The challenge-phage assay reveals differences in the binding equilibria of mutant Escherichia coli Trp super-repressors in vivo.

The phenotypes of four mutant Escherichia coli Trp repressor proteins with increased activities have been examined in vivo using the challenge-phage assay, an assay based on a positive genetic selection for DNA binding. These proteins, which differ by single amino acid changes from the wild type (Glu13-->Lys, Glu18-->Lys, Glu49-->Lys and Ala77-->Val), require less L-tryptophan than wild-type repressor for activation in vivo, and are super-aporepressors. However, none of the four mutant repressors binds DNA in a corepressor-independent manner. Three of the four mutant repressors (with Glu-->Lys changes) are more active when complexed with tryptophan, and are superholorepressors. Challenge-phage assays with excess tryptophan rank the mutant holorepressors in the same order as determined by binding studies in vitro. Challenge-phage assays with limiting tryptophan reveal additional phenotypic differences among the mutant proteins. These results show that the challenge-phage assay is a robust assay for measuring the relative affinities of specific protein-DNA interactions in vivo.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.     

Systematic analysis of the entire second extracellular loop of the V(1a) vasopressin receptor: key residues, conserved throughout a G-protein-coupled receptor family, identified

The roles of extracellular residues of G-protein-coupled receptors (GPCRs) are not well defined compared with residues in transmembrane helices. Nevertheless, it has been established that extracellular domains of both peptide-GPCRs and amine-GPCRs incorporate functionally important residues. Extracellular loop 2 (ECL2) has attracted particular interest, because the x-ray structure of bovine rhodopsin revealed that ECL2 projects into the binding crevice within the transmembrane bundle. Our study provides the first comprehensive investigation into the role of the individual residues comprising the entire ECL2 domain of a small peptide-GPCR. Using the V(1a) vasopressin receptor, systematic substitution of all of the ECL2 residues by Ala generated 30 mutant receptors that were characterized pharmacologically. The majority of these mutant receptor constructs (24 in total) had essentially wild-type ligand binding and intracellular signaling characteristics, indicating that these residues are not critical for normal receptor function. However, four aromatic residues Phe(189), Trp(206), Phe(209), and Tyr(218) are important for agonist binding and receptor activation and are highly conserved throughout the neurohypophysial hormone subfamily of peptide-GPCRs. Located in the middle of ECL2, juxtaposed to the highly conserved disulfide bond, Trp(206) and Phe(209) project into the binding crevice. Indeed, Phe(209) is part of the Cys-X-X-X-Ar (where Ar is an aromatic residue) motif, which is well conserved in both peptide-GPCRs and amine-GPCRs. In contrast, Phe(189) and Tyr(218), located at the extreme ends of ECL2, may be important for determining the position of the ECL2 cap over the binding crevice. This study provides mechanistic insight into the roles of highly conserved ECL2 residues.     

Control of conformational equilibria in the human B2 bradykinin receptor. Modeling of nonpeptidic ligand action and comparison to the rhodopsin structure

A prototypic study of the molecular mechanisms of activation or inactivation of peptide hormone G protein-coupled receptors was carried out on the human B2 bradykinin receptor. A detailed pharmacological analysis of receptor mutants possessing either increased constitutive activity or impaired activation or ligand recognition allowed us to propose key residues participating in intramolecular interaction networks stabilizing receptor inactive or active conformations: Asn(113) and Tyr(115) (TM III), Trp(256) and Phe(259) (TM VI), Tyr(295) (TM VII) which are homologous of the rhodopsin residues Gly(120), Glu(122), Trp(265), Tyr(268), and Lys(296), respectively. An essential experimental finding was the spatial proximity between Asn(113), which is the cornerstone of inactive conformations, and Trp(256) which plays a subtle role in controlling the balance between active and inactive conformations. Molecular modeling and mutagenesis data showed that Trp(256) and Tyr(295) constitute, together with Gln(288), receptor contact points with original nonpeptidic ligands. It provided an explanation for the ligand inverse agonist behavior on the WT receptor, with underlying restricted motions of TMs III, VI, and VII, and its agonist behavior on the Ala(113) and Phe(256) constitutively activated mutants. These data on the B2 receptor emphasize that conformational equilibria are controlled in a coordinated fashion by key residues which are located at strategic positions for several G protein-coupled receptors. They are discussed in comparison with the recently determined rhodopsin crystallographic structure.     

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.     

Conserved Extracellular Cysteine Pair in the M3 Muscarinic Acetylcholine Receptor Is Essential for Proper Receptor Cell Surface Localization but Not for G Protein Coupling

Most G protein-coupled receptors contain a conserved pair of extracellular cysteine residues that are predicted to form a disulfide bond linking the first and second extracellular loops. Previous studies have shown that this disulfide bond may be critical for ligand binding, receptor activation, and/or proper receptor folding. However, the potential importance of the two conserved cysteine residues for proper receptor cell surface localization has not been investigated systematically. To address this issue, we used the rat M3 muscarinic receptor as a model system. Most studies were carried out with a modified version of this receptor subtype (lacking potential N-glycosylation sites and the central portion of the third intracellular loop) that could be readily detected via western blot analysis. Cys-->Ala mutant receptors were generated, transiently expressed in COS-7 cells, and then examined for their subcellular distribution and functional properties. ELISA and immunofluorescence studies showed that the presence of both conserved cysteine residues (corresponding to C140 and C220 in the rat M3 muscarinic receptor sequence) is required for efficient expression of the M3 muscarinic receptor on the cell surface. On the other hand, these residues were found not to be essential for protein stability (determined via immunoblotting) and receptor-mediated G protein activation (studied in second messenger assays). These results shed new light on the functional role of the two extracellular cysteine residues present in most G protein-coupled receptors.     

Conservative and nonconservative mutations in proteins: anomalous mutations in a transport receptor analyzed by free energy and quantum chemical calculations.

Experimental studies on a bacterial sulfate receptor have indicated anomalous relative binding affinities for the mutations Ser130-->Cys,Ser130-->Gly, and Ser130-->Ala. The loss of affinity for sulfate in the former mutation was previously attributed to a greater steric effect on the part of the Cys side chain relative to the Ser side chain, whereas the relatively small loss of binding affinity for the latter two mutations was attributed to the loss of a single hydrogen bond. In this report we present quantum chemical and statistical thermodynamic studies of these mutations. Qualitative results from these studies indicate that for the Ser130-->Cys mutation the large decrease in binding affinity is in part caused by steric effects, but also significantly by the differential work required to polarize the Cys thiol group relative to the Ser hydroxyl group. The Gly mutant cobinds a water molecule in the same location as the Ser side chain resulting in a relatively small decrease in binding affinity. Results for the Ala mutant are in disagreement with experimental results but are likely to be limited by insufficient sampling of configuration space due to physical constraints applied during the simulation.     

Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM.     

Quantitative measurements of the cooperativity in an EF-hand protein with sequential calcium binding.

Positive cooperativity, defined as an enhancement of the ligand affinity at one site as a consequence of binding the same type of ligand at another site, is a free energy coupling between binding sites. It can be present both in systems with sites having identical ligand affinities and in systems where the binding sites have different affinities. When the sites have widely different affinities such that they are filled with ligand in a sequential manner, it is often difficult to quantify or even detect the positive cooperativity, if it occurs. This study presents verification and quantitative measurements of the free energy coupling between the two calcium binding sites in a mutant form of calbindin D9k. Wild-type calbindin D9k binds two calcium ions with similar affinities and positive cooperativity--the free energy coupling, delta delta G, is around -8 kJ.mol-1 (Linse S, et al., 1991, Biochemistry 30: 154-162). The mutant, with the substitution Asn 56-->Ala, binds calcium in a sequential manner. In the present work we have taken advantage of the variations among different metal ions in terms of their preferences for the two binding sites in calbindin D9k. Combined studies of the binding of Ca2+, Cd2+, and La3+ have allowed us to conclude that in this mutant delta delta G < -6.4 kJ.mol-1, and that Cd2+ and La3+ also bind to this protein with positive cooperativity. The results justify the use of the (Ca2+)1 state of the Asn 56-->Ala mutant, as well as the (Cd2+)1 state of the wild type, as models for the half-saturated states along the two pathways of cooperative Ca2+ binding in calbindin D9k.     

A highly conserved tryptophan residue in the fourth transmembrane domain of the A1 adenosine receptor is essential for ligand binding but not receptor homodimerization

Dimerization between G protein-coupled receptors (GPCRs) is a clearly established phenomenon. However, limited information is currently available on the interface essential for this process. Based on structural comparisons and sequence homology between rhodopsin and A₁ adenosine receptor (A₁R), we initially hypothesized that four residues in transmembrane (TM) 4 and TM5 are involved in A₁R homodimerization. Accordingly, these residues were substituted with Ala by site-directed mutagenesis. Interestingly, the mutant protein displayed no significant decrease in homodimer formation compared with wild-type A₁R, as evident from coimmunoprecipitation and BRET² analyses (improved bioluminescence resonance energy transfer system offered by Perkin-Elmer Life Sciences), but lost ligand binding activity almost completely. Further studies disclosed that this effect was derived from the mutation of one particular residue, Trp132, which is highly conserved among many GPCRs. Confocal immunofluorescence and cell-surface biotinylation studies revealed that the mutant receptors localized normally at transfected cell membranes, signifying that loss of ligand binding was not because of defective cellular trafficking. Molecular modeling of the A₁R-ligand complex disclosed that Trp132 interacted with several residues located in TM3 and TM5 that stabilized agonist binding. Thus, loss of interactions of Trp with these residues may, in turn, disrupt binding to agonists. Our study provides strong evidence of the essential role of the highly conserved Trp132 in TM4 of adenosine receptors.     

The role and predicted propensity of conserved proline residues in the 5-HT3 receptor

5-HT3 receptors possess a number of highly conserved proline residues. We changed each of these to alanine, expressed the mutants as homomeric 5-HT3A receptors in HEK293 cells, and analyzed them with radioligand binding, electrophysiology, and immunocytochemistry. Mutation of Pro56, Pro104, Pro123, and Pro170 resulted in ablation of radioligand binding, whereas mutation of Pro257 and Pro301 did not. Only the latter were expressed at the plasma membrane but were non-functional. Thus the former, which are in the N-terminal domain, may be involved in forming correct receptor structure, while those in the transmembrane region (Pro257 and Pro301) are necessary for the function of the protein. To explore the conformational preference (propensity) of these residues we examined the proportion of cis-prolines and the influence of adjacent residues in known protein structures. 4.7% of prolines in the protein data base were in the cis conformation, and the distribution of amino acids adjacent to cis-prolines was not randomly distributed. Comparison of the proportion of each amino acid residue adjacent to a cis-proline revealed that aromatic and bend-facilitating residues were favored while those with beta-branched chains were not. Thus five residues (Gly, Pro, Tyr, Trp, Phe) and three residues (Pro, Tyr, Phe) were found more frequently than expected before and after cis-prolines respectively, whereas five residues (Val, Ile, Leu, Asp, Thr) and two residues (Asp, Glu) were found less frequently. Of the 20 proline residues in the 5-HT3A receptor subunit only Pro170 has adjacent residues that are favorable. Mutating these to non-favorable residues resulted in ablation of ligand binding, whereas replacement with alternative favorable residues did not. We therefore propose that Pro170, which is part of the characteristic cys-loop found in this family of proteins, may be in the cis conformation.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

Site-directed mutagenesis of the m3 muscarinic receptor: identification of a series of threonine and tyrosine residues involved in agonist but not antagonist binding.

The hydrophobic core of all muscarinic receptors contains several conserved serine, threonine and tyrosine residues, most of which do not occur in any other G-protein coupled receptor. Since these amino acids can serve as potential hydrogen bond donors or acceptors, we have tested the hypothesis that they may be involved in the selective binding of muscarinic ligands. To eliminate the OH groups present in these residues, we have created nine single point mutations in the rat m3 muscarinic receptor by converting serine and threonine residues to alanine, and tyrosine residues to phenylalanine. The ligand binding and functional properties of these receptors were studied after transient expression in COS-7 cells. Six out of the nine mutant receptors (threonine and tyrosine mutations) showed strong reductions (approximately 10- to 40-fold lower than the wild-type receptor) in agonist binding affinities and reduced potencies in agonist-induced activation of phosphoinositide hydrolysis. Their antagonist binding properties, however, were similar to those of the wild-type m3 receptor. Despite their location on different transmembrane domains (III, V, VI and VII), all six mutations are positioned at a similar level (one to two helical turns away from the membrane surface) within the outer leaflet of the plasma membrane and may thus define the plain in which muscarinic agonists (but not antagonists) bind to their target receptor.     

Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides.

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutants at Ser277 of Xenopus cdc2 protein kinase induce oocyte maturation in the absence of the positive regulatory phosphorylation site Thr161.

The cdc2 protein kinase is an important regulatory protein for both meiosis and mitosis. Previously, we demonstrated that simultaneous mutation of Thr14-->Ala14 and Tyr15-->Phe15 in the Xenopus cdc2 protein results in an activated cdc2 mutant that induces maturation in resting oocytes. In addition, we confirmed the importance of the positive regulatory phosphorylation site, Thr161, by demonstrating that cdc2 mutants containing additional mutations of Thr161-->Ala161 or Glu161 are inactive in the induction of oocyte maturation. Here, we have analyzed the importance of an additional putative cdc2 phosphorylation site,Ser277. Single mutation of Ser277-->Asp277 or Ala277 had no effect on activity, and these mutants were unable to induce Xenopus oocyte maturation. However, the double mutant Ala161/Asp277 was capable of inducing oocyte maturation, suggesting that mutation of Ser277-->Asp277 could compensate for the mutation of Thr161-->Ala161. The Asp277 mutation could also compensate for the Ala161 mutation in the background of the activating mutations Ala14/Phe15. Although mutants containing the compensatory Ala161 and Asp277 mutations were capable of inducing oocyte maturation, these mutant cdc2 proteins lacked detectable in vitro kinase activity. Tryptic phosphopeptide mapping of mutant cdc2 protein and comparison with in vitro synthesized peptides indicated that Ser277 is not a major site of phosphorylation in Xenopus oocytes; however, we cannot rule out the possibility of phosphorylation at this site in a biologically active subpopulation of cdc2 molecules. The data presented here, together with prior reports of Ser277 phosphorylation in somatic cells, suggest an important role for Ser277 in the regulation of cdc2 activity. The regulatory role of Ser277 most likely involves its indirect effects on the nearby residue Arg275, which participates in a structurally important ion pair with Glu173, which lies in the same loop as Thr161 in the cdc2 protein.     

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.     

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.     

Weakening of the interface between adjacent catalytic chains promotes domain closure in Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase from Escherichia coli is a dodecameric enzyme consisting of two trimeric catalytic subunits and three dimeric regulatory subunits. Asp-100, from one catalytic chain, is involved in stabilizing the C1-C2 interface by means of its interaction with Arg-65 from an adjacent catalytic chain. Replacement of Asp-100 by Ala has been shown previously to result in increases in the maximal specific activity, homotropic cooperativity, and the affinity for aspartate (Baker DP, Kantrowitz ER, 1993, Biochemistry 32:10150-10158). In order to determine whether these properties were due to promotion of domain closure induced by the weakening of the C1-C2 interface, we constructed a double mutant version of aspartate transcarbamoylase in which the Asp-100-->Ala mutation was introduced into the Glu-50-->Ala holoenzyme, a mutant in which domain closure is impaired. The Glu-50/Asp-100-->Ala enzyme is fourfold more active than the Glu-50-->Ala enzyme, and exhibits significant restoration of homotropic cooperativity with respect to aspartate. In addition, the Asp-100-->Ala mutation restores the ability of the Glu-50-->Ala enzyme to be activated by succinate and increases the affinity of the enzyme for the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA). At subsaturating concentrations of aspartate, the Glu-50/Asp-100-->Ala enzyme is activated more by ATP than the Glu-50-->Ala enzyme and is also inhibited more by CTP than either the wild-type or the Glu-50-->Ala enzyme. As opposed to the wild-type enzyme, the Glu-50/Asp-100-->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Glu-50/Asp-100-->Ala enzyme by solution X-ray scattering indicates that the double mutant exists in the same T quaternary structure as the wild-type enzyme in the absence of ligands and in the same R quaternary structure in the presence of saturating PALA. However, saturating concentrations of carbamoyl phosphate and succinate only convert a fraction of the Glu-50/Asp-100-->Ala enzyme population to the R quaternary structure, a behavior intermediate between that observed for the Glu-50-->Ala and wild-type enzymes. Solution X-ray scattering was also used to investigate the structural consequences of nucleotide binding to the Glu-50/Asp-100-->Ala enzyme.