transgenic approaches to define the functional role of dual site phospholamban phosphorylation

Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca2+-ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser16 and Thr17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser16 --> Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wild-type phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser16 is a prerequisite for Thr17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the beta-agonist stimulatory responses in the mammalian heart.     

Hybridization of tRNAs of Drosophila melanogaster to polytene chromosomes

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila silivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC, tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5-7, 72F1-2, 83F-84A; tRNA3Met, 46A1-2, 61D1-2, 70F1-2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84d3-4, 92b1-9; tRNA4Val, 56D3-7, 70BC.     

Suramin is an active site-directed, reversible, and tight-binding inhibitor of protein-tyrosine phosphatases

The effect of suramin, a well known antitrypanosomal drug and a novel experimental agent for the treatment of several cancers, on protein-tyrosine phosphatases (PTPases) has been examined. Suramin is a reversible and competitive PTPase inhibitor with Kis values in the low microM range, whereas the Kis for the dual specificity phosphatase VHR is at least 10-fold higher. Although suramin can also inhibit the activity of the potato acid phosphatase at a slightly higher concentration, it is 2-3 orders of magnitude less effective against the protein Ser/Thr phosphatase 1alpha and the bovine intestinal alkaline phosphatase. Suramin binds to the active site of PTPases with a binding stoichiometry of 1:1. Furthermore, when suramin is bound to the active site of PTPases, its fluorescence is enhanced approximately by 10-fold. This property has allowed the determination of the binding affinity of suramin for PTPases and several catalytically impaired mutant PTPases by fluorescence titration techniques. Thus, the active site Cys to Ser mutants bind suramin with similar affinity as the wild type, while the active site Arg to Ala mutant exhibits a 20-fold reduced affinity toward suramin. Interestingly, the general acid deficient Asp to Ala mutant PTPases display an enhanced affinity toward suramin, which is in accord with their use as improved "substrate-trapping" agents. That suramin is a high affinity PTPase inhibitor is consistent with the observation that suramin treatment of cancer cell lines leads to an increase in tyrosine phosphorylation of several cellular proteins. Given the pleiotropic effects of suramin on many enzyme systems and growth factor-receptor interactions, the exact in vivo actions of suramin require further detailed structure-activity investigation of suramin and its structural analogs.     

Mutation of serine-46 to aspartate in the histidine-containing protein of Escherichia coli mimics the inactivation by phosphorylation of serine-46 in HPrs from gram-positive bacteria

Histidine-containing protein (HPr) is a phosphocarrier protein of the bacterial phosphoenolpyruvate:sugar phosphotransferase system. HPr is phosphorylated at the active site residue, His15, by phosphoenolpyruvate-dependent enzyme I in the first enzyme reaction in the process of phosphoryl transfer to sugar. In many Gram-positive bacterial species HPr may also be phosphorylated at Ser46 by an ATP-dependent protein kinase but not in the Gram-negative Escherichia coli and Salmonella typhimurium. One effect of the phosphorylation at Ser46 is to make HPr a poor acceptor for phosphorylation at His15. In Bacillus subtilis HPr, the mutation Ser46Asp mimics the effects of phosphorylation. A series of mutations were made at Ser46 in E. coli HPr: Ala, Arg, Asn, Asp, Glu, and Gly. The two acidic replacements mimic the effects of phosphorylation of Ser46 in HPrs from Gram-positive bacteria. In particular, when mutated to Asp46, the His 15 phosphoacceptor activity (enzyme I Km/Kcat) decreases by about 2000-fold (enzyme I Km, 4 mM HPr; Kcat, approximately 30%). The alanine and glycine mutations had near-wild-type properties, and the asparagine and arginine mutations yielded small changes to the Km values. The crystallographic tertiary structure of Ser46Asp HPr has been determined at 1.5 A resolution, and several changes have been observed which appear to be the effect of the mutation. There is a tightening of helix B, which is demonstrated by a consistent shortening of hydrogen bond lengths throughout the helix as compared to the wild-type structure. There is a repositioning of the Gly54 residue to adopt a 3(10) helical pattern which is not present in the wild-type HPr. In addition, the higher resolution of the mutant structure allows for a more definitive placement of the carbonyl of Pro11. The consequence of this change is that there is no torsion angle strain at residue 16. This result suggests that there is no active site torsion angle strain in wild-type E. coli HPr. The lack of substantial change at the active center of E. coli HPr Ser46Asp HPr suggests that the effect of the Ser46 phosphorylation in HPrs from Gram-positive bacteria is due to an electrostatic interference with enzyme I binding.     

Naive human T cells develop into Th1 or Th0 effectors and exhibit cytotoxicity early after stimulation with Leishmania-infected macrophages

In MS2 assembly of phage particles results from an interaction between a coat protein dimer and a stem-loop of the RNA genome (the operator hairpin). Amino acid residues Thr45, which is universally conserved among the small RNA phages, and Thr59 are part of the specific RNA binding pocket and interact directly with the RNA; the former through a hydrogen bond, the latter through hydrophobic contacts. The crystal structures of MS2 protein capsids formed by mutants Thr45Ala and Thr59Ser, both with and without the 19 nt wild-type operator hairpin bound, are reported here. The RNA hairpin binds to these mutants in a similar way to its binding to wild-type protein. In a companion paper both mutants are shown to be deficient in RNA binding in an in vivo assay, but in vitro the equilibrium dissociation constant is significantly higher than wild-type for the Thr45Ala mutant. The change in binding affinity of the Thr45Ala mutant is probably a direct consequence of removal of direct hydrogen bonds between the protein and the RNA. The properties of the Thr59Ser mutant are more difficult to explain, but are consistent with a loss of non-polar contact.     

Contribution of cutinase serine 42 side chain to the stabilization of the oxyanion transition state

Cutinase from the fungus Fusarium solani pisi is a lipolytic enzyme able to hydrolyze both aggregated and soluble substrates. It therefore provides a powerful tool for probing the mechanisms underlying lipid hydrolysis. Lipolytic enzymes have a catalytic machinery similar to those present in serine proteinases. It is characterized by the triad Ser, His, and Asp (Glu) residues, by an oxyanion binding site that stabilizes the transition state via hydrogen bonds with two main chain amide groups, and possibly by other determinants. It has been suggested on the basis of a covalently bond inhibitor that the cutinase oxyanion hole may consist not only of two main chain amide groups but also of the Ser42 O gamma side chain. Among the esterases and the serine and the cysteine proteases, only Streptomyces scabies esterase, subtilisin, and papain, respectively, have a side chain residue which is involved in the oxyanion hole formation. The position of the cutinase Ser42 side chain is structurally conserved in Rhizomucor miehei lipase with Ser82 O gamma, in Rhizopus delemar lipase with Thr83 O gamma 1, and in Candida antartica B lipase with Thr40 O gamma 1. To evaluate the increase in the tetrahedral intermediate stability provided by Ser42 O gamma, we mutated Ser42 into Ala. Furthermore, since the proper orientation of Ser42 O gamma is directed by Asn84, we mutated Asn84 into Ala, Leu, Asp, and Trp, respectively, to investigate the contribution of this indirect interaction to the stabilization of the oxyanion hole. The S42A mutation resulted in a drastic decrease in the activity (450-fold) without significantly perturbing the three-dimensional structure. The N84A and N84L mutations had milder kinetic effects and did not disrupt the structure of the active site, whereas the N84W and N84D mutations abolished the enzymatic activity due to drastic steric and electrostatic effects, respectively.     

The agonistic binding site at the histamine H2 receptor. I. Theoretical investigations of histamine binding to an oligopeptide mimicking a part of the fifth transmembrane alpha-helix

Mutation studies on the histamine H2 receptor were reported by Gantz et al. [J. Biol. Chem., 267 (1992) 20840], which indicate that both the mutation of the fifth transmembrane Asp186 (to Ala186) alone or in combination with Thr190 (to Ala190) maintained, albeit partially, the cAMP response to histamine. Recently, we have shown that histamine binds to the histamine H2 receptor as a monocation in its proximal tautomeric form, and, moreover, we suggested that a proton is donated from the receptor towards the tele-position of the agonist, thereby triggering the biological effect [Nederkoorn et al., J. Mol. Graph., 12 (1994) 242; Eriks et al., Mol. Pharmacol., 44 (1993) 886]. These findings result in a close resemblance with the catalytic triad (consisting of Ser, His and Asp) found in serine proteases. Thr190 resembles a triad's serine residue closely, and could also act as a proton donor. However, the mutation of Thr190 to Ala190-the latter is unable to function as a proton donor-does not completely abolish the agonistic cAMP response. At the fifth transmembrane alpha-helix of the histamine H2 receptor near the extracellular surface, another amino acid is present, i.e. Tyr182, which could act as a proton donor. Furthermore, Tyr182 lies within the proximity of Asp186, so an alternative couple of amino acids, Tyr182 and Asp186, could constitute the histamine binding site at the fifth alpha-helix instead of the (mutated) couple Asp186 and Thr190. In the first part of our present study, this hypothesis is investigated with the aid of an oligopeptide with an alpha-helical backbone, which represents a part of the fifth transmembrane helix. Both molecular mechanics and ab initio data lead to the conclusion that the Tyr182/Asp186 couple is most likely to act as the binding site for the imidazole ring present in histamine.     

Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations

To stabilize Aspergillus awamori glucoamylase (GA), three proline substitution mutations were constructed. When expressed in Saccharomyces cerevisiae, Ser30-->Pro (S30P) stabilized the enzyme without decreased activity, whereas Asp345-->Pro (D345P) did not significantly alter and Glu408-->Pro (E408P) greatly decreased enzyme thermostability. The S30P mutation was combined with two previously identified stabilizing mutations: Gly137-->Ala, and Asn20-->Cys/Ala27-->Cys (which creates a disulfide bond between positions 20 and 27). The combined mutants demonstrated cumulative stabilization as shown by decreased irreversible thermoinactivation rates between 65 and 80 degrees C. Additionally, two of the combined mutants outperformed wild-type GA in high-temperature (65 degrees C) saccharifications of DE 10 maltodextrin and were more active than the wild-type enzyme when assayed using maltose as substrate.     

Mutations in Bartonella bacilliformis gyrB confer resistance to coumermycin A1

This study describes the first isolation and characterization of spontaneous mutants conferring natural resistance to an antibiotic for any Bartonella species. The Bartonella bacilliformis gyrB gene, which encodes the B subunit of DNA gyrase, was cloned and sequenced. The gyrB open reading frame (ORF) is 2,079 bp and encodes a deduced amino acid sequence of 692 residues, corresponding to a predicted protein of approximately 77.5 kDa. Sequence alignment indicates that B. bacilliformis GyrB is most similar to the GyrB protein from Bacillus subtilis (40.1% amino acid sequence identity) and that it contains the longest N-terminal tail (52 residues) of any GyrB characterized to date. The cloned B. bacilliformis gyrB was expressed in an Escherichia coli S30 cell extract and was able to functionally complement a temperature-sensitive E. coli Cour gyrB mutant (strain N4177). We isolated and characterized spontaneous mutants of B. bacilliformis resistant to coumermycin A1, an antibiotic that targets GyrB. Sequence analysis of gyrB from 12 Cour mutants of B. bacilliformis identified single nucleotide transitions at three separate loci in the ORF. The predicted amino acid substitutions resulting from these transitions are Gly to Ser at position 124 (Gly124-->Ser), Arg184-->Gln, and Thr214-->Ala or Thr214-->Ile, which are analogous to mutated residues found in previously characterized resistant gyrB genes from Borrelia burgdorferi, E. coli, Staphylococcus aureus, and Haloferax sp. The Cour mutants are three to five times more resistant to coumermycin A1 than the wild-type parental strain.     

Cysteine-scanning mutagenesis of helix IV and the adjoining loops in the lactose permease of Escherichia coli: Glu126 and Arg144 are essential. off

Cys-scanning mutagenesis has been applied to the remaining 45 residues in lactose permease that have not been mutagenized previously (from Gln100 to Arg144 which comprise helix IV and adjoining loops). Of the 45 single-Cys mutants, 26 accumulate lactose to > 75% of the steady state observed with Cys-less permease, and 14 mutants exhibit lower but significant levels of accumulation (35-65% of Cys-less permease). Permease with Phe140-->Cys or Lys131-->Cys exhibits low activity (15-20% of Cys-less permease), while mutants Gly115-->Cys, Glu126-->Cys and Arg144-->Cys are completely unable to accumulate the dissacharide. However, Cys-less permease with Ala or Pro in place of Gly115 is highly active, and replacement of Lys131 or Phe140 with Cys in wild-type permease has a less deleterious effect on activity. In contrast, mutant Glu126-->Cys or Arg144-->Cys is inactive with respect to both uphill and downhill transport in either Cys-less or wild-type permease. Furthermore, mutants Glu126-->Ala or Gln and Arg144-->Ala or Gln are also inactive in both backgrounds, and activity is not rescued by double neutral replacements or inversion of the charged residues at these positions. Finally, a mutant with Lys in place of Arg144 accumulates lactose to about 25% of the steady state of wild-type, but at a slow rate. Replacement of Glu126 with Asp, in contrast, has relatively little effect on activity. None of the effects can be attributed to decreased expression of the mutants, as judged by immunoblot analysis. Although the activity of most of the single-Cys mutants is unaffected by N-ethylmaleimide, Cys replacement at three positions (Ala127, Val132, or Phe138) renders the permease highly sensitive to alkylation. The results indicate that the cytoplasmic loop between helices IV and V, where insertional mutagenesis has little effect on activity [McKenna, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11954-11958], contains residues that play an important role in permease activity and that a carboxyl group at position 126 and a positive charge at position 144 are absolutely required.