Figure 1.
ATPase activity of the MalFGK2 complex in nanodiscs.
(A) The MalFGK2 complex in nanodisc (lane 1) and detergent solution (lane 2) was analyzed by BN-PAGE and CN-PAGE followed by Coomassie blue staining of the gel. Molecular weight markers: BSA (67/134 kDa); catalase (232 kDa); ferritin (440 kDa). On CN-PAGE, the MalFGK2 complex precipitates as a protein smear at the top of the gel. (B) The ATPase activity supported by Nd-MalFGK2 was compared to detergent solubilized MalFGK2 and MalFGK2 in proteoliposomes (2 µM each) at 37°C in the presence of MalE (2 µM) or maltose (1 mM) in TSGM Buffer (50 mM Tris-HCl pH 8, 50 mM NaCl, 5% glycerol, 5 mM MgCl2). The reported values were derived from 3 independent experiments.
Figure 2.
High-affinity binding of MalE to the MalFGK2 complex.
(A) Nd-MalFGK2 (4 µM) was incubated with MalE (1 µM) or [125I]-MalE (∼10,000 c.p.m., 1 µM) in TSGM buffer containing nucleotides (1 mM) and maltose (1 mM) as indicated. After incubation (10 min, 37°C), samples were analyzed by CN-PAGE followed by Coomassie blue staining (bottom part) and autoradiography (upper part). (B) The indicated amount of Nd-MalFGK2 was incubated with MalE or [125I]-MalE in the presence or absence of maltose (2 mM) in TSGM buffer containing AMP-PNP (1 mM). After incubation (10 min, 37°C), samples were analyzed by CN-PAGE followed by Coomassie blue staining (bottom part) then autoradiography (upper part). (C) Nd-MalFGK2 (4 µM) was incubated with [125I]-MalE (∼10,000 c.p.m., 1 µM) in TSGM buffer containing AMP-PNP (1 mM) and the indicated amount of maltose. After incubation (10 min, 37°C), samples were analyzed by CN-PAGE and autoradiography. (D) Nd-MalFGK2 (4 µM) was immobilized onto Ni-NTA Sepharose beads and incubated with [125I]-MalE (∼10,000 c.p.m., 1 µM) in TSGM buffer containing AMP-PNP (1 mM) in the absence or presence of maltose (1 mM). After incubation (10 min, room temperature), bound MalE was eluted and revealed by SDS-PAGE and autoradiography. (E) Structure of the complex MalFGK2-(E159Q) with MalE. The position MalE-31 and MalF-177 are indicated in red. (F) Time course fluorescence quenching between MalE (20nM) and Nd-MalFGK2 (90 nM) in the presence or absence of maltose (1mM) and AMP-PNP (1mM). (G) Equilibrium titration of MalE (20 nM) fluorescence quenching with up to 1.5 µM Nd-MalFGK2. When the data were fitted to one-site binding equation, the dissociation constant in the presence of AMP-PNP was ∼79 nM. The dissociation constant in the presence of AMP-PNP and maltose was ∼390 nM. When the data were fitted to a competitive one-site binding equation, in which maltose-bound MalE do not bind to the transporter and maltose acts as a competitor, the dissociation constant of transporter-bound MalE for maltose was 127 µM.
Table 1.
Dissociation constants of MalE for Nd-MalFGK2 determined in the presence or absence of AMP-PNP and maltose.
Figure 3.
MalE has low affinity for maltose when bound to MalFGK2.
The indicated amount of MalE was incubated with Nd-MalFGK2 (0.5 µM) and [14C]-maltose (10 µM, 57 µCi/µmol) in TSGM buffer containing AMP-PNP (1 mM). After incubation (10 min, 37°C), samples were analyzed by CN-PAGE and (A) autoradiography or (B) Coomassie blue staining. (C) The binding of MalE to the transporter was monitored by fluorescence quenching, using MalE (20 nM) and Nd-MalFGK2 (70 nM). At the indicated time (arrow), 1 mM maltooligosaccharides were added to the reaction mixture. (D) Equilibrium titration to determine the maltose affinity of the MalE-FGK2 complex using MalE (20 nM) and Nd-MalFGK2 (70 nM). The derived dissociation constant was 120 µM. (E) Nd-MalFGK2 (4 µM) was incubated with [125I]-MalE (∼10,000 c.p.m., 1 μM) in TSGM buffer containing AMP-PNP (1 mM) and the indicated maltooligosaccharides (1 mM). After incubation (10 min, 37°C), samples were analyzed by CN-PAGE and autoradiography.
Figure 4.
Binding of the MalE mutants to MalFGK2. (A)
Wild type MalE and mutants (1 µM each) were mixed with [14C]-maltose in TSG buffer. Samples were analyzed by CN-PAGE and autoradiography. (B) [125I]-labeled MalE and variants were incubated with Nd-MalFGK2 (4 µM) in TSGM buffer containing AMP-PNP and maltose (2 mM) as indicated. After incubation (10 min, 37°C), samples were analyzed by CN-PAGE and autoradiography. (C) The ATPase activity supported by the MalE mutants (1 µM each) was determined in the presence of Nd-MalFGK2 (2 µM) and maltose (1 mM). The reported values were derived from 3 independent experiments.
Figure 5.
Binding of MalE to the MalFGK2 complex in proteoliposomes.
(A) [125I]-MalE was incubated with MalFGK2 proteoliposomes (2 µM) in TM buffer (20 mM Tris-HCl pH 8.0, 10 mM MgCl2) with or without AMP-PNP (1 mM) and maltose (1 mM) as described in [26]. The fraction of MalE bound to MalFGK2 was isolated by ultra-centrifugation. The samples were subjected to SDS-PAGE followed by Coomassie blue. (B) Autoradiography of the same gel. (C) The co-sedimentation assay was performed using MalE and variants in the presence of AMP-PNP. (D) Autoradiography of the same gel. (E) MalFGK2 in proteoliposomes (10 µM) was incubated with ATTO655-labeled MalE-31C (0.5 µM) in the presence of AMP-PNP. The samples were applied on a sucrose density gradient containing the indicated maltooligosaccharides (1 mM). Equal fractions were collected and analyzed by SDS-PAGE and fluorescence assay. The control experiments showed that MalE did not co-sediment with MalFGK2 in the absence of AMP-PNP (sample 2), but very well in the absence of maltose (sample 1). (F) Quantification of MalE bound to MalFGK2 in proteoliposomes. The amount of MalE bound to MalFGK2 without maltooligosaccharides was set to 100%.
Figure 6.
Regulation of the MalK transport ATPase by maltose.
(A) ATP hydrolysis was measured with MalFGK2 proteoliposomes (37°C, 10 min) in the absence or presence of maltose (2 mM) using MalE and variants (2 µM each). (B) Steady-state transport ATPase using MalE variants (2 µM each) as a function of the maltose concentration. Left panel and right panel are the same curve but fitted to different x-axis. The data were fitted to the Michaelis-Menten equation to determine the maximal velocity Vmax and Kt of the transport ATPase reaction. The calculated values derived from 3 independent experiments are presented in Table 2.
Table 2.
Kinetic parameters of the transport reaction and affinity of MalE variants for maltose.
Figure 7.
Maltose transport in intact cells.
(A) Strain HS3309 (ΔMalE) transformed with pLH1 encoding for the indicated MalE protein was plated on M9-maltose (left) or MacConkey-maltose (right) agar plate. The color on MacConkey plates reflects maltose transport and fermentation after 10 h. The plasmid vector was used as a negative control. (B) The transport assay using [14C]-maltose and strain HS3309 was performed as described in materials and methods. At the indicated time, cells were spotted on PVDF membrane and maltose import was detected by autoradiography. The intensity of each dot was determined by using ImageQuant (GE healthcare).
Figure 8.
(A) In the conventional model, MalE captures maltose with high affinity (Kd ∼2 µM), then associates with the P-closed transporter with low affinity (Kd ∼50–100 µM). The association triggers the P-open state, the opening of MalE and the release of maltose in the translocation cavity. The transporter returns to the P-closed state upon ATP hydrolysis. MalE dissociates and return to the periplasm. (B) In the auto-regulation model, MalE binds with high affinity (Kd ∼79 nM) to the P-open ATPase active transporter. Maltose is then captured by the complex of MalE-MalFGK2 (Kd ∼120 µM) and rapidly transported (Kt ∼2 µM) upon hydrolysis of ATP. When the concentration of maltose exceeds the import capacity, MalE acquires its closed-liganded conformation and looses affinity for MalFGK2. This regulation limits the maximal rate of transport (“transporter set point”). The function of closed-liganded MalE is to retain maltose in the periplasm (“retention effect”).