The hinge helix of the ternary complex is folded

Since chemical shifts are sensitive reporters of local chemical environment and local structure, they can be used to determine the structural state of the hinge helix in the ternary complex. Unfortunately, the high molecular weight of both LacI-operator complexes precluded comprehensive chemical shift assignment—even with TROSY methodologies and NUS at the limit of sparseness (5% sampling). As described in Chapter 3, only 72% of all expect amide cross peaks could be assigned for the 1H-15N TROSY HSQC of the binary complex. Though this coverage is quite limited, it was sufficient to identify resonances of critical residues in the DBD and hinge helix.

Figure 5.1 Overlay of 1H-15N TROSY HSQC spectra of the LacI-Osymbinary complex (black) and

LacI-Osym-IPTG, ternary complex (red). All of the data were collected at 750 MHz and 35˚C.

IPTG-binding to the binary complex induces significant perturbations in chemical shift as illustrated in Figure 5.1. Mapping resonance assignments of the binary complex spectrum to the ternary complex spectrum was difficult to do unequivocally. Fortunately, the 1H-15N TROSY HSQC of the ternary complex yielded nearly identical chemical shifts to the corresponding spectrum of the isolated RD bound to IPTG. This indicates that the RD of the ternary complex is structurally identical to that of the isolated RD bound to IPTG. This offers a significant advantage with respect to spectral analysis. Resonances that arise from the RD can be identified immediately and the remaining resonances are likely to arise from the DBD.

In order to facilitate identification of DBD resonances in the spectrum of the ternary complex, additional reference spectra of different constructs of LacI were included in the analysis. Figure 5.2 illustrates how spectra of the RD-IPTG and DBD-Osym complexes could be used to

Figure 5.2 Strategy for assigning DBD resonances in the 1H-15N TROSY HSQC spectrum of the ternary complex (black). DBD resonances are identified by comparison of 1H-15N TROSY HSQC spectrum of the RD-IPTG complex (green) and the 1H-15N TROSY HSQC spectrum of the DBD-

Osym complex (cyan). Black peaks that overlay with green peaks arise from the RD whereas black

peaks that overlay with cyan peaks arise from the DBD. All of the data were collected at 750 MHz and 25˚C. Negative peaks are shown in yellow.

The above strategy enabled identification of DBD resonances in the spectrum of the ternary complex. But it is the hinge region that is of interest here. In order to identify hinge residues and discern secondary structure, chemical shifts of the ternary complex were also compared to those of the LacI-IPTG complex in the absence of operator. In Chapter 4, it was shown that an overwhelming majority of DBD and hinge resonances could be assigned for the 1_H-15_{N TROSY HSQC spectrum of the LacI-IPTG complex. The structural modeling presented in}

that chapter confirmed that the hinge of the LacI-IPTG complex is unfolded. Therefore, hinge resonances of the LacI-IPTG complex are indicative of an unfolded conformation. If the ternary

complex exhibits an unfolded hinge, it should yield resonances consistent with those of the LacI- IPTG complex. If not, it should yield resonances more consistent with the LacI-Osym complex or

DBD-Osym complex, where the hinge is known to be folded from previous structural

characterization [32, 75, 82, 86].

When comparing spectra of proteins with and without DNA, one potential variable that must be considered is the effect that DNA has on chemical shifts. For example, if a hinge resonance happens to be in close proximity to the operator, it may exhibit a unique chemical shift that is dominated by the chemical properties of the DNA rather than protein secondary structure. This would render the resonance incomparable to those from the spectra of the LacI-IPTG complex. Fortunately, this issue has been addressed by the Kaptein group who characterized the isolated LacI DBD bound to a variant operator in which the hinge helix only formed on one of the two half-sites [87]. It should be clarified that in this complex both half-sites are still bound to protein [87]. The 1H-15N HSQC spectrum of that complex indicated that the unfolded hinge resonances were nearly identical to those from the apo DBD. These data validate our approach. Figure 5.3 shows the amino acid sequence of the hinge helix and Figure 5.4 shows a comparison of 1H-15N HSQC spectra of the ternary complex, LacI-IPTG, RD-IPTG, and DBD-Osym. Note that

hinge residues G58 and the strictly conserved A53 of the ternary complex clearly exhibit a resonance frequency more consistent with those from the spectrum of DBD-Osym (folded hinge)

rather than those from the spectrum of LacI-IPTG (unfolded hinge). This indicates that the ternary complex exhibits a folded hinge.

Figure 5.3 Amino acid sequence of the hinge region of LacI (residues 46-62). The hinge helix is shown as a blue bar and the corresponding residues involved in helix formation are shown beneath it. Residues in red and marked with asterisks are strictly conserved across the ten nearest relatives of LacI from LacI/GalR family based on sequence alignment using ClustalW [267].

Figure 5.4 Strategy for determining the conformation of the hinge helix of the ternary complex. The 1H-15N TROSY HSQC spectrum of the ternary complex (black) is overlaid with the corresponding spectra for the RD-IPTG complex (green), DBD-Osym complex (cyan), and the

LacI-IPTG complex (red). (A) Shift for folded conformation of G58. (B) Shift for unfolded conformation of G58. (C) Shift for folded conformation for A53. (D) Shift for unfolded conformation of A53. All of the data were collected at 750 MHz and 25˚C.

The data above suggest that the hinge helix remains folded in the ternary complex. Like the previous SAXS study, this suggests that the mechanism of induction does not involve local unfolding of the hinge helix.

In order to further characterize the nature of the interaction between the hinge helix and operator in the ternary complex, we focused our attention to L56. As described in Chapter 1, the side chain of L56 intercalates directly into the minor groove of the operator. This interaction is essential for high affinity binding [268] and L56 is strictly conserved across the LacI/GalR family (Figure 5.3). The resonance of L56 could not be assigned unambiguously in the 1H-15N TROSY HSQC of either the binary or ternary complexes. Therefore, NMR experiments which probe the 1_H-13_{C resonances of methyl-bearing side chains were employed to characterize L56. These}

experiments are advantageous because the side chain of L56 mediates the interaction with operator. The resonances of both methyl groups of L56 were identified from comparison of the stereo-specifically assigned 1H-13C HSQC spectrum of the DBD-Osym complex as shown in Figure

5.5a. In the methyl spectrum of the ternary complex, both resonances are well resolved. Importantly, the resonances exhibit notable line broadening, suggesting that the L56 side chain is indeed in very close proximity to the protonated operator DNA, further corroborating that the assignments are correct.

Figure 5.5 L56 is intercalated into the operator in the ternary complex. (A) Crystal structure of Lac-Osym complex (PDB: 1EFA). L56 is shown as red sticks. (B) Assignments of the 1H-13C

HMQC spectrum of the ternary complex (black) were made via comparison with the assigned 1H- 13_{C HSQC of the DBD-O}

sym complex (red). L56 assignments are shown in red type. (C-D) 13C R1ρ

measurements of the well resolved and unambiguously assigned L56 δ2 side chain resonance for the binary (C) and ternary complex (D).

In order to probe the dynamics properties of the L56 side chain in the binary and ternary complexes, carbon T1ρ relaxation experiments were performed. Relaxation samples were labeled

Experiments were performed in D2O in order to minimize remote protons. This was found to result in stronger peak intensities for L56 relative to samples in H2O (data not shown) which permitted more quantitatively accurate characterization.

These relaxation measurements are sensitive to the local mobility of the probe (higher mobility probes yield slower relaxation rates) as well as the density of remote proteins nearby. Since our LacI NMR samples are perdeuterated, the vast majority of remote protons that could influence these measurements arise from non-exchangeable sites of the operator. Therefore, it is reasonable to expect that any significant IPTG-induced distortions in the orientation or mobility of the L56 side chain would be detectable. Figure 5.5b shows that the 13C R1ρrates of L56 in the

binary and ternary complexes are within error (24.946 ± 0.403 s-1 for binary and 25.906 ± 1.264 s-

1 _{for ternary). This suggests that the L56 side chain is not changed significantly as a result of}

IPTG-binding and further confirms that the hinge helix is folded.