Laura M. L. Hawka,
Clifford T. Geea,
Andrew K. Urickab,
Haitao Hub and
William C. K. Pomerantz*a
aDepartment of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, MN 55455, USA. E-mail: wcp@umn.edu
bDiscovery Chemistry Research & Technologies, Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
First published on 29th September 2016
Protein-observed 19F (PrOF) NMR is an emerging tool for ligand discovery. To optimize the efficiency of PrOF NMR experiments, paramagnetic relaxation enhancement through the addition of chelated Ni(II) was used to shorten longitudinal relaxation time without causing significant line broadening. Thus enhancing relaxation time leads to shorter experiments without perturbing the binding of low- or high-affinity ligands. This method allows for time-efficient screening of potential ligands for a wide variety of proteins in the growing field of fragment-based ligand discovery.
In NMR experiments, the recycle delay, during which the spectrometer is idle while the magnetization relaxes after a radio frequency pulse, comprises up to 80% or more of the total experiment time (Fig. 1B).6 Shorter recycle delays allow more scans per unit time, leading to more efficient instrument use. One strategy employed to shorten recycle delays is the addition of a paramagnetic metal to decrease the longitudinal relaxation time (T1) of 1H, 13C, and 15N nuclei in labelled proteins while minimizing unfavourable line-broadening from decreasing transverse relaxation time (T2).6–11 The application of paramagnetic additives to 19F NMR has also been described in the context of NMR with molecular oxygen.12 However, the specialized NMR tubes required would not be ideal for routine small molecule screening. These studies did not explore if the beneficial effects of paramagnetic metal additives are retained in the presence of bound ligands of varying affinity, a question we examine herein.
In this work, we explore the application of paramagnetic relaxation enhancement (PRE) to PrOF NMR, utilizing a solvent-accessible side-chain labelling scheme. This labelling scheme is well-suited to studying protein–protein interaction interfaces, which contain a high proportion of aromatic amino acids13 that can readily be biosynthetically replaced with their fluorinated counterparts. The appended fluorine atoms on the side chains can be more solvent exposed than backbone amides, and thus more accessible to paramagnetic additives in the bulk solvent. We demonstrate that PRE can reduce experiment times significantly by shortening the recycle delay with minimal line broadening from T2 effects, ultimately leading to a marked enhancement in sensitivity. In the context of low-complexity molecules commonly used in fragment-based ligand discovery screening, we further show improvements by using this method for rapidly obtaining dissociation constants for weak-binding fragments. In addition to fragment screening, this method may also be useful for proteins available in limited abundance such as GPCRs,14,15 as the signal to noise increases by the square root of the number of scans. Importantly, this approach is also compatible with proteins containing metal binding hexahistidine affinity tags and with various fluorinated amino acids.
NMR studies of fluorinated biomolecules have been applied to diverse biophysical processes, including protein folding and ligand binding.16,17 The high sensitivity (83% that of proton), 100% natural abundance, absence of background in biological systems, and large chemical shift range all serve to make 19F NMR an attractive tool for characterizing biological systems. Several recent studies have screened for ligand binding by PrOF NMR.4,18–21 Although the 19F NMR spectra can be readily acquired,5 library screening by this method nevertheless requires a significant amount of experiment time. Shortened experiments would increase throughput in a small molecule NMR screen where the number of experiments may be large.
To determine the optimal chelated Ni(II) concentration for protein studies, we explored the effect of a range of concentrations of chelated Ni(II) on the relaxation properties of 4FF (Fig. S1A†) as well as 5-fluoroindole (Fig. S1B†), the side chain present in proteins that have been 19F-labelled at their tryptophan residues. The T1 values of both of these molecules at Ni(II) concentrations up to 320 mM for 4FF and 200 mM for 5-fluoroindole were determined by inversion recovery experiments. values were determined from the peakwidth at half height. Because of the importance of peakwidth for resolving spectral resonances,
was used rather than T2. For both 4FF and 5-fluoroindole, the highest Ni(II) concentration tested led to a more than a 100-fold decrease in T1 values (Table 1).
also decreased with increasing chelated Ni(II) concentration, leading to approximately a 5-fold increase in peak width at 200 mM chelated Ni(II). Concentrations of 20–50 mM provide an optimal balance between T1 reduction and
reduction and agree well with the 50 mM Ni(II) used by Cai et al. in 1H, 15N, and 13C experiments.6
Compound | Ni-DTPA (mM) | T1 (ms) | (ms) |
---|---|---|---|
a Small molecules used in this study were 5-fluoroindole and 4-fluorophenylalanine (4FF). T1 and ![]() |
|||
5-Fluoroindole | 0 | 4890 | 208 |
20 | 528 | 107 | |
50 | 202 | 80 | |
4FF | 0 | 1870 | 77 |
20 | 216 | 97 | |
40 | 113 | 80 | |
W75 (+JQ1) | 0 | 508 (552) | 7.5 (7.1) |
20 | 269 (399) | 7.2 (6.2) | |
40 | 294 (346) | 6.4 (6.9) | |
W120 (+JQ1) | 0 | 721 (685) | 8.4 (6.1) |
20 | 146 (212) | 8.0 (7.2) | |
40 | 86 (156) | 8.4 (8.1) | |
W81 (+JQ1) | 0 | 837 (772) | 8.8 (7.0) |
20 | 196 (289) | 8.6 (5.4) | |
40 | 192 (149) | 8.6 (2.9) |
We then moved to testing the effects of Ni-DTPA on a model protein, the first domain of the bromodomain-containing protein Brd4, hereafter referred to as Brd4. Bromodomains are epigenetic regulatory domains, inhibition of which has therapeutic implications for cancer, heart disease, and inflammation,24,25 and are often targeted in fragment-screening campaigns.26–28 For these studies, Brd4 was biosynthetically 19F-labelled using the amino acid precursor 5-fluoroindole at all three tryptophan sites (W75, W81, and W120; Fig. 1A) due to the enrichment of aromatic amino acids at protein–protein interaction interfaces.13,29 When small molecules or ε-acetylated histone substrates bind to Brd4, we have observed a significant perturbation of the W81 resonance, which is located in the binding pocket. The resonance corresponding to W75, which is located beneath the binding pocket, experiences minor perturbations due to the environmental sensitivity of fluorine. The resonance corresponding to W120, which is located 36 Å from the binding site, generally shows minimal perturbation. T1 measurements were carried out on 5-fluorotryptophan (5FW)-labelled Brd4 solutions titrated with Ni-DTPA without significantly affecting resonance width (Fig. S2†).
As expected, increasing concentrations of Ni-DTPA led to decreases in the T1 (Table 1 and Fig. S3†). The T1 of the fluorine appended to W120 decreases the most rapidly and continues decreasing as more Ni-DTPA is added, suggesting that this fluorine is most accessible to the paramagnetic additive (80% reduction in T1 at 20 mM Ni-DTPA), with W81 being intermediate in accessibility (75% reduction). The fluorine on W75 (50% reduction) is least accessible, and its T1 value is less affected by Ni-DTPA. While the level of accessibility to paramagnetic additives consistent with T1 measurements differs from the level of solvent exposure determined by the program GetArea,30 the accessibility of the appended fluorine atom may differ from that of the residue as a whole. The extent of T1 reduction upon addition of Ni-DTPA correlates with the apparent solvent exposure of the 5-position of the indole ring of the labelled tryptophans (Fig. S4†). These results are consistent with solvent accessible nuclei experiencing the strongest PRE effects.
The fluorines appended to W75 and W81 reach their T1 minima at 20 mM metal; doubling the metal concentration to 40 mM had little effect on T1 values. Therefore, 20 mM Ni-DTPA was used for further experiments to minimize the Q-damping effects resulting from additional ions in solution and concomitant loss of sensitivity.31
In the absence of Ni-DTPA, experiments were carried out with a recycle delay of 1 second (1.2 times the measured T1 value of W81, the residue most affected by ligand binding in the binding pocket), with a total experiment time of five minutes (280 scans) at modest protein concentrations of 50 μM. With 20 mM Ni-DTPA present, the recycle delay was shortened to 0.24 seconds for a consistent value of 1.2 times the measured T1 of W81. In this case, the experiment time was reduced to two minutes (350 scans) at the same protein concentration with similar signal-to-noise for the W81 resonance (11.8 with Ni-DTPA vs. 10.9 without), a 60% reduction in experiment time (Fig. 2). The signal-to-noise ratio may be further improved if Q-damping effects could be mitigated. The T1 value of W120 (0.15 s) is more reduced than that of W81 (0.20 s) by the addition of 20 mM Ni-DTPA. Consequently, the relative intensity of W120 increases with the addition of 20 mM Ni-DTPA because the net magnetization has more fully relaxed back to the ground state before application of the next pulse. In contrast, W75 has a longer T1 value (0.27 s) than W81 and thus its resonance is truncated by the short recycle delay of this experiment. Because W75 is farther from the binding pocket, the experiment was optimized for maximizing signal-to-noise per unit time of W81. Because binding sites must be accessible to their binding partners, amino acid side chains located at protein–protein interaction interfaces should also be accessible to paramagnetic additives and thus subject to substantial PRE effects, allowing for shortened recycle delays and experiment times.
The chemical shift of W75 of Brd4 resonance is slightly perturbed upon titration of Ni-DTPA, moving 0.04 ppm in the presence of 20 mM Ni-DTPA and 0.07 ppm in the presence of 40 mM Ni-DTPA, which are above the threshold for significance of 0.03 ppm.19 However, binding is non-specific and the change in chemical shift does not reach a saturation point up to 200 mM Ni-DTPA (Fig. S5†). Similarly, the chemical shift of fluorine resonances can change substantially upon titration of deuterated solvent, underscoring the large environmental sensitivity of the fluorine nucleus.32 Protein stability does not undergo large perturbations upon titration of Ni-DTPA, as measured by differential scanning fluorimetry, a thermal shift assay. Changes in melting temperature were ≤1.2 °C by this method (Fig. S6†). To assess the functional effect of Ni-DTPA on binding, fluorescence anisotropy was used to quantitate the binding affinity between a fluorescently labelled BET bromodomain ligand BI-BODIPY19,33 and Brd4 (Fig. S7†). Binding was minimally perturbed, with only minor perturbations to the dissociation constant upon increasing concentrations of Ni-DTPA (Table S1†). These results reduce concerns over the perturbing nature of the paramagnetic additive.
Having optimized conditions for running a time-efficient NMR experiment on a fluorinated protein, we next evaluated the time-enhancement effects of these conditions on ligand binding of both fragments and potent molecules. To this end, we tested two Brd4 inhibitors, compounds 1 and 2 (Fig. 3A), uncovered in our small molecule discovery NMR fragment screens. Both 1 and 2 bind to 5FW-Brd4 (Fig. 3B). For molecules like 1 and 2 that bind in the fast exchange regime, Kd values can be obtained by plotting changes in chemical shift against ligand concentration and fitting the resulting data to a binding isotherm that accounts for ligand depletion.16 For both fragments, the resonance for W81 was the most perturbed, W75 was slightly perturbed, and W120 remained unaffected (Fig. 3C and D). Fitting of the data for 1 to a binding isotherm yielded a Kd value of 81 ± 8 μM in the absence of paramagnetic metal and 87 ± 9 μM in the presence of 20 mM Ni-DPTA based on the chemical shift perturbation of W81. For 2, the Kd values were 390 ± 60 μM without paramagnetic additive and 440 ± 30 μM with 20 mM Ni-DTPA. Additional bromodomain ligands were shown to bind to 5FW-Brd4 in the presence and absence of Ni-DTPA (Fig. S8†). These experiments were acquired at a higher magnetic field strength (564 MHz) allowing for a one-minute experiment time. We conclude that moderate concentrations of Ni-DTPA minimally perturb the binding of low-affinity molecules to this fluorinated protein and can thus be applied for fragment-based screens.
An experimental time reduction of one to two minutes allows for facile Kd determinations of fragments. Affinity determinations are valuable in fragment screening for prioritizing hits. We found that because low-affinity fragments have a short residence time on the protein, the 19F-labelled residues are subject to PRE effects similar to those in the absence of fragment, allowing a full titration (nine samples) to be carried out in less than 20 minutes of experiment time. Without the advantage of pulse sequences like those used for 2D heteronuclear experiments such as SOFAST-HMQC which result in seven minute experiment times for similar sized proteins,5,34 these 19F experiments represent some of the fastest PrOF NMR experiments at the modest concentrations used.
We next sought to determine whether this method was compatible with more potent binders with longer residence times on the protein. We previously showed 19F NMR spectra of 5FW-Brd4 exhibit slow exchange kinetics with known ligand (+)-JQ1 (Kd = 77 nM),35 consistent with a long residence time on the protein. In our hands, the spectra acquired with one equivalent of (+)-JQ1 are qualitatively similar in the presence and absence of Ni-DTPA (Fig. S9†). However, the presence of this tight binding ligand limits the interaction of the paramagnetic additive and the labelled residue. Therefore, the T1 experiences a smaller reduction in the presence of (+)-JQ1, consistent the r−6 distance dependence of relaxation times as described by the Solomon–Bloembergen equations.6 Despite the changes of T1 values, the W81 resonance is similarly perturbed by (+)-JQ1 in the presence and absence of Ni-DTPA, and spectra remain consistent with a tight-binding molecule in slow exchange.
Finally, to assess the generality of using PRE to shorten PrOF NMR experiments, we tested the compatibility of Ni-DTPA with three additional 19F-labelled protein constructs. We tested proteins with hexahistidine affinity tags due to their ability to chelate metals. Encouragingly, spectra of hexahistidine-tagged 5FW-Brd4 recorded in the presence of 40 mM Ni-DTPA had linewidths similar to those of 5FW-Brd4 without its affinity tag, indicating that the Ni-DTPA is compatible with the presence of a hexahistidine tag despite this sequence's high affinity for Ni(II) (Fig. S10†). Ni-DTPA was also tested and is compatible with 3-fluorotyrosine (3FY)-labelled Brd4 (Fig. S11†) and 3FY-labelled KIX (Fig. S12†). Because 3FY KIX shows larger perturbations in its NMR spectrum than 5FW Brd4 upon addition of Ni-DPTA, the capacity of KIX for binding small molecules in the presence of Ni-DTPA was tested by both fluorescence anisotropy and PrOF NMR. Fluorescence anisotropy showed a small but significant dose-dependent increase in the Kd of the interaction between a fluorescein-labelled peptide of the transcriptional activation domain of MLL and the KIX protein at increasing concentrations of Ni-DPTA (Fig. S13 and Table S2†). For 20 mM Ni-DTPA, the increases in Kd were within two-fold. Using PrOF NMR, the Kd measured for the binding of KIX ligand naphthol-AS-E phosphate36 in the presence of 20 mM Ni-DTPA was 187 ± 50 μM, similar to the previously reported Kd of 115 ± 15 μM (Fig. S14†).18
In addition to the proteins studied here, Cai et al. also measured PRE effects on the 15N-labeled protein SUMO-1 using a DO2A (1,7-dicarboxymethyl-1,4,7,10-tetraazacyclododecane) chelator for Ni(II) rather than DTPA.6 PRE effects from chelated Ni(II) have also been applied to 13C-ribosome nascent chains.10 Finally, the 13C, 15N-labelled intrinsically disordered protein α-synuclein, which is known to bind metals, has also been subjected to PRE conditions with chelated Ni(III) with minimal changes observed in its spectra.7 These results support a broader use with proteins.
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Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra21226c |
This journal is © The Royal Society of Chemistry 2016 |