Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue

Abstract

Many mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI. When DNA is released, LacI shows a 20 A increase in length that was previously attributed to unfolding of the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants exhibit altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function.

Figure 1. Model of the LLhP homodimer and positions of specificity determinants

(A) The model of apo-LLhP was constructed as described in Materials and Methods and rendered with Chimera (50). One monomer is colored light gray and the other is dark gray. One DNA-binding domain is indicated with the dotted box; the two DNA-binding domains of a dimer are required for binding operator DNA. One regulatory domain is indicated within the dashed oval. The binding site for co-repressor hypoxanthine is indicated with a green circle in a cleft between the N- and C-subdomains (which are respectively at the top and bottom of the regulatory domain.) (B) The linkers of the LLhP dimer are shown, with the beginning and end indicated with arrows. The orientation is rotated ∼90° relative to (A). The central helical region of the linker is often called the “hinge helix”. The positions of linker specificity determinants for wild-type sites I48, Q55, and S61 are shown in ball-and-stick representation in green and magenta. Gly58 is indicated with a magenta ribbon for the backbone. None of the linker specificity determinants directly contact DNA in either the LacI or PurR structures. (C) The model for apo-LLhP (white) was aligned with the magenta structure of co-repressed PurR bound to DNA (1wet; (28)) using Chimera (50). Co-repressor is not shown in this figure. Note that the DNA-binding domains of apo-LLhP have moved relative to those of the DNA-bound PurR. Intriguingly, molecular dynamics simulations of the LacI DNA-binding domain show similar motions (8). Flexibility in the region spanning amino acids 45−50 is required for this motion, suggesting a possible mechanism for altering DNA-binding affinity by polymorphisms at position 48.

Figure 2. Trends in K_d and allosteric response for LLhP variants binding to alternative lac operators

The symbols and colors in legend shown are used for all three panels. For panel (A), “+” indicates the presence of saturating hypoxanthine. (A) Affinities of LacI and LLhP variants for lacO¹, lacO^sym, and lacO^disC. LacI (dashed black line) shows steeper discrimination between the 3 operators than does LLhP (solid black line). With the exception of LLhP+HX+lacO^sym, unmodified LLhP, the 48 variants, and the 55 variants have different affinities but the same selectivities (solid, parallel lines). The LLhP variants at position 61 show a different pattern of DNA selectivity (dotted lines). (B) Allosteric ratios of LLhP variants report the magnitude of change in K_d when the repressor binds hypoxanthine. Data for S61C and S61M are slightly offset to aid visual inspection. (C) Midpoints of operator capture for LLhP variants reflect the amount of hypoxanthine required to elicit the full allosteric change.

Figure 3. Solution X-ray scattering by LLhP

(A) I(Q) versus Q for apo-LLhP (green), LLhP-lacO¹ (red) and LLhP-lacO¹-HX (black). The inset shows a Guinier plot of the low-Q data and the linear fits obtained (apo is green; LLhP-lacO¹ is red; LLhP-lacO¹-HX is black). The scattering data are shown normalized to constant monitor counts, and for the Guinier plots, the apo-LLhP data are multiplied by 3 to allow for easier display. (B) P(r) versus r (large symbols) calculated using the data in (A), using the same key. Difference plots are also shown in small symbols near the origin Y=0 for the P(r) profiles of LLhP-lacO¹ with and without hypoxathine (black). For comparison, a difference plot is also shown for the LacI R3 dimer bound to lacO¹ with and without IPTG (from (23); blue).

Figure 4. In vivo repression versus in vitro DNA-binding affinity for LLhP variants

In vivo data are reported in (4). Data shown on this plot include measurements made in both the presence and absence of co-repressor for LLhP variants in the current study. The dashed line reflects the best fit (linear regression) of the entire data set; R² = 0.40. The solid line reflects the best fit when S61C(+) and S61M(+) are not included; R² = 0.79. Since K_d values for G58T and G58V could not be reasonably estimated, these data are not included on this plot. However, the G58 variants show good correlation between poor repression and weak DNA binding.