Arginine 197 of lac repressor contributes significant energy to inducer binding. Confirmation of homology to periplasmic sugar binding proteins
Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill, B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray crystallographic structure of the arabinose-bindi...
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Veröffentlicht in: | The Journal of biological chemistry 1991-12, Vol.266 (34), p.22998-23002 |
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Zusammenfassung: | Based on primary sequence homology between the lactose repressor protein and periplasmic sugar-binding proteins (Müller-Hill,
B. (1983) Nature 302, 163-164), a hypothetical sugar-binding site for the lac repressor was proposed using the solved x-ray
crystallographic structure of the arabinose-binding protein (ABP) (Sams, C. F., Vyas, N. K., Quiocho, F. A., and Matthews,
K. S. (1984) Nature 310, 429-430). By analogy to Arg151 in the ABP sugar site, Arg197 is predicted to play an important role
in lac repressor binding to inducer sugars. Hydrogen bonding occurs between Arg151 and the ring oxygen and 4-hydroxyl of the
sugar ligand, two backbone carbonyls, and a side chain in ABP, and similar interactions in the lac repressor would be anticipated.
To test this hypothesis, Arg197 in the lac repressor protein was altered by oligonucleotide-directed site-specific mutagenesis
to substitute Gly, Leu, or Lys. Introduction of these substitutions at position 197 had no effect on operator binding parameters
of the isolated mutant proteins, whereas the affinity for inducer was dramatically decreased, consistent with in vivo phenotypic
behavior obtained by suppression of nonsense mutations at this site (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol.
212, 295-318). Inducer binding affinity was reduced approximately 3 orders of magnitude for Leu, Gly, or Lys substitutions,
corresponding to a loss of 50% of the free energy of binding. The pH shift characteristic of wild-type repressor is conserved
in these mutants. Circular dichroic spectra demonstrated no significant alterations in secondary structure for these mutants.
Thus, the primary effect of substitution for Arg197 is a very significant decrease in the affinity for inducer sugars. Arginine
is uniquely able to make the multiple contacts found in the ABP sugar site, and we conclude that this residue plays a similar
role in sugar binding for lactose repressor protein. These results provide experimental validation for the proposed homology
between ABP and the lac repressor and suggest that homology with ABP may be employed to generate additional insight into the
structure and function of this regulatory protein. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(18)54453-7 |