Gain of function mutations in membrane region M2C2 of KtrB open a gate controlling K+ transport by the KtrAB system from Vibrio alginolyticus

Abstract

KtrB, the K(+)-translocating subunit of the Na(+)-dependent bacterial K(+) uptake system KtrAB, consists of four M(1)PM(2) domains, in which M(1) and M(2) are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M(2C) is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M(2C1) and M(2C3), which are connected by a nonhelical M(2C2) region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M(2C2) led to a huge gain of function of K(+) uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in V(max) for K(+) transport. Na(+) translocation by KtrB was not affected. Partial to complete deletions of M(2C2) also led to enhanced V(max) values for K(+) uptake via KtrB. However, several deletion variants also exhibited higher K(m) values for K(+) uptake and at least one deletion variant, KtrB(Delta326-328), also transported Na(+) faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M(2C2) forms a flexible structure within the membrane allowing M(2C3) to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M(2C2) forms a flexible gate controlling K(+) translocation at the cytoplasmic side of KtrB, and (ii) that M(2C2) is required for the interaction between KtrA and KtrB.

FIGURE 1.

Region M_2C of VaKtrB. A, schematic representation of how M_2C is supposed to fold across the membrane according to Ref. , subdivision of M_2C into regions M_2C1 to M_2C3. B, conserved residues in regions P_C and M_2C2. Increasing degree of conservation of single residues is indicated by the background changing from white to black. The absolutely conserved VaKtrB residues Gly²⁹⁰ (selectivity-filter residue (9, 14)), Gly³¹⁴ and Lys³²⁵ (both M_2C2 residues) are marked by vertical arrows.

FIGURE 2.

Growth experiments and KtrB expression of cells containing single amino acid changes in M_2C2. A, strain LB2003/pEL903–100 encoding Cys-less KtrB. B, strain LB2003/pKtrB_G314S. C, strain LB2003/KtrB_G325R. D, strain LB2003/pKtrB_V326C; K⁺ concentrations: 1 mm, ♦; 3 mm, ■; 7 mm, ▴; 10 mm, ▵; 30 mm, □; and 115 mm, ◇. E, description of the results of the growth experiments with the different KtrB variants (upper panel), and their relative amounts present in the cells (lower part). KtrB and KtrB(cl) refer to native and Cys-less KtrB, respectively. The other KtrB variants are given with the position at which the changed amino acid occurs followed by this amino acid. All KtrB variants were His-tagged. > 0.1 mm indicates that the cells grew well at all K⁺ concentrations tested; ND, not determined.

FIGURE 3.

K⁺ uptake by cells containing KtrB variants with single amino acid changes. Plasmid-containing cells of strain LB2003 were grown and induced for ktrB expression with 0.02% l-arabinose, according to protocol 1. For the K⁺ uptake experiment, cells were suspended at 1 mg (dry weight)/ml of medium containing 200 mm NaHepes, pH 7.5, 0.2% glycerol, and 0.02% l-arabinose. The suspension was shaken at room temperature. After 10 min, KCl was added at following concentrations: ●, 0.5 mm; ▴, 1 mm; ■, 2 mm; and ♦, 5 mm. For each data point a 1.0 ml sample was taken from the suspension and its cell K⁺ content was determined by flame photometry. A, strain LB2003/pEL903-100; B, strain LB2003/pKtrB _K325C; C, strain LB2003/pKtrB_K325R; and D, strain LB2003/pKtrB_T318C.

FIGURE 4.

Survey of the kinetic parameters of K⁺ uptake (A and B) and Na⁺ uptake (C and D) by cells containing KtrB variants with single amino acid changes. K⁺ was determined as outlined in the legend to Fig. 3. For Na⁺ uptake plasmid containing cells of strain TO114 were grown, induced for ktrB expression and depleted of K⁺ and Na⁺ as described under “Experimental Procedures” and in Ref. . For the Na⁺ uptake experiment, cells were suspended at 1 mg (dry weight)/ml of medium containing 200 mm triethanolamine Hepes, pH 7.2, 0.2% glycerol, and 0.02% l-arabinose. The suspension was shaken at room temperature. After 10 min, NaCl was added at 1.3 mm, 1.9 mm, 5 mm, or 7 mm. Cell samples of 1 ml were taken at different time points and analyzed for their Na⁺ contents by flame photometry. The nomenclature of the His-tagged KtrB variants with single amino acid changes was as in Fig. 2.

FIGURE 5.

Alkaline phosphatase activities of KtrB-PhoA proteins with fusions in region M_2C of KtrB.

FIGURE 6.

Kinetics of K⁺ uptake by strains containing KtrB with deletions in region M_2C. Plasmid-containing cells of strain LB2003 were grown at 3 mm K⁺ according to protocol 2, except for strain LB2003/pEL903-100, which was grown at 10 mm K⁺, and for strain LB2003/pKtrB_Δ326–328, which was grown at 30 mm K⁺ according to protocol 1. The l-arabinose concentration used is indicated in the figure. The bottom panel gives the relative amounts of KtrB variants present in the cell suspensions used for the uptake assays. His-tagged KtrB variants labeled with KtrB, Δ314–328, Δ329–339, Δ314–317, Δ314–320, Δ314–323, Δ326–328, Δ324–328, and Δ318–329 are encoded by plasmid pEL903-100 and its derivatives pKtrB_Δ314–328, pKtrB_Δ329–339, pKtrB_Δ314–317, pKtrB_Δ314–320, pKtrB_Δ314–323, pKtrB_Δ326–328, pKtrB_Δ324–328, and pKtrB_Δ318–329, respectively.

FIGURE 7.

Na⁺ uptake by strains containing KtrB with deletions in region M_2C2. Plasmid-containing cells of strain TO114 were grown at 3 mm K⁺ and 0.02% l-arabinose in the triethanolamine-Hepes based medium described under the “Experimental Procedures.” Na⁺ uptake after the addition of 3 mm NaCl to the cell suspension was determined as outlined in the legend to Fig. 4. The ordinate gives the initial rate of Na⁺ uptake after the addition of NaCl to the cell suspension. The bottom panel gives the relative amounts of KtrB variants present in the different suspensions used for the uptake experiments. Labeling of the KtrB variants is as outlined in the legend to Fig. 6.

FIGURE 8.

Isolation of KtrA-KtrB complexes by affinity chromatography. Complexes were isolated as described under “Experimental Procedures.” Lanes 1 and 2, His₁₀-KtrA-KtrB encoded by plasmid pIH301; lanes 3 and 4, His₁₀-KtrA-KtrB_G316S encoded by plasmid pKtrAB_G316S; lanes 5 and 6, His₁₀-KtrA-KtrB_Δ314–328 encoded by plasmid pKtrAB_Δ314–328; lanes 7 and 8, His₁₀-KtrA-KtrB_Δ326–328 encoded by plasmid pKtrAB_Δ326–328; lanes 9 and 10, His₁₀-KtrA-KtrB_Δ318–329 encoded by plasmid pKtrAB_Δ318–329. Odd lanes, proteins of the solution with which His₁₀-KtrA bound to the NTA column was washed; even lanes, proteins in the 500 mm imidazole solution with which His₁₀-KtrA was eluted from the column. The proteins on the gel are stained with Coomassie Brilliant Blue, which stains the integral membrane KtrB poorly.

FIGURE 9.

Model explaining how M_2C2 functions as a gate for K⁺ transport through KtrB. Region M_2C2 is proposed to form a gate, which in its closed form prevents K⁺ translocation from the selectivity filter region (indicated by the two glycine residues) to the cytoplasm. A salt bridge between VaKtrB-M_2C2 residue Lys³²⁵ and Asp²²² from membrane span M_2B (13) will keep the gate closed. K⁺ permeates from the periplasm into KtrB, where it is dehydrated to the enter the selectivity filter region (11). From this position, K⁺ opens the gate by electrostatic repulsion of residue Lys³²⁵, leading to a weakening of the Asp²²²–Lys³²⁵ salt bridge and thereby allowing K⁺ to move to the cytoplasm, followed by closing of the M_2C2 gate. The gain of function amino acid changes in M_2C2 allow K⁺ to slip through the gate. The presence of a second gate for K⁺ at the periplasmic site of KtrB (42) and a role for Na⁺ in the K⁺ transport cycle are not included in this simplified model.