lac Repressor headpiece binds specifically to half of the lac operator: a proton nuclear magnetic resonance study

Abstract

The complex formation of the N-terminal domain (headpiece) of the Escherichia coli lac repressor and a synthetic 14-base-pair lac operator fragment has been investigated by 1H NMR. Titration shifts in the imino-proton region of the DNA spectrum and in the aromatic region of the headpiece spectrum are examined in detail and interpreted where possible. The assignment of the resonances in the complex follows in part from the titration data and is completed by nuclear Overhauser measurements. The shift of the His-29 C-2 resonance has been used to assess the binding strength of the complex. Evidence is presented for the presence of a high-affinity site on the lac operator fragment (KD less than or equal to 2 X 10(-5) M), which shows features in common with one of the specific binding sites on the complete lac operator, and for the presence of a second, nonspecific binding site with lower affinity. The influence of this second site on the interpretation of the binding data is discussed.

Structural aspects of protein-DNA recognition

“…spectra of the repressor and operator fragments on forming the complex. For example, the pK of His-29 increases on forming the specific complex, consistent with proximity to a negative potential (Scheek et al, 1983). Experiments were also carried out to probe the surface accessibility of aromatic residues, which showed that Tyr-7 and Tyr-17, which are in the recognition helix, and His-29, become buried in the protein-DNA complex, suggesting that these residues are in the protein-DNA interface (Stob et al, 1988).…”

“…Each subunit consists of two autonomously folded domains, the larger of which contains the binding site for the inducer allolactose. The smaller N-terminal domain can be easily cleaved with different proteases to yield the monomeric 51-59-residue head-piece (Geisler & Weber, 1977) which has specific DNA binding activity (Scheek et al, 1983), but which has lost its allosteric response to allolactose. However, n.m.r.…”

Secondary structure of the lac repressor DNA-binding domain by two-dimensional 1H nuclear magnetic resonance in solution.

“…The regions interacting specifically with operator DNA are located in the domains formed in each subunit by the 51 or 59 NH2-terminal amino acids (11)(12)(13). This DNA-binding domain, hereafter referred to as the "headpiece," retains its structure and the specific DNA-binding capacity when isolated from the intact repressor by enzymatic cleavage (12)(13)(14)(15)(16)(17).…”

“…Nuclear magnetic resonance (NMR) in solution has therefore been the method of choice for studies of this protein and its interactions with the operator DNA (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27). NMR investigations have so far concentrated on observations of the amino acid side-chain proton resonances.…”

“…2D proton NMR spectra of lac repressor headpiece lac repressor headpiece, residues 1-51, was isolated from E. coli strain BMH74-12 and was purified and characterized as described (17). Fig.…”