Role of Conserved Gly-Gly Pairs on the Periplasmic Side of LacY

Abstract

On the periplasmic side of LacY, two conserved Gly-Gly pairs in helices II and XI (Gly46 and Gly370, respectively) and helices V and VIII (Gly159 and Gly262, respectively) allow close packing of each helix pair in the outward (periplasmic)-closed conformation. Previous studies demonstrate that replacing one Gly residue in each Gly-Gly pair with Trp leads to opening of the periplasmic cavity with abrogation of transport activity, but an increased rate of galactoside binding. To further investigate the role of the Gly-Gly pairs, 11 double-replacement mutants were constructed for each pair at positions 46 (helix II) and 262 (helix VIII). Replacement with Ala or Ser results in decreased but significant transport activity, while replacements with Thr, Val, Leu, Asn, Gln, Tyr, Trp, Glu, or Lys exhibit very little or no transport. Remarkably, however, the double mutants bind galactoside with affinities 10-20-fold higher than that of the pseudo-WT or WT LacY. Moreover, site-directed alkylation of a periplasmic Cys replacement indicates that the periplasmic cavity becomes readily accessible in the double-replacement mutants. Molecular dynamics simulations with the WT and double-Leu mutant in the inward-open/outward-closed conformation provide support for this interpretation.

Figure 1

Structure of LacY. (A) Structure of LacY (PDB entry 2CFQ) viewed from the side. The Cα atoms of the Gly46-Gly370 pair (blue) and the Gly159-Gly262 pair (red) are shown as spheres. Helices V and VIII are labeled in red and helices II and XI in blue. The Cα atom of Asn245 is shown as a yellow sphere. (B) Cytoplasmic view. (C) Periplasmic view.

Figure 2

(A) Lactose transport of E. coli T184 cells expressing single-Cys245 LacY (●) or single-Cys245 LacY with double Ala (□) and Ser (△) replacements at positions 46 and 262, or no permease (○), was measured with [¹⁴C]lactose (10 mCi/mmol) at 0.4 mM for given times as described in Materials and Methods. The lactose transport by double-replacement mutants with Val, Leu, Thr, Asn, Gln, Tyr, Trp, Glu, or Lys is colored red, with bars indicating the activity spread of the different mutants. All mutants contain the given double replacement and a single Cys in place of Asn245. (B) Steady-state level of lactose accumulation of each mutant presented as the percentage of the Gly-Gly pseudo-WT. (C) Expression of the pseudo-WT and the double-replacement mutants as determined by Western blotting with the penta-His antibody–HRP conjugate. From left to right: Gly-Gly single Cys245 (1), empty vector (2), and double replacements with Ala (3), Glu (4), Lys (5), Leu (6), Asn (7), Gln (8), Ser (9), Thr (10), Val (11), or Tyr (12). Two micrograms of total membrane protein was analyzed in each lane by SDS–PAGE.

Figure 3

(A) Binding of α-NPG to purified single Cys245 (●) and single Cys245 with Ala (△), Ser (□), Thr (○), Leu (*), Glu (✩), Gln (◊) or Tyr (▽) double replacements at positions 46 and 262 measured by addition of 30 mM melibiose to samples preincubated with α-NPG at given concentrations. Binding curves were obtained by plotting the change in fluorescence as a function of α-NPG concentration. (B) Binding of α-NPG to purified WT LacY (●) and the double-Lys replacements at positions 46 and 262 in the WT background (○).

Figure 4

(A) RSO membrane vesicles containing single Cys245 or single Cys245 with Ala, Val, Leu, Ser, Thr, Glu, Lys, Asn, Gln, Tyr, or Trp double replacements at positions 46 and 262 labeled with 40 μM TMRM for given times in the absence or presence of 2 mM α-NPG. For each mutant, the TMRM labeling intensity (top panel) was obtained by scanning the SDS–PAGE gel with a phosphorimager, and protein (bottom panel) was visualized by silver staining. (B) TMRM labeling rates of Cys245 in single-Cys245 and double-Ala, -Ser, -Thr, -Val, -Leu, -Glu, -Asn, -Gln, -Lys, -Tyr, or -Trp replacements at positions 46 and 262 obtained by plotting the relative labeling in the presence (●) or absence (○) of 2 mM α-NPG as a function of labeling time. For each mutant, the relative labeling of 20 s in the presence of sugar point was set to 1.

Figure 5

Dynamics and structural rearrangements in wild-type and double-Leu mutant LacY. (A) Root-mean-square fluctuations (rmsf) extracted from the full simulations of wild-type (black) and double-Leu (red) LacY. The arrows mark sequence regions of significant differences. (B) Cα–Cα interatomic distances tracked between positions 46–370 and 159–262 in the wild-type and mutant proteins. (C) Superimposed frames corresponding to the ends of both the wild-type (blue) and mutant (cyan) simulations. The protein is viewed from the periplasmic side, and the mutated leucine residues are shown in licorice representation. The dashed black lines highlight the separation between the II–XI and V–VIII helix pairs.