Conformational effects of amino acid substitutions in the P-glycoprotein of themdr 1 gene

The P-glycoprotein of themdr 1 gene is responsible for the phenomenon of multidrug resistance in human cells. The presumed drug-binding site of the wild-type P-glycoprotein contains a glycine at position 185. A mutant P-glycoprotein which contains valine at this position causes cells to retain resistance to colchichine, but to lose cross-resistance to other drugs such as the chemotherapeutic agents vinblastine and Adriamycin. This has been hypothesized to be due to a conformational change in the protein induced by the amino acid substitution. Using conformational energy analysis, we have determined the allowed three-dimensional structures for the wild-type and mutant proteins in the region of position 185. The results indicate that the wild-type protein adopts a unique left-handed conformation at position 185 which is energetically unfavorable for the protein withl-amino acids (including valine) at this position. This conformational change induced by amino acid substitutions for Gly 185 could explain the differences in binding to the P-glycoprotein of various drugs and, hence, the differences in drug resistance exhibited by various cell lines expressing these proteins.     

A mutant lactose repressor with altered inducer and operator binding parameters

The lactose repressor protein from the mutant Escherichia coli BG185 contains valine at position 81 instead of alanine. Spectroscopic, chemical and direct binding measurements demonstrate that the BG185 protein exhibits properties similar to the wild-type repressor-inducer complex. Kinetic measurements of inducer binding to BG185 repressor yielded rate constants that were more than two orders of magnitude smaller than those observed for wild-type repressor; these results suggest that the structural transitions required for inducer binding are markedly impaired by the mutation. The fluorescence spectral shift in response to inducer binding was identical for mutant and wild-type proteins. This identity indicates direct effects of inducer binding on the tryptophan(s) near the sugar binding site rather than environmental changes consequent to conformational shifts. Analogy to the bacterial sugar binding proteins suggest that the Ala to Val change at position 81 in BG185 repressor yields a molecule that is fixed in a closed, sugar-binding conformation.     

Effect of single amino acid substitutions at the same position on stability of a two-domain protein

In order to elucidate the role of individual amino acid residues on the conformational stability of a protein, the stabilities of the wild-type tryptophan synthase α-subunit from Escherichia coil and its five mutant proteins substituted by single amino acid residues at the same position 49 were compared. The five mutant proteins have glutamine, methionine, valine, serine, or tyrosine in place of glutamic acid of the wild-type protein at position 49. Denaturation of these proteins, which consist of two domains, by guanidine hydrochloride can be analyzed as a two-step process. We obtained the equilibrium constants between the native and the denatured forms and between the native and the stable intermediate forms for the above six proteins in the absence of denaturant at three pH values.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of theneu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an α-helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all α-helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Effect of single amino acid substitutions on the protease susceptibility of tryptophan synthase alpha subunit

In order to explore the correlation between protease susceptibility and conformational stability of a protein, the proteolytic degradation by trypsin, subtilisin and pronase P of the wild-type alpha subunit of tryptophan synthase from Escherichia coli and of its two mutant proteins was studied by measuring circular dichroism at 222 nm at various pH values at 37 degrees C. The mutant proteins are substituted by Gln or Met in place of Glu at position 49. The single amino acid substitutions at position 49 significantly affected susceptibility of this protein to the three proteases. Dependence of protease susceptibility of the wild-type and the two mutant proteins on pH was characteristic of each protein and similar for the three proteases. Comparison of the present results with the conformational stabilities of the three proteins previously measured shows that the order of resistance to the proteases among the three proteins coincides with the order of the values of unfolding Gibbs energy change, suggesting that protein degradation depends upon the conformational stability of a protein.     

Conformational changes induced by the transforming amino acid substitution in the transmembrane domain of the neu oncogene-encoded p185 protein

Theneu oncogene is frequently found in certain types of human carcinomas and has been shown to be activated in animal models by nitrosourea-induced mutation. The activating mutation in theneu oncogene results in the substitution of a glutamic acid for a valine at position 664 in the transmembrane domain of the encoded protein product of 185 kda (designated p185), which, on the basis of homology studies, is presumed to be a receptor for an as yet unidentified growth factor. It has been proposed that activating amino acid substitutions in this region of p185 lead to a conformational change in the protein which causes signal transduction via an increase in tyrosine kinase activity in the absence of any external signal. Using conformational energy analysis, we have determined the preferred three-dimensional structures for the transmembrane decapeptide (residues 658–667) of the p185 protein with valine and glutamic acid at the critical position 664. The results indicate that the global minimum energy conformation of the decapeptide from the normal protein with Val at position 664 is an -helix with a sharp bend (CD* conformation at residues 664 and 665) in this region, whereas the global minimum conformation for the decapeptide from the mutant transforming protein with Glu at position 664 assumes an all -helical configuration. Furthermore, the second highest energy conformation for the decapeptide from the normal protein is identical to the global minimum energy conformation for the decapeptide from the transforming protein, providing a possible explanation why overexpression of the normal protein also has a transforming effect. These results suggest there may be a normal and a transforming conformation for theneu-encoded p185 proteins which may explain their differences in transforming activity.     

Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation

A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K(m) for colchicine and a lower K(m) for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.     

Characterization of the azidopine and vinblastine binding site of P-glycoprotein.

To determine the number of drug binding sites that exist on the multidrug transporter, P-glycoprotein, we used azidopine, a dihydropyridine photoaffinity compound that reverses multidrug resistance and labels P-glycoprotein. Azidopine labels P-glycoprotein in two distinct locations: one labeled site is within the amino half of P-glycoprotein between amino acid residues 198 and 440, and the other site is within the carboxy half of the protein. Vinblastine is a cytotoxic drug that is used in cancer chemotherapy and is a substrate for transport by P-glycoprotein. We found that vinblastine inhibits azidopine labeling to approximately the same extent at each labeled site on P-glycoprotein. Because several studies have shown that amino acid residue 185 of P-glycoprotein plays a critical role in some aspects of drug binding and transport, we also studied the effect that amino acid residue 185 has on azidopine labeling. These studies show that azidopine labels both sites equivalently in both wild-type (G185) and mutant (V185) P-glycoproteins. We conclude from our results that the two halves of P-glycoprotein approach each other to form a single binding site for these drugs.     

Membrane proteins associated with amino acid transport by yeast (Saccharomyces cerevisiae).

Cells of the wild-type yeast (Saccharomyces cerevisiae) strain Y185, grown under conditions that de-repress the formation of a general amino acid permease ('Gap') system, bind delta-N-chloroacetyl[1-(14)C]ornithine; L- and D-amino acid substrates of the general amino acid permease system protect against this binding. The protein responsible is released from the cells by homogenization or by preparation of protoplasts; it is not released by osmotic shock. This protein is virtually absent from the wild-type strain when it is grown under conditions that repress the general amino acid permease system, and is also absent from a Gap- mutant Y185-His3, selected by its resistance to D-amino acids. This mutant and repressed wild-type cells also fail to form a number of membrane proteins elaborated by de-repressed wild-type cells. It is possible that all these proteins are components of the general amino acid permease system.     

A single amino acid change in acetolactate synthase confers resistance to valine in tobacco

The metabolic control of branched chain amino acid (BCAA) biosynthesis involves allosteric regulation of acetolactate synthase (ALS) by the end-products of the pathway, valine, leucine and isoleucine. We describe here the molecular basis of valine resistance. We cloned and sequenced an ALS gene from the tobacco mutant Val^r-1 and found a single basepair substitution relative to the wild-type allele. This mutation causes a serine to leucine change in the amino acid sequence of ALS at position 214. We then mutagenized the wild-type allele of the ALS gene ofArabidopsis and found that it confers valine resistance when introduced into tobacco plants. Taken together, these results suggest that the serine to leucine change at position 214 of ALS is responsible for valine resistance in tobacco.This paper is dedicated to the memory of Jean-Pierre Bourgin, who died on October 29, 1994, at the age of 50     

An altered pattern of cross-resistance in multidrug-resistant human cells results from spontaneous mutations in the mdr1 (P-glycoprotein) gene

Multidrug resistance in human cells results from increased expression of the mdr1 (P-glycoprotein) gene. Although the same gene is activated in cells selected with different drugs, multidrug-resistant cell lines can be preferentially resistant to their selecting agent. The mdr1 cDNA sequence from vinblastine-selected KB cells, which are uniformly resistant to different lipophilic drugs, was compared with the corresponding sequence from colchicine-selected KB cells preferentially resistant to colchicine. These sequences differ at three positions, resulting in a single amino acid change in P-glycoprotein. These differences result from mutations that occurred during colchicine selection. The appearance of these mutations coincides with the emergence of preferential resistance to colchicine. We have constructed biologically active mdr1 cDNA clones that express either wild-type or mutant P-glycoprotein. Multi-drug-resistant transfectants obtained with the mutant sequence were characterized by increased relative resistance to colchicine compared with transfectants obtained with wild-type sequence. mdr1 mutations are therefore responsible for preferential resistance to colchicine in multidrug-resistant KB cells.     

Isolation and characterization of an Escherichia coli seryl-tRNA synthetase mutant with a large increase in Km for serine.

A mutant of Escherichia coli resistant to serine hydroxamate which has a large increase in Km for serine of seryl-tRNA synthetase is described. The mutant serS gene was cloned and sequenced and was found to contain a single-base-pair mutation, resulting in the substitution of the residue alanine 262 by valine in motif 2. The methyl side chain of alanine 262 is not exposed at the active site, and molecular modeling indicated that replacement of alanine 262 by valine does not significantly affect the configuration of amino acids at the active site. This finding suggests that the residue at this position may be involved in a conformational change (possibly induced by ATP binding) which is necessary for optimal binding of the cognate amino acid.     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus

Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single, nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution of glutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.     

Comparison of CD spectra in the aromatic region on a series of variant proteins substituted at a unique position of tryptophan synthase alpha-subunit

CD spectra in the aromatic region of a series of the mutant alpha-subunits of tryptophan synthase from Escherichia coli, substituted at position 49 buried in the interior of the molecule, were measured at pH 7.0 and 25 degrees C. These measurements were taken to gain information on conformational change produced by single amino acid substitutions. The CD spectra of the mutant proteins, substituted by Tyr or Trp residue in place of Glu residue at position 49, showed more intense positive bands due to one additional Tyr or Trp residue at position 49. The CD spectra of other mutant proteins also differed from that of the wild-type protein, despite the fact that the substituted residues at position 49 were not aromatic. Using the spectrum of the wild-type protein (Glu49) as a standard, the spectra of the other mutants were classified into three major groups. For 10 mutant proteins substituted by Ile, Ala, Leu, Met, Val, Cys, Pro, Ser, His, or Gly, their CD values of bands (due to Tyr residues) decreased in comparison with those of the wild-type protein. The mutant protein substituted by Phe also belonged to this group. These substituted amino acid residues are more hydrophobic than the original residue, Glu. In the second group, three mutant proteins were substituted by Lys, Gln, or Asn, and the CD values of tyrosyl bands increased compared to those of the wild-type proteins. These residues are polar. In the third group, the CD values of tyrosyl bands of two mutant proteins substituted by Asp or Thr were similar to those of the wild-type protein, except for one band at 276.5 nm. These results suggested that the changes in the CD spectra for the mutant proteins were affected by the hydrophobicity of the residues at position 49.     

Effect of amino acid residues on conformational stability in eight mutant proteins variously substituted at a unique position of the tryptophan synthase alpha-subunit

To elucidate the role of individual amino acid residues in stabilizing the conformation of a protein, the stabilities of wild-type tryptophan synthase alpha-subunit from Escherichia coli and seven mutant proteins substituted by single amino acid residues at position 49, which is buried in the interior of the protein, were compared. The mutant proteins have Gln, Met, Val, Tyr, Leu, Ser, or Lys in place of Glu in the wild-type protein. The dissociation constant, pK, of the Glu residue at position 49 for the wild-type protein was determined to be 7.5 from a titration curve obtained by comparison of two-dimensional isoelectric focusing electrophoresis of the wild-type and mutant proteins. Our results indicate that 1) the conformational stabilities of the proteins studied increase linearly with hydrophobicity of the substituting residues (except Tyr), with the coefficient of this linear dependence being 2.0, 3.4, or 1.3 at pH 5.5, 7.0, or 9.0, respectively; and 2) Lys or Glu at position 49 serve as a destabilizing factor when ionized.     

Functional characterization of a glycine 185-to-valine substitution in human P-glycoprotein by using a vaccinia-based transient expression system.

Human P-glycoprotein (Pgp) is a 170-kDa plasma membrane protein that confers multidrug resistance to otherwise sensitive cells. A mutation in Pgp, G185-->V, originally identified as a spontaneous mutation, was shown previously to alter the drug resistance profiles in cell lines that are stably transfected with the mutant MDR1 cDNA and selected with cytotoxic agents. To understand the mechanism by which the V185 mutation leads to an altered drug resistance profile, we used a transient expression system that eliminates the need for drug selection to attain high expression levels and allows for the rapid characterization of many aspects of Pgp function and biosynthesis. The mutant and wild-type proteins were expressed at similar levels after 24-48 h in human osteosarcoma (HOS) cells by infection with a recombinant vaccinia virus encoding T7 RNA polymerase and simultaneous transfection with a plasmid containing MDR1 cDNA controlled by the T7 promoter. For both mutant and wild-type proteins, photolabeling with [3H]azidopine and [125I]iodoarylazidoprazosin, drug-stimulated ATPase activity, efflux of rhodamine 123, and accumulation of radiolabeled vinblastine and colchicine were evaluated. In crude membrane preparations from HOS cells, a higher level of basal Pgp-ATPase activity was observed for the V185 variant than for the wild-type, suggesting partial uncoupling of drug-dependent ATP hydrolysis by the mutant. Several compounds, including verapamil, nicardipine, tetraphenylphosphonium, and prazosin, stimulated ATPase activities of both the wild-type and mutant similarly, whereas cyclosporin A inhibited the ATPase activity of the mutant more efficiently than that of the wild-type. This latter observation explains the enhanced potency of cyclosporin A as an inhibitor of the mutant Pgp. No differences were seen in verapamil-inhibited rhodamine 123 efflux, but the rate of accumulation was slower for colchicine and faster for vinblastine in cells expressing the mutant protein, as compared with those expressing wild-type Pgp. We conclude that the G185-->V mutation confers pleiotropic alterations on Pgp, including an altered basal ATPase activity and altered interaction with substrates and the inhibitor cyclosporin A.     

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.     

Effect of cavity-modulating mutations on the stability of Escherichia coli ribonuclease HI.

The size of the cavity around Ser68 of Escherichia coli ribonuclease HI was modulated by amino acid substitutions to examine the effects on the stability of the enzyme. Five mutant proteins, Ser68----Gly, Ser68----Ala, Ser68----Thr, Ser68----Val and Ser68----Leu, were constructed. Each of the mutant proteins exhibited at least 40% of the enzyme activity of the wild-type protein. The stabilities of the mutant proteins were determined from urea-denaturation and thermal-denaturation curves. Among the five mutations, only the Ser----Val mutation resulted in an increase in the stability of the enzyme. The melting temperature, tm, at pH 3.0 of the mutant protein Ser68----Val was increased by 1.9 degrees C. Its free-energy change of unfolding in the absence of urea, delta G(H2O), and the midpoint of the denaturation curve, [D]1/2, were also increased by 5.4 kJ/mol and 0.18 M, respectively. The increase in the stability of the enzyme is probably due to the filling of the cavity space around Ser68 by valine. However, the mutation of Ser68 to glycine or leucine residues resulted in a considerable decrease in stability. In these cases, some conformational changes occur, as suggested by the CD and 1H-NMR spectra of these mutant proteins.