Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low Km) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low Km of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys160 of LAT1 and Cys110 of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl2 with a Ki of 0.56+/-0.11 microM. The inhibitory effect of HgCl2 for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl2 for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys88 and Cys439, altered amino acid transport. The Vmax was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys439 residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

Site-directed mutagenesis of cysteine residues of large neutral amino acid transporter LAT1

The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood-brain barrier (BBB). Owing to the high affinity (low K_m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K_m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys¹⁶⁰ of LAT1 and Cys¹¹⁰ of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl₂ with a K_i of 0.56 ± 0.11 μM. The inhibitory effect of HgCl₂ for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl₂ for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys⁸⁸ and Cys⁴³⁹, altered amino acid transport. The V_max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys⁴³⁹ residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane.     

The unique ligand-binding pocket for the human prostacyclin receptor. Site-directed mutagenesis and molecular modeling

The human prostacyclin receptor is a seven-transmembrane alpha-helical G-protein coupled receptor, which plays important roles in both vascular smooth muscle relaxation as well as prevention of blood coagulation. The position of the native ligand-binding pocket for prostacyclin as well as other derivatives of the 20-carbon eicosanoid, arachidonic acid, has yet to be determined. Through the use of prostanoid receptor sequence alignments, site-directed mutagenesis, and the 2.8-A x-ray crystallographic structure of bovine rhodopsin, we have developed a three-dimensional model of the agonist-binding pocket within the seven-transmembrane (TM) domains of the human prostacyclin receptor. Upon mutation to alanine, 11 of 29 candidate residues within TM domains II, III, IV, V, and VII exhibited a marked decrease in agonist binding. Of this group, four amino acids, Arg-279 (TMVII), Phe-278 (TMVII), Tyr-75 (TMII), and Phe-95 (TMIII), were identified (via receptor amino acid sequence alignment, ligand structural comparison, and computer-assisted homology modeling) as having direct molecular interactions with ligand side-chain constituents. This binding pocket is distinct from that of the biogenic amine receptors and rhodopsin where the native ligands (also composed of a carbon ring and a carbon chain) are accommodated in an opposing direction. These findings should assist in the development of novel and highly specific ligands including selective antagonists for further molecular pharmacogenetic studies of the human prostacyclin receptor.     

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.     

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.     

Site-directed mutagenesis of the YCDTDS amino acid motif of the phi 29 DNA polymerase

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains amino acid consensus sequences common to other alpha-like DNA polymerases. Using site-directed mutagenesis we have studied the functional significance of the most conserved C-terminal segment mainly represented by the YCDTDS motif. A series of single point mutants has been constructed and the corresponding proteins have been overproduced and characterized. Measurements, on crude fractions, of the activity of the mutant proteins in the formation of the protein p3-dAMP initiation complex and in an in situ DNA polymerase assay, indicate that the YCDTDS domain is involved both in initiation and in elongation reactions.     

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 bovine FGF-2 cDNA allows the production of the human-form of FGF-2 in Escherichia coli

Human fibroblast growth factor-2 can be used to induce angiogenesis in ischemias and wound healing. Site-directed mutagenesis of bovine FGF-2 cDNA was performed in order to produce the human-form of FGF-2 in E. coli. The mitogenic, angiogenic and neurotrophic activities of the recovered protein were analysed by [³H]thymidine uptake to DNA of cultured fibroblasts, rabbit ear dermal ulcers wound healing and neuronal differentiation of PC12 cells.     

Unraveling the amino acid sequence crucial for heparin binding to collagen V

We have previously shown that a recombinant 12-kDa fragment of the collagen alpha1(V) chain (Ile(824)-Pro(950)), referred to as HepV, binds to heparin and heparan sulfate (Delacoux, F., Fichard, A., Geourjon, C., Garrone, R., and Ruggiero, F. (1998) J. Biol. Chem. 273, 15069-15076). No consensus sequence was found in the alpha1(V) primary sequence, but a cluster of 7 basic amino acids (in the Arg(900)-Arg(924) region) was postulated to contain the heparin-binding site. The contribution of individual basic amino acids within this sequence was examined by site-directed mutagenesis. Further evidence for the precise localization of the heparin-binding site was provided by experiments based on the fact that heparin can protect the alpha1(V) chain heparin-binding site from trypsin digestion. The results parallel the alanine scanning mutagenesis data, i.e. heparin binding to the alpha1(V) chain involved Arg(912), Arg(918), and Arg(921) and two additional neighboring basic residues, Lys(905) and Arg(909). Our data suggest that this extended sequence functions as a heparin-binding site in both collagens V and XI, indicating that these collagens use a novel sequence motif to interact with heparin.     

Improving the binding affinity of an antibody using molecular modeling and site-directed mutagenesis

Activated Factor X releases F1.2, a 271-amino acid peptide, from the amino terminus of prothrombin during blood coagulation. A nine-amino acid peptide, C9 (DSDRAIEGR), corresponding to the carboxyl terminus of F1.2 was synthesized and used to produce a monoclonal antibody, TA1 (K(D)) 1.22 x 10(-6) M). To model the TA1 antibody, we entered the sequence information of the cloned TA1 Fv into the antibody modeling program, ABM, which combines homology methods, conformational search procedures, and energy screening and has proved to be a reliable and reproducible antibody modeling method. Using a novel protein fusion procedure, we expressed the C9 peptide fused to the carboxyl terminus of the PENI repressor protein from Bacillus licheniformis in Escherichia coli. We constructed fusion proteins containing alanine substitutions for each amino acid in the C9 epitope. Binding studies, using the C9 alanine mutants and TA1, and spatial constraints predicted by the modeled TA1 binding cleft enabled us to establish a plausible conformation for C9 complexed with TA1. Furthermore, based on binding results of conservative amino acid substitutions in C9 and mutations in the antibody, we were able to refine the complex model and identify antibody mutations that would improve binding affinity.     

Site-directed mutagenesis of epoxide hydrolase to probe catalytic amino acid residues and reaction mechanism

Epoxide hydrolase from Rhodococcus opacus catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to L(+)-tartrate. It shows low but significant similarity to haloacid dehalogenase and haloacetate dehalogenase (16–23% identity). To identify catalytically important residues, we mutated 29 highly conserved charged and polar amino acid residues (except for one alanine). The replacement of D18, D193, R55, K164, H190, T22, Y170, N134 and A188 led to a significant loss in the enzyme activity, indicating their involvement in the catalysis. Single and multiple turnover reaction studies show that the enzyme reaction proceeded through the two-step mechanism involving the formation of a covalent intermediate. We discuss the roles of these residues and propose its possible reaction mechanism.     

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acid involved in its ribosomal binding and recycling

Starting from a synthetic modular gene (infA) encoding Escherichia coli translation initiation factor IF1, we have constructed mutants in which amino acids are deleted from the carboxyl terminus or in which His29 or His34 are replaced by Tyr or Asp residues. The mutant proteins were overproduced, purified and tested in vitro for their properties in several partial reactions of the translation initiation pathway and for their capacity to stimulate MS2 RNA-dependent protein synthesis. The results allow for the conclusion that: (i) Arg69 is part of the 30S ribosomal subunit binding site of IF1 and its deletion results in the substantial loss of all IF1 function; (ii) neither one of its two histidines is essential for the binding of IF1 to the 30S ribosomal subunit, for the stimulation of fMet-tRNA binding to 30S or 70S ribosomal particles or for MS2 RNA-dependent protein synthesis; but (iii) His29 is involved in the 50S subunit-induced ejection of IF1 from the 30S ribosomal subunit.     

Design, synthesis, FGF-1 binding, and molecular modeling studies of conformationally flexible heparin mimetic disaccharides

Disaccharide mimetics of a heparin sequence that binds to fibroblast growth factors were prepared by coupling a D-galactose donor with a methyl beta-D-gluco- or xylopyranoside acceptor. When fully sulfated, the glucose or xylose moieties exist in solution in equilibrium between the (4)C1 and (1)C4 conformers, as confirmed by 1H NMR spectroscopy, thus mimicking the conformationally flexible L-iduronic acid found in heparin. Docking calculations showed that the predicted locations of disaccharide sulfo groups in the binding site of FGF-1 are consistent with the positions observed for co-crystallized heparin-derived oligosaccharides. Predicted binding affinities are in accord with experimental Kd values obtained from binding assays and are similar to the predicted values for a model heparin disaccharide.     

Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme

The condensation step of fatty acid elongation is the addition of a C₂ unit from malonyl-CoA to an acyl primer catalyzed by one of two families of enzymes, the 3-ketoacyl-CoA synthases and the ELO-like condensing enzymes. 3-Ketoacyl-CoA synthases use a Claisen-like reaction mechanism while the mechanism of the ELO-catalyzed condensation reaction is unknown. We have used site-directed mutagenesis of Dictyostelium discoideum EloA to identify residues important to catalytic activity and/or structure. Mutation of highly conserved polar residues to alanine resulted in an inactive enzyme strongly suggesting that these residues play a role in the condensation reaction.     

Site-directed mutagenesis studies with EcoRV restriction endonuclease to identify regions involved in recognition and catalysis.

Guided by the X-ray structure analysis of a crystalline EcoRV-d(GGGATATCCC) complex (Winkler, in preparation), we have begun to identify functionally important amino acid residues of EcoRV. We show here that Asn70, Asp74, Ser183, Asn185, Thr186, and Asn188 are most likely involved in the binding and/or cleavage of the DNA, because their conservative substitution leads to mutants of no or strongly reduced activity. In addition, C-terminal amino acid residues of EcoRV seem to be important for its activity, since their deletion inactivates the enzyme. Following the identification of three functionally important regions, we have inspected the sequences of other restriction and modification enzymes for homologous regions. It was found that two restriction enzymes that recognize similar sequences as EcoRV (DpnII and HincII), as well as two modification enzymes (M.DpnII and, in a less apparent form, M.EcoRV), have the sequence motif -SerGlyXXXAsnIleXSer- in common, which in EcoRV contains the essential Ser183 and Asn188 residues. Furthermore, the C-terminal region, shown to be essential for EcoRV, is highly homologous to a similar region in the restriction endonuclease SmaI. On the basis of these findings we propose that these restriction enzymes and to a certain extent also some of their corresponding modification enzymes interact with DNA in a similar manner.     

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 active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad.

Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326----Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gln or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.     

Characterization of the structure of RAMP1 by mutagenesis and molecular modeling

Receptor activity modifying proteins (RAMPs) are a family of single-pass transmembrane proteins that dimerize with G-protein-coupled receptors. They may alter the ligand recognition properties of the receptors (particularly for the calcitonin receptor-like receptor, CLR). Very little structural information is available about RAMPs. Here, an ab initio model has been generated for the extracellular domain of RAMP1. The disulfide bond arrangement (Cys27-Cys82, Cys40-Cys72, and Cys57-Cys104) was determined by site-directed mutagenesis. The secondary structure (alpha-helices from residues 29-51, 60-80, and 87-100) was established from a consensus of predictive routines. Using these constraints, an assemblage of 25,000 structures was constructed and these were ranked using an all-atom statistical potential. The best 1000 conformations were energy minimized. The lowest scoring model was refined by molecular dynamics simulation. To validate our strategy, the same methods were applied to three proteins of known structure; PDB:1HP8, PDB:1V54 chain H (residues 21-85), and PDB:1T0P. When compared to the crystal structures, the models had root mean-square deviations of 3.8 A, 4.1 A, and 4.0 A, respectively. The model of RAMP1 suggested that Phe93, Tyr100, and Phe101 form a binding interface for CLR, whereas Trp74 and Phe92 may interact with ligands that bind to the CLR/RAMP1 heterodimer.     

A site-directed mutagenesis study to identify amino acid residues involved in the catalytic function of the restriction endonuclease EcoRV.

We have used site-directed mutagenesis of the EcoRV restriction endonuclease to change amino acid side chains that have been shown crystallographically to be in close proximity to the scissile phosphodiester bond of the DNA substrate. DNA cleavage assays of the resulting mutant proteins indicate that the largest effects on nucleolytic activity result from substitution of Asp74, Asp90, and Lys92. We suggest on the basis of structural information, mutagenesis data, and analogies with other nucleases that Asp74 and Asp90 might be involved in Mg2+ binding and/or catalysis and that Lys92 probably stabilizes the pentacovalent phosphorus in the transition state. These amino acids are part of a sequence motif, Pro-Asp...Asp/Glu-X-Lys, which is also present in EcoRI. In both enzymes, it is located in a structurally similar context near the scissile phosphodiester bond. A preliminary mutational analysis with EcoRI indicates that this sequence motif is of similar functional importance for EcoRI and EcoRV. On the basis of these results, a proposal is made for the mechanism of DNA cleavage by EcoRV and EcoRI.     

Arginine kinase from Nautilus pompilius, a living fossil. Site-directed mutagenesis studies on the role of amino acid residues in the Guanidino specificity region

Arginine kinases were isolated from the cephalopods Nautilus pompilius, Octopus vulgaris, and Sepioteuthis lessoniana, and the cDNA-derived amino acid sequences have been determined. Although the origin and evolution of cephalopods have long been obscure, this work provides the first molecular evidence for the phylogenetic position of Cephalopoda in molluscan evolution. A crystal structure for Limulus arginine kinase showed that four amino acid residues (Ser(63), Gly(64), Val(65), and Tyr(68)) are hydrogen-bonded with the substrate arginine. We introduced three independent mutations, Ser(63) --> Gly, Ser(63) --> Thr, and Tyr(68) --> Ser, in Nautilus arginine kinase. One of the mutants had a considerably reduced substrate affinity, accompanied by a decreased V(max). In other mutants, the activity was lost almost completely. It is known that substantial conformational changes take place upon substrate binding in arginine kinase. We hypothesize that the hydrogen bond between Asp(62) and Arg(193) stabilizes the closed, substrate-bound state. Site-directed mutagenesis studies strongly support this hypothesis. The mutant (Asp(62) --> Gly or Arg(193) --> Gly), which destabilizes the maintenance of the closed state and/or perhaps disrupts the unique topology of the catalytic pocket, showed only a very weak activity (0.6-1.5% to the wild-type).