Secondary Structure of the lac Repressor Headpiece

Abstract

The secondary structure of the short tryptic headpiece of the lac repressor has been investigated by the analysis of its infrared and circular dichroic spectra. For the latter we used the method of Provencher and Glockner [Biochemistry (1981) 20, 33-37], which seems to be at present the most successful for the determination of the p content of proteins. Nevertheless our results indicate that in the case of the lac repressor headpiece this method overestimates the amount of [j structure. We find that the headpiece contains an important helical content of about 50:,;, depending slightly on the ionic strength. A decomposition of the infrared spectrum in a sum of Gaussian curves reveals clearly the absence of a vibrational band around 1630 cm-', excluding thus the presence of a multi-stranded 8-pleated sheet. The only structure compatible with the infrared results seems to be a twostranded antiparallel fl sheet, as judged from our results on the fl-sheet model-compound gramicidin s. The unusually strong intensity of the amide I' band is in favour of the existence of such a structure. The quantitative analysis of both infrared and circular dichroism spectra indicates the presence of a certain (but different) amount of fl structure. Comparing these results with several secondary structure predictions, part of the helical residues should be located between Leu-45 and (at least) Arg-35, and an eventual two-stranded j? sheet should be situated in the N-terminal part of the headpiece.In the Escherichia coli lac operon the expression of the structural genes is negatively controlled by the interaction of a protein, the lac repressor, with the luc operator [l, 21. Intact repressor has a molecular weight of 154000 and is a tetramer of four identical subunits of 360 amino acids. Upon limited proteolysis of lac repressor in high-ionic-strength buffer one obtains four small N-terminal 'headpieces' (residues 1 -51, 1 -56 or I -59 depending on the protease used and the duration of hydrolysis) and a tetrameric 'core' [3,4]. There is little doubt now that the headpieces constitute the major, if not the entire, part of the DNA-binding domain of the lac repressor. It has been shown that the isolated headpiece has nonspecific affinity for DNA [3,5,6] and induces the same conformational change as does the intact lac repressor upon nonspecific binding (our unpublished results). Ogata and Gilbert [7,8] have shown that the pattern of modification of the Iuc operator by dimethyl sulfate is very similar whether it is induced by the intact protein or by the headpiece. It is thus of considerable interest to determine the structure of the headpiece in order to understand how it interacts with the DNA.Information on the global shape of the headpiece has recently been obtained by neutron-scattering measurements, suggesting an elongated form with a long dimension of about 5 nm and a cross-sectional diameter of 1.5-2 nm [9]. On the other hand, local information, concerning mainly the four tyrosine residues, has been obtained by 'H and I9F NMR [lo-1...