Protein Science (1992), I , 1652-1660. Cambridge University Press. Printed in the USA. Copyright 0 1992 The Protein Society
Escherichia coli K12 arabinose-binding protein mutants with altered transport properties
D.G. KEHRES AND R.W. HOGG Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 August 13, 1992) (RECEIVED May 27, 1992; REVISED MANUSCRIPT RECEIVED
Abstract The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed withwild-type AraG and AraH from a plasmid, in a strainlacking the chromosomalaraFGH operon, and the whole cell uptake parameters, V,,(maximum initial velocity of arabinose entry) and Ken (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal Knvalues, 3 mutants had V,, and Kenvalues twice wild type, and 11 mutants had V,, and Kenvalues 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutantbinding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex. Keywords: Escherichia coli; periplasmic transport; protein-protein interactions; site-directed mutagenesis
Periplasmic transport systems are widely used for nutrient uptake by gram-negative bacteria. They belong to an emerging superfamily of proteins found in yeast, insects, and humans as well as inbacteria, which are termed traffic ATPases, because they all translocate substrates across cellular membranesat the expense of ATP (Ames& Joshi, 1990). The high affinity arabinose uptake system of Escherichia coli K12, araFGH, is a periplasmic transport system that detects and scavenges micromolarconcentrations of the pentose sugar L-arabinose for use as a carbon and energy source(Hogg, 1977). Nucleotide sequencing (Scripture et al., 1987), expression studies (Horazdovsky & Hogg, 1987), and genetic reconstitution (Horazdovsky, 1989) have shown that araFGH encodes the arabinose-binding protein, ABP (AraF), a probable ATPase subunit (AraG), and an integral membrane subunit (AraH). The threeReprint requests to: R.W. Hogg, Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
dimensional structure of the ABP has been determined to a resolution of 1.8 A (Gilliland & Quiocho, 1981; Quiocho & Vyas, 1984). The availabilityof detailed structural information aboutABP and the genetic simplicityof the system as a whole (only three gene products instead of the four or five found in most other cases), makearaFGH a good system for investigating structural aspects of periplasmic transport and of traffic ATPase function in general. As a first step in understanding the mechanism of arabinose periplasmic transport, we are defining the binding protein surface(s) responsible for contacting the AraG: AraH complex. ABP consists of two lobes connected by a three-crossover hinge- the P domain contains mostly N-terminal residues and the Q domain mostly C-terminal residues (Fig. 1; Kinemage 1). Each domain is a fivestranded parallel beta-sheet with alpha-helices connecting each strand to the next. Short loops between the C-terminal end of each beta-strand and theN-terminal end of the following alpha-helix define the surface of a deep
1652
Arabinose-binding protein mutants
1653
N
Fig. 1. Alpha-carbon backbone of arabinose-binding protein. This view is directly into the arabinose-binding cleft, showing the P domain (right) and Q domain (left), and the three crossovers comprising the “hinge” at the base of the cleft (center). Every 10th amino acid is shown asa circle, and every 50th is numbered. Bold lines indicate the second betastrand, interior loop, and second alpha-helix of each domain.
arabinose-binding cleft. Inspectionof the crystal structure suggests that bound arabinose can dissociate only by a reversal of the “hinge fold” cleft closure motion that occurs upon binding (Ma0 et al., 1982). The most likely interaction surface on ABP, therefore, is the one formed just outside the mouth of the binding cleft by juxtaposition of amino acids from both the P and Q domains, which would be substantially farther apart in the unliganded (open) form than in the liganded (closed)form of the protein. Several side chains on the perimeter of the cleft mouth project prominently from the mean level of this surface. Transportintermediates might involve more extended or even entirely different regions of ABP, but the cleft mouth surface is likely to be involved in at least the early interactions with AraG:AraH. We report here the isolation and the whole cell transport properties of 38 ABP cleft mouth surface mutants generated by a combination of random and site-specific mutagenesis. Results
Mutagenesis and identification of mutants
concentrations to determine V,(maximum initial velocity of arabinose entry) and K,, (concentration of arabinose yielding half-maximal entry). One specifically generated mutation, K1751, when assayed for arabinose uptake, had a V, less than half of wild type (Table l), which suggestedthat the region of the ABP cleft mouth surface containing this exposed lysine was important in transport. K175 is located in the second interior loop of the Q domain. Thesecond beta-strand, turn, and second alpha-helixform the longest contiguous stretch of exposed amino acids on the cleft mouth surface of each domain (see Fig. 1; Kinemage 1). Therefore long “doped” primer oligonucleotides were synthesizedto saturate these regions withmutations (Fig. 2). The 54-nucleotide P domain primer, P1, could potentially mutate codons between glycine32 through alanine 49, and the 45nucleotide Q domain primer, 4 2 , could mutate codons proline 165 through isoleucine 179.
Table 1. Arabinose-bindingprotein mutants and their transport properties
Mutation Controls Wild type N205V
V ,a (pmol/30 s/ 10’ cells) 260 90
Mutants analyzed in detail E13A 460 D42E 290 D42A 130 E44A 300 E34D/K45R 290 K45N 170 40 K37T/D42N/K45N D67A 140 K69A 480 E144A 70 T174P 1.7 510 K1751 50 K175R 160 70 K175Q D178A 130 D178H 60 D 184A 140
(pM)
Uptake phenotype
Kd (pM)
1.0 13.6
Wild type Severe down
2 20
1.3
up Silent Mild down Silent Silent Mild down Severe down Mild down Up Severe down up Severe down Mild down Severe down Mild down Severe down Mild down
Escherichia coli K12 arabinose-binding protein mutants with altered transport properties
D.G. KEHRES AND R.W. HOGG Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 August 13, 1992) (RECEIVED May 27, 1992; REVISED MANUSCRIPT RECEIVED
Abstract The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed withwild-type AraG and AraH from a plasmid, in a strainlacking the chromosomalaraFGH operon, and the whole cell uptake parameters, V,,(maximum initial velocity of arabinose entry) and Ken (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal Knvalues, 3 mutants had V,, and Kenvalues twice wild type, and 11 mutants had V,, and Kenvalues 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutantbinding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex. Keywords: Escherichia coli; periplasmic transport; protein-protein interactions; site-directed mutagenesis
Periplasmic transport systems are widely used for nutrient uptake by gram-negative bacteria. They belong to an emerging superfamily of proteins found in yeast, insects, and humans as well as inbacteria, which are termed traffic ATPases, because they all translocate substrates across cellular membranesat the expense of ATP (Ames& Joshi, 1990). The high affinity arabinose uptake system of Escherichia coli K12, araFGH, is a periplasmic transport system that detects and scavenges micromolarconcentrations of the pentose sugar L-arabinose for use as a carbon and energy source(Hogg, 1977). Nucleotide sequencing (Scripture et al., 1987), expression studies (Horazdovsky & Hogg, 1987), and genetic reconstitution (Horazdovsky, 1989) have shown that araFGH encodes the arabinose-binding protein, ABP (AraF), a probable ATPase subunit (AraG), and an integral membrane subunit (AraH). The threeReprint requests to: R.W. Hogg, Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
dimensional structure of the ABP has been determined to a resolution of 1.8 A (Gilliland & Quiocho, 1981; Quiocho & Vyas, 1984). The availabilityof detailed structural information aboutABP and the genetic simplicityof the system as a whole (only three gene products instead of the four or five found in most other cases), makearaFGH a good system for investigating structural aspects of periplasmic transport and of traffic ATPase function in general. As a first step in understanding the mechanism of arabinose periplasmic transport, we are defining the binding protein surface(s) responsible for contacting the AraG: AraH complex. ABP consists of two lobes connected by a three-crossover hinge- the P domain contains mostly N-terminal residues and the Q domain mostly C-terminal residues (Fig. 1; Kinemage 1). Each domain is a fivestranded parallel beta-sheet with alpha-helices connecting each strand to the next. Short loops between the C-terminal end of each beta-strand and theN-terminal end of the following alpha-helix define the surface of a deep
1652
Arabinose-binding protein mutants
1653
N
Fig. 1. Alpha-carbon backbone of arabinose-binding protein. This view is directly into the arabinose-binding cleft, showing the P domain (right) and Q domain (left), and the three crossovers comprising the “hinge” at the base of the cleft (center). Every 10th amino acid is shown asa circle, and every 50th is numbered. Bold lines indicate the second betastrand, interior loop, and second alpha-helix of each domain.
arabinose-binding cleft. Inspectionof the crystal structure suggests that bound arabinose can dissociate only by a reversal of the “hinge fold” cleft closure motion that occurs upon binding (Ma0 et al., 1982). The most likely interaction surface on ABP, therefore, is the one formed just outside the mouth of the binding cleft by juxtaposition of amino acids from both the P and Q domains, which would be substantially farther apart in the unliganded (open) form than in the liganded (closed)form of the protein. Several side chains on the perimeter of the cleft mouth project prominently from the mean level of this surface. Transportintermediates might involve more extended or even entirely different regions of ABP, but the cleft mouth surface is likely to be involved in at least the early interactions with AraG:AraH. We report here the isolation and the whole cell transport properties of 38 ABP cleft mouth surface mutants generated by a combination of random and site-specific mutagenesis. Results
Mutagenesis and identification of mutants
concentrations to determine V,(maximum initial velocity of arabinose entry) and K,, (concentration of arabinose yielding half-maximal entry). One specifically generated mutation, K1751, when assayed for arabinose uptake, had a V, less than half of wild type (Table l), which suggestedthat the region of the ABP cleft mouth surface containing this exposed lysine was important in transport. K175 is located in the second interior loop of the Q domain. Thesecond beta-strand, turn, and second alpha-helixform the longest contiguous stretch of exposed amino acids on the cleft mouth surface of each domain (see Fig. 1; Kinemage 1). Therefore long “doped” primer oligonucleotides were synthesizedto saturate these regions withmutations (Fig. 2). The 54-nucleotide P domain primer, P1, could potentially mutate codons between glycine32 through alanine 49, and the 45nucleotide Q domain primer, 4 2 , could mutate codons proline 165 through isoleucine 179.
Table 1. Arabinose-bindingprotein mutants and their transport properties
Mutation Controls Wild type N205V
V ,a (pmol/30 s/ 10’ cells) 260 90
Mutants analyzed in detail E13A 460 D42E 290 D42A 130 E44A 300 E34D/K45R 290 K45N 170 40 K37T/D42N/K45N D67A 140 K69A 480 E144A 70 T174P 1.7 510 K1751 50 K175R 160 70 K175Q D178A 130 D178H 60 D 184A 140
(pM)
Uptake phenotype
Kd (pM)
1.0 13.6
Wild type Severe down
2 20
1.3
up Silent Mild down Silent Silent Mild down Severe down Mild down Up Severe down up Severe down Mild down Severe down Mild down Severe down Mild down
