Altering residues N125 and D149 impacts sugar effector binding and allosteric parameters in Escherichia coli lactose repressor.

摘要: The lactose repressor protein (LacI) is a negative transcription regulator in E. coli and has long served as model system to study allosteric mechanisms (1-4). In the presence of lactose, metabolite allolactose produced by β-galactosidase, binds LacI, elicits conformational change that releases lac operon operator DNA, thereby allowing structural genes required transport utilize (3-7). LacI can bind multiple sugars at same effector binding site with varying outcomes on DNA-binding affinity (5, 7-10). Isopropyl-β,D-thiogalactoside (IPTG) (Figure 1A) highly effective inducer for (1, 5, 10) widely used experiments instead natural allolactose. o-Nitrophenyl-β,D-fucoside (ONPF) serves an anti-inducer, increasing ~3-fold, whereas 2-phenylethyl-β,D-galactoside (PhEG) weaker compared IPTG 10). FIGURE 1 (A) Structures IPTG, ONPF, PhEG. (B) Structure dimeric bound Osym DNA ONPF (not shown). from PDB file 1EFA (12). tetramer identical monomers, dimer (shown here without C-terminal tetramerization ... The signal must be passed ligand through structure (~40 A) reach remote (3, 4, 11, 12) 1B). Numerous residues are actively involved transmitting between these sites (11-15). Crystallographic structures demonstrated D149 directly contacts targeted molecular dynamics (TMD) simulation follow changes upon identified pathway, including interactions D149/S193 D149/N125 (11-13). Mutation allow disulfide formation D149C S193C importance flexibility response (14). TMD also N125 transiently moves closer during transition 1C). This simulated interaction indicates might function key residue along pathway N-subdomain interface. In high resolution crystal sugar ligands, form N-terminal end site. IPTG-bound structure, direct contact made D149, waters include engaged intensive water-mediated hydrogen bond network 1D) (8). may not only secure binding, but stabilize altered orientation N- C-subdomains induced state. observed effectors1 demonstrate different orientations distinct account their differential consequences ONPF-bound 1E), extended present, although hydrogen-bond nitro moiety both possible based distance From structures, participate ways. To further understand role response, we have explored using stochastic boundary (SBMD) (16, 17) were able transient characteristic two side chains. We therefore individually2 simultaneously Ala assess impact removing capacity Cys determine whether could them. results 125 149 together critical functional information within LacI.