In this study, site-directed mutagenesis in combination with cross-linking, active transport, binding assays and site-directed alkylation was employed to study the role of Asp68 and its interaction with Lys131. N2
split LacY with mutations D68C/K131C does not cross-link spontaneously, but does so completely at a rapid rate when catalyzed by Cu-Ph induced oxidation or treatment with homobifunctional MTS reagents (). Thus, although the two positions are relatively close physically, as shown in the crystal structure 9; 10
where the distance between the functional groups is ~4.2 Å (), and mutant the distance should be somewhat greater in the double-Cys, which may explain the lack of spontaneous cross-linking. In any event, the double-Cys mutant cross-links in the presence of Cu-Ph or methanethiosulfonate reagents, and binding of sugar probably does not change the rate of cross-linking.
All replacements for Asp68 tested inactivate, particularly Glu, while Lys131 is more permissive; K131R LacY transports at least as well as wild-type, and K131A or K131C single mutants have low but significant activity. Despite the sensitivity of Asp68 to replacement, high transport activity is observed with second-site suppressors 25
, indicating that this carboxyl side chain is not absolutely required for symport. Double neutral replacements for Asp68 and Lys131, as described in this study, also rescue low but significant transport activity (). Importantly, flow dialysis studies show clearly that loss of function in Asp68 mutants is not due to a defect in sugar binding (). Rather, site-directed alkylation of single-Cys replacements in the cytoplasmic or potential periplasmic opening in LacY () indicates that loss of function in the Asp68 mutants is due to inability of sugar binding to increase the open probability of the periplasmic pathway, which severely impairs a key step in the transport cycle.
In addition to the Asp68/Lys131 charge pair in LacY, two other important pairs have been described, Asp237 (helix VII)/Lys358 (helix XI) and Asp240 (helix VII)/Lys319 (helix X) 34; 35; 37; 38; 39; 40
. The Asp237/Lys358 charge pair, which is important for membrane insertion and stability of LacY, is promiscuous. Thus, Asp237 can be replaced with Glu, carboxymethyl-Cys or sulfonylethylthio-Cys with Lys or Arg at position 358 with retention of good activity. Similarly, LacY tolerates replacement of Lys358 with Arg or ammoniumethylthio-Cys with Asp or Glu at position 237. Remarkably, moreover, LacY with Lys, Arg, or ammoniumethylthio-Cys in place of Asp237 is highly active when Lys358 is replaced with Asp or Glu, thereby demonstrating that the polarity of the charge interaction can be reversed without loss of activity 35; 37
. In contrast, the Asp240/Lys319 charge pair, which comprises part of the H-bond/charge pair network involved in H+
translocation and coupling, behaves similarly to the Asp68/Lys131 charge pair except that single neutral replacement of Lys131 retains significant activity (). But as with Asp240, replacement with Glu abolishes lactose transport, and activity is not rescued by replacing Lys319 with Arg nor will either pair tolerate interchange.
Unlike Asp237/Lys358, the interaction between Asp68 and Lys131, like that of the Asp240/Lys319 pair, exhibits marked sensitivity to changes in the nature of the side chains. Even short extension of either Asp residue by replacement with Glu inactivates transport. Since Asp68 appears to interact relatively weakly with Lys131, replacement of the former with the longer Glu side chain may result in a stronger charge-pair interaction, which inhibits the dynamics of LacY. If the interaction between helices II and IV is dynamic and Asp68 and Lys131 undergo association-dissociation during turnover of LacY, the strength of the interaction would be important for optimal activity. Thus, alterations in the length of the side chains at these positions may favor or disfavor interaction depending upon steric constraints.
All crystallographic structures of LacY to date are in the same inward-facing conformation 9; 10; 11
. However, many independent biochemical and biophysical studies show that LacY exists in multiple conformations, which are essential for the overall transport cycle 41
. Site-directed thiol cross-linking 28
, shows that helices II and VII cross-link in a manner suggesting that ligand binding induces a translational or scissors-like movement between the two helices. Since helix II is kinked at Phe59, there is probably also clockwise rotation of the C-terminal portion of helix II with Asp68 (, stereo view). Consequently, replacement of Asp68 with Glu may result in a stronger interaction with Lys131, which interferes with rotation of helices II and IV.
Mapping the cross-linked pairs V125C/F354C and I129C/T350C on helices IV and XI 31
reveals that positions 125 and 129 are on the side of helix IV facing away from helix XI (). In order for these pairs to cross-link, helix IV likely rotates counterclockwise towards helix XI (, stereo view). This idea is supported by experimental results obtained from double electron-electron resonance distance measurements with Cys pairs labeled with nitroxide probes 14
. Ligand-induced distance changes between neighboring positions in helix IV (136 and 137) exhibit behavior that is inconsistent with rigid body movements. The major population in the absence of sugar exhibits the same interspin distance between nitroxide probes at positions 136 and 137 on helix IV paired with nitroxides at either position 340 in helix X (41 Å) or position 401 in helix XII (44 Å), but shifts differently upon ligand binding (41 Å to 30 Å for S136C/Q340C and to 23 Å for N137C/Q340C; 44 Å to 40 Å for S136C/S401C and to 31 Å for N137C/S401C). This behavior may be due to a combination of partial unwinding and counterclockwise rotation of C-terminal end of helix IV. The rotation of helices II and IV against each other may also move Glu130 and Lys69 into closer proximity with formation of a weak charge pair or H-bond that acts to stabilize the outward-facing conformer (, stereo view). Of the 417 amino acid residues in LacY, only six side chains are absolutely irreplaceable with respect to active transport: Glu126 (helix IV) and Arg144 (helix V), which are critical for substrate binding, and Glu269 (helix VIII), Arg302 (helix IX), His322 and Glu325 (helix X), which are essential for H+
translocation/coupling 4; 5
. Asp68 is required for transport, but not absolutely irreplaceable, and the residue is not involved in substrate binding and probably not directly involved in H+
translocation or coupling between sugar and H+
translocation. Even the most conservative replacement with Glu blocks sugar-induced opening of the periplasmic cavity. Second-site mutations that restore transport activity are located at the periplasmic end of helices II, VII and XI where the helices are tightly packed (). Moreover, the second-site suppressors of Asp68 mutations are generally replacements with relatively bulky side-chains on opposing faces of helices II and XI, suggesting that the mutations restore transport activity by loosening the tight packing between helices II, VII and XI in the periplasmic region. Packing of helices II and XI in the crystal structures 9; 10; 11
also shows that these helices are tightly packed at the location of the conserved Gly46 and Gly370 pair (). Upon sugar-induced rotation of helices II and IV, interaction between Gly46 and Gly370 would be weakened, and tight contact between the two helices would be abolished.
Finally, LacY exhibits pseudo-symmetry between the N- and C-terminal helix bundles. Therefore, it is highly noteworthy that helices VIII and X in the C-terminal bundle exhibit the same relationship to each other as helices II and IV in the N-terminal bundle (). Clearly, if sugar binding induces movement of helices VIII and X in the same manner as suggested for helices II and IV, their concerted movement plays an important role in opening the periplasmic pathway.
Figure 8 Congruence of helix-pairs II/IV and VIII/X in LacY. A) Cytoplasmic view of LacY (helix II, red; helix IV, blue; helix VIII, orange; helix X, green; remaining helices, gray). B) Side-view of helix pair II/IV. C) Side-view of helix pair VIII/X. D) Superimposition (more ...)
In any event, the Asp68 mutants probably exist in a novel locked conformation in the presence of sugar, and these mutants are candidates for crystallization trials to trap another conformational state(s) of LacY.