Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.     

Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.     

Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.     

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between Cys316 and engineered cysteines in cytoplasmic loop 1

A dark state tertiary structure in the cytoplasmic domain of rhodopsin is presumed to be the key to the restriction of binding of transducin and rhodopsin kinase to rhodopsin. Upon light-activation, this tertiary structure undergoes a conformational change to form a new structure, which is recognized by the above proteins and signal transduction is initiated. In this and the following paper in this issue [Cai, K., Klein-Seetharaman, J., Altenbach, C., Hubbell, W. L., and Khorana, H. G. (2001) Biochemistry 40, 12479-12485], we probe the dark state cytoplasmic domain structure in rhodopsin by investigating proximity between amino acids in different regions of the cytoplasmic face. The approach uses engineered pairs of cysteines at predetermined positions, which are tested for spontaneous formation of disulfide bonds between them, indicative of proximity between the original amino acids. Focusing here on proximity between the native cysteine at position 316 and engineered cysteines at amino acid positions 55-75 in the cytoplasmic sequence connecting helices I-II, disulfide bond formation was studied under strictly defined conditions and plotted as a function of the position of the variable cysteines. An absolute maximum was observed for position 65 with two additional relative maxima for cysteines at positions 61 and 68. The observed disulfide bond formation rates correlate well with proximity of these residues found in the crystal structure of rhodopsin in the dark. Modeling of the engineered cysteines in the crystal structure indicates that small but significant motions are required for productive disulfide bond formation. During these motions, secondary structure elements are retained as indicated by the lack of disulfide bond formation in cysteines that do not face toward Cys316 in the crystal structure model. Such motions may be important in light-induced conformational changes.     

The amino terminus of the fourth cytoplasmic loop of rhodopsin modulates rhodopsin-transducin interaction

Rhodopsin is a seven-transmembrane helix receptor that binds and catalytically activates the heterotrimeric G protein transducin (G(t)). This interaction involves the cytoplasmic surface of rhodopsin, which comprises four putative loops and the carboxyl-terminal tail. The fourth loop connects the carboxyl end of transmembrane helix 7 with Cys(322) and Cys(323), which are both modified by membrane-inserted palmitoyl groups. Published data on the roles of the fourth loop in the binding and activation of G(t) are contradictory. Here, we attempt to reconcile these conflicts and define a role for the fourth loop in rhodopsin-G(t) interactions. Fluorescence experiments demonstrated that a synthetic peptide corresponding to the fourth loop of rhodopsin inhibited the activation of G(t) by rhodopsin and interacted directly with the alpha subunit of G(t). A series of rhodopsin mutants was prepared in which portions of the fourth loop were replaced with analogous sequences from the beta(2)-adrenergic receptor or the m1 muscarinic receptor. Chimeric receptors in which residues 310-312 were replaced could not efficiently activate G(t). The defect in G(t) interaction in the fourth loop mutants was not affected by preventing palmitoylation of Cys(322) and Cys(323). We suggest that the amino terminus of the fourth loop interacts directly with G(t), particularly with Galpha(t), and with other regions of the intracellular surface of rhodopsin to support G(t) binding.     

Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein autoprotease.

The structural proteins of Sindbis virus are translated as a polyprotein precursor that is cleaved upon translation. The capsid protein is postulated to be a serine protease that releases itself from the N terminus of the nascent polyprotein by autoproteolysis. We have tested the importance in autoproteolysis of His-141, Asp-147, and Ser-215, previously postulated to form the catalytic triad of the protease, and of Asp-163. Several site-specific mutations were constructed at each of these positions, and the release of the capsid protein during translation in a cell-free system was examined. Because proteolysis occurs in cis during translation, the kinetics of release cannot be determined in this system, but the extent of proteolysis can be ascertained. Ser-215 appears to be the catalytic serine of the proteinase. Cys or Thr could substitute inefficiently for Ser-215, but substitution with Ala or Ile led to complete loss of activity. His-141 was also important for proteolysis. Substitution with Ala or Pro led to total loss of activity. Surprisingly, substitution with Arg resulted in complete proteolysis in vitro. Changes at the two Asp residues resulted in complete proteolysis of the substrate in vitro. All mutations that resulted in at least partial cleavage in vitro were incorporated into a full-length clone of Sindbis virus and an attempt was made to recover mutant virus. All changes tested were lethal for the virus except Asp-163 to Asn. Thus, production of infectious virus is either a more sensitive measure of the catalytic rate than the extent of in vitro cleavage, or these residues have necessary functions in addition to their possible role in proteolysis.     

Site-directed mutagenesis reveals roles for conserved amino acid residues in the hexameric DNA helicase DnaB from Bacillus stearothermophilus

Site-directed mutagenesis studies on conserved amino acid residues within motifs H1, H1a, H2 and H3 of the hexameric replicative helicase DnaB from Bacillus stearothermophilus revealed specific functions associated with these residues. In particular, residues that coordinate a bound Mg²⁺ in the active site (T217 and D320) are important for the function of the enzyme but are not required for the formation of stable hexamers. A conserved glutamic acid (E241) in motif H1a is likely to be involved in the activation of a water molecule for in line attack on the γ-phosphate of the bound nucleotide during catalysis. A conserved glutamine (Q362) in motif H3 acts as a γ-phosphate sensor and mediates the conformational coupling of nucleotide- and DNA-binding sites. The nature of the residue at this position is also important for the primase-mediated activation of DnaB, suggesting that primase uses the same conformational coupling pathway to induce its stimulatory effect on the activity of DnaB. Together, these mutations reveal a conservation of many aspects of biochemical activity in the active sites of monomeric and hexameric helicases.     

Site-directed mutagenesis of firefly luciferase active site amino acids: a proposed model for bioluminescence color.

Under physiological conditions firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen. In nature, bioluminescence emission by beetle luciferases is observed in colors ranging from green (approximately 530 nm) to red (approximately 635 nm), yet all known luciferases use the same luciferin substrate. In an earlier report [Branchini, B. R., Magyar, R. M., Murtiashaw, M. H., Anderson, S. M., and Zimmer, M. (1998) Biochemistry 37, 15311-15319], we described the effects of mutations at His245 on luciferase activity. In the context of molecular modeling results, we proposed that His245 is located at the luciferase active site. We noted too that the H245 mutants displayed red-shifted bioluminescent emission spectra. We report here the construction and purification of additional His245 mutants, as well as mutants at residues Lys529 and Thr343, all of which are stringently conserved in the beetle luciferase sequences. Analysis of specific activity and steady-state kinetic constants suggested that these residues are involved in luciferase catalysis and the productive binding of substrates. Bioluminescence emission spectroscopy studies indicated that point mutations at His245 and Thr343 produced luciferases that emitted light over the color range from green to red. The results of mutational and biochemical studies with luciferase reported here have enabled us to propose speculative mechanisms for color determination in firefly bioluminescence. An essential role for Thr343, the participation of His245 and Arg218, and the involvement of bound AMP are indicated.     

The second cytoplasmic loop of metabotropic glutamate receptor functions at the third loop position of rhodopsin.

G protein-coupled receptors identified so far are classified into at least three major families based on their amino acid sequences. For the family of receptors homologous to rhodopsin (family 1), the G protein activation mechanism has been investigated in detail, but much less for the receptors of other families. To functionally compare the G protein activation mechanism between rhodopsin and metabotropic glutamate receptor (mGluR), which belong to distinct families, we prepared a set of bovine rhodopsin mutants whose second or third cytoplasmic loop was replaced with either the second or third loop of Gi/Go- or Gq-coupled mGluR (mGluR6 or mGluR1). Among these mutants, the mutants in which the second or third loop was replaced with the corresponding loop of mGluR exhibited no G protein activation ability. In contrast, the mutant whose third loop was replaced with the second loop of Gi/Go-coupled mGluR6 efficiently activated Gi but not Gt: this activation profile is almost identical with those of the mutant rhodopsins whose third loop was replaced with those of the Gi/Go-coupled receptors in family 1 [Yamashita et al. (2000) J. Biol. Chem. 275, 34272-34279]. The mutant whose third loop was replaced with the second loop of Gq-coupled mGluR1 partially retained the Gi coupling ability of rhodopsin, which is in contrast to the fact that all the rhodopsin mutants having the third loops of Gq-coupled receptors in family 1 exhibit no detectable Gi activation. These results strongly suggest that the molecular architectures of rhodopsin and mGluR are different, although the G protein activation mechanism involving the cytoplasmic loops is common.     

Site-directed mutagenesis of the conserved residues in component I of Bacillus subtilis heptaprenyl diphosphate synthase.

Heptaprenyl diphosphate synthase of Bacillus subtilis is composed of two dissociable heteromeric subunits, component I and component II. Component II has highly conserved regions typical of (E)-prenyl diphosphate synthases, but it shows no prenyltransferase activity alone unless it is combined with component I. Alignment of amino acid sequences for component I and the corresponding subunits of Bacillus stearothermophilus heptaprenyl diphosphate synthase and Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase shows three regions of high similarity. To elucidate the role of these regions of component I during catalysis, 13 of the conserved amino acid residues in these regions were selected for substitution by site-directed mutagenesis. Kinetic studies indicated that substitutions of Val-93 with Gly, Leu-94 with Ser, and Tyr-104 with Ser resulted in 3-10-fold increases of K(m) values for the allylic substrate and 5-15-fold decreases of V(max) values compared to those of the wild-type enzyme. The three mutated enzymes, V93G, L94S, and Y104S, showed little binding affinity to the allylic substrate in the membrane filter assay. Furthermore, product analyses showed that D97A yielded shorter chain prenyl diphosphates as the main product, while Y103S gave the final product with a C(40) prenyl chain length. These results suggest that some of the conserved residues in region B of component I are involved in the binding of allylic substrate as well as determining the chain length of the enzymatic reaction product.     

Site-directed mutagenesis of amino acid residues involved in the glutathione binding of human glutathione S-transferase P1-1.

The four residues of human glutathione S-transferase P1-1 whose counterparts were indicated by X-ray crystallography to reside in the GSH-binding site of pig glutathione S-transferase P1-1 were individually replaced with threonine or alanine by site-directed mutagenesis to obtain mutants R13T, K44T, Q51A, and Q64A. The kinetic parameters, susceptibilities to an inhibitor, S-hexyl-GSH, and affinities for GSH-Sepharose of the latter were compared with those of the wild-type enzyme, and pKa of the thiol group of GSH bound in R13T was shown to be equivalent to that in the wild type. From the results, Lys44, Gln51, and Gln64 were deduced to contribute to the binding of GSH. On the other hand, Arg13 seems to be essential for the enzymatic activity as mainly involved in the construction of a proper structure of the active site.     

Stability of dark state rhodopsin is mediated by a conserved ion pair in intradiscal loop E-2

The rhodopsin crystal structure reveals that intradiscal loop E-2 covers the 11-cis-retinal, creating a "retinal plug." Recently, we noticed the ends of loop E-2 are linked by an ion pair between residues Arg-177 and Asp-190, near the highly conserved disulfide bond. This ion pair appears biologically significant; it is conserved in almost all vertebrate opsins and may occur in other G-protein-coupled receptors. We report here that the Arg-177/Asp-190 ion pair is critical for the folding and stability of dark state rhodopsin. We find ion pair mutants that regenerate with retinal are functionally and spectrally wild-type-like yet thermally unstable in their dark state because of rapid hydrolysis of the retinal Schiff base linkage. Surprisingly, Arrhenius analysis indicates that the activation energies for the hydrolysis process are similar between the ion pair mutants and wild-type rhodopsin. Furthermore, the ion pair mutants do not show increased reactivity toward hydroxylamine, suggesting that their instability is not caused by an increased exposure to bulk solvent. Our results indicate that the loop E-2 ion pair is important for rhodopsin stability and thus suggest that retinitis pigmentosa observed in patients with Asp-190 mutations may in part be the result of thermally unstable rhodopsin proteins.     

Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.

Site-directed mutagenesis and molecular dynamics simulations of the alpha 1B-adrenergic receptor (AR) were combined to explore the potential molecular changes correlated with the transition from R (inactive state) to R (active state). Using molecular dynamics analysis we compared the structural/dynamic features of constitutively active mutants with those of the wild type and of an inactive alpha 1B-AR to build a theoretical model which defines the essential features of R and R. The results of site-directed mutagenesis were in striking agreement with the predictions of the model supporting the following hypothesis. (i) The equilibrium between R and R depends on the equilibrium between the deprotonated and protonated forms, respectively, of D142 of the DRY motif. In fact, replacement of D142 with alanine confers high constitutive activity to the alpha 1B-AR. (ii) The shift of R143 of the DRY sequence out of a conserved 'polar pocket' formed by N63, D91, N344 and Y348 is a feature common to all the active structures, suggesting that the role of R143 is fundamental for mediating receptor activation. Disruption of these intramolecular interactions by replacing N63 with alanine constitutively activates the alpha 1B-AR. Our findings might provide interesting generalities about the activation process of G protein-coupled receptors.     

Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1.

Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.     

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between cysteine pairs engineered in cytoplasmic loops 1, 3, and 4

To probe proximities between amino acids in the cytoplasmic domain by using mutants containing engineered cysteine pairs, three sets of rhodopsin mutants have been prepared. In the first two sets, a cysteine was placed, one at a time, at positions 311-314 in helix VIII, while the second cysteine was fixed at position 246 (set I) and at position 250 (set II) at the cytoplasmic end of helix VI. In the third set, one cysteine was fixed at position 65 while the second cysteine was varied between amino acid positions 306 and 321 located at the cytoplasmic end of helix VII and throughout in helix VIII. Rapid disulfide bond formation in the dark was found between the cysteine pairs in mutants A246C/Q312C,A246C/K311C and in mutants H65C/C316, H65C/315C and H65C/312C. Disulfide bond formation at much lower rates was found in mutants A246C/F313C, V250C/Q312C, H65C/N310C, H65C/K311C, H65C/F313C, and H65C/R314C; the remaining mutants showed no significant disulfide bond formation. Comparisons of the results from disulfide bond formation in solution with the distances observed in the rhodopsin crystal structure showed that the rates of disulfide bond formation in most cases were consistent with the amino acid proximities as revealed in crystal structure. However, deviations were also found, in particular, in the set containing fixed cysteine at position Cys246 and cysteines at positions 311-314. The results implicate significant effects of structural dynamics on disulfide bond formation in solution.     

Hydrophobic amino acids at the cytoplasmic ends of helices 3 and 6 of rhodopsin conjointly modulate transducin activation

Rhodopsin is the visual photoreceptor responsible for dim light vision. This receptor is located in the rod cell of the retina and is a prototypical member of the G-protein-coupled receptor superfamily. The structural details underlying the molecular recognition event in transducin activation by photoactivated rhodopsin are of key interest to unravel the molecular mechanism of signal transduction in the retina. We constructed and expressed rhodopsin mutants in the second and third cytoplasmic domains of rhodopsin – where the natural amino acids were substituted by the human M3 acetylcholine muscarinic receptor homologous residues – in order to determine their potential involvement in G-protein recognition. These mutants showed normal chromophore formation and a similar photobleaching behavior than WT rhodopsin, but decreased thermal stability in the dark state. The single mutant V138^3.53 and the multiple mutant containing V227^5.62 and a combination of mutations at the cytoplasmic end of transmembrane helix 6 caused a reduction in transducin activation upon rhodopsin photoactivation. Furthermore, combination of mutants at the second and third cytoplasmic domains revealed a cooperative role, and partially restored transducin activation. The results indicate that hydrophobic interactions by V138^3.53, V227^5.62, V250^6.33, V254^6.37 and I255^6.38 are critical for receptor activation and/or efficient rhodopsin–transducin interaction.     

Site-directed mutagenesis of the Klebsiella pneumoniae nitrogenase. Effects of modifying conserved cysteine residues in the alpha- and beta-subunits.

The five conserved cysteine residues present in the alpha-subunit and the three conserved cysteine residues present in the beta-subunit of nitrogenase component 1 were individually changed to alanine. Mutations in the alpha-subunit at positions 63, 89, 155 and 275 and in the beta-subunit at positions 69, 94 and 152 all resulted in a loss of diazotrophic growth and component 1 activity and loss of the normal e.p.r. signal of the component 1 protein. Component 2 activity was retained. Replacement of cysteine-184 in the alpha-subunit with alanine greatly diminished, but did not eliminate, diazotrophic growth and component 1 activity. Substitution of serine for cysteine at position 152 in the beta-subunit, in contrast with the substitution of alanine at this position, resulted in the formation of active component 1. Replacement of the non-conserved cysteine-112 in the beta-subunit with alanine did not greatly perturb diazotrophic growth or the activity of component 1. Extracts prepared from a mutant, with cysteine-275 of the alpha-subunit replaced by alanine, complemented extracts of a mutant unable to synthesize the iron-molybdenum cofactor of nitrogenase, indicating that the alanine-275 substitution increases the availability of cofactor. Furthermore extracts of this mutant exhibited an e.p.r. signal similar to that of extracted iron-molybdenum cofactor. These data suggest a role for cysteine-275 as a ligand to the cofactor.     

Site-directed mutagenesis analysis of amino acids critical for activity of the type I signal peptidase of the archaeon Methanococcus voltae

Site-directed mutagenesis studies of the signal peptidase of the methanogenic archaeon Methanococcus voltae identified three conserved residues (Ser52, His122, and Asp148) critical for activity. The requirement for one conserved aspartic acid residue distinguishes the archaeal enzyme from both the Escherichia coli and yeast Sec11 enzymes.