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Localization of ligands within human carbonic anhydrase II using 19F pseudocontact shift analysis† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc05683h

Unraveling the native structure of protein–ligand complexes in solution enables rational drug design.


Introduction
In order to localize and study ligands bound to proteins, sources of structural restraints are required. In contrast to X-ray crystallography 1 and cryo-electron microscopy, 2 paramagnetic nuclear magnetic resonance (NMR) spectroscopy can deliver valuable structural information in solution. [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21] Among other effects associated with the introduction of paramagnetic metal centres complexed in a chelating tag, as e.g. residual dipolar couplings (RDC) 17,[22][23][24] or paramagnetic relaxation enhancement (PRE), 13,[25][26][27] pseudocontact shis (PCS) stand out as an ideal tool to deliver long range information on the localization and orientation of ligand molecules within proteins taking into account the 3D structural restraints that can be obtained using this method and the favourable distance dependence of r À3 . [28][29][30][31][32][33][34] To unlock the opportunities that PCS of protein-bound ligands offer, the NMR signals of the ligand have to be determined unambiguously. Proton resonances of the ligand usually overlap with the protein signals, thus, rendering a successful assignment difficult. This holds in particular true for ligands that bind rmly to the protein, since they exhibit the same rotational correlation time as the protein and their lines are therefore broadened to a similar extent. This usually renders a discrimination between protein and ligand signals difficult. Whereas isotope ltered NMR experiments suffer from a low signal-to-noise ratio and long measurement times, isotopic labelling of the ligand carbon-and hetero atoms includes labour intensive chemical modication of the ligand and the effort has to be repeated for each ligand under investigation.
However, due to the 100% natural abundance of NMR active isotopes such as 19 F or 31 P, no further isotope enrichment is necessary for the acquisition of NMR spectra of molecules containing these elements. In particular uorine is of great interest, since whereas in 1970 only 2% of drugs incorporated uorine, the percentage of drugs containing at least one uorine atom in 2014 amounted to 25%. 35 If protein ligands containing 19 F are used, their 19 F chemical shi can be obtained directly from one-dimensional 19 F NMR spectra, superseding isotopic labelling of ligands and solvent signal.
We selected three sulphonamide ligands for human carbonic anhydrase II (hCA II) in order to test their localization within the protein using PCS NMR spectroscopy (Fig. 1). The tested ligands are all derivatives of the well-established class of sulphonamide ligands that bind with their sulphonamide moiety to the zinc ion in the active site of the protein and exhibit nanomolar affinities to the human carbonic anhydrase II. [36][37][38][39] In order to unambiguously localize ligands within a protein using PCS NMR spectroscopy, four different anisotropy tensors need to be determined from four different tagging sites or from less tagging sites by using different lanthanide chelating tags (Fig. 2). Intersection of the isosurfaces of two anisotropy tensors lead to a remaining curve (Fig. 2B). Intersection of this curve with a third isosurface leads to two points as remaining possibilities for the localization of the ligand (Fig. 2C). To determine the position exclusively in a strict mathematic view, a fourth isosurface is needed (Fig. 2D). However, in practice only three tensors suffice, since the remaining other possibility for the position of the ligand can usually be excluded due to chemical and structural reasons, i.e. the remaining "ghost site" lies in a very dense region or completely outside of the investigated protein.
With the information about the anisotropy tensors in hand, compounds can then be screened and their position localized by measuring only one-dimensional 19 F NMR spectra. We envisioned to introduce ve serine to cysteine mutations suitable to serve as tagging sites into hCA II and express the different mutants in different labelling schemes, i.e. uniformly 15 N labelled, selectively 15 N leucine labelled, as well as one mutant 2 H 13 C 15 N. Whereas the 15 N labelled sample can be used to record 1 H-15 N HSQC spectra for determination of the pseudocontact shis needed for the determination of the anisotropy tensors, selectively 15 N labelled samples are a convenient starting point for the evaluation of the anisotropy tensors caused by lanthanide chelating tags. Leucine stands out as an ideal amino acid type for selective labelling, since it is the most abundant amino acid in proteins and the selective labelling of recombinant proteins is straightforward and inexpensive. For hCA II, the 26 Leu residues (10%) show a favourable distribution in the primary sequence, as well as in 1 H-15 N spectra. The triple labelled mutant was expressed for backbone assignment and a diamagnetic lutetium tag was attached to the protein in order to avoid ambiguities in the assignment caused by a small number of residues that shi upon tagging. Upon expression, tagging and determination of the anisotropy tensors, uorine pseudocontact shis can then be analysed in order to localize the selected ligands within the protein.
In order to obtain sufficiently large PCS for the localization of ligands in a protein with a molecular weight and size in the range of hCA II (diameter: 40-56Å, molecular weight: 30 kDa), 39 sufficiently rigid tags have to be used. Lanthanide chelating tags based on a sterically overcrowded DOTA-M8 scaffold 11 as well as the recently reported M7PyThiazole-DOTA 21 provided suitable tools for our intent.
By application of the envisioned methodology, we demonstrate that the position of ligands within a protein can be determined solely by measurement of one-dimensional 19 F NMR experiments and analysis of the obtained 19 F PCS over a distance range of 22-38Å. Human carbonic anhydrase II is expressed in three different labelling schemes for ve different single-cysteine constructs and three ligands are tested. During the drug candidate screening process, the presented method signicantly reduces NMR measurement times compared to NOE-based approaches and eliminates the need for isotope labelled protein as well as chemical modication of the ligand. Furthermore, we observe differences in the solution structure of one ligand (F2-Inh) when compared to the crystal structure and establish an angle score as a measure of the suitability of chosen anisotropy tensors.

Materials and methods
Extensive protocols of the expression of uniformly 15 N labelled hCA II mutants, selectively 15 N leucine labelled hCA II mutants, uniformly 2 H 13 C 15 N labelled hCA II mutants, tagging reactions, NMR experiments and pseudocontact shi analysis are available in the ESI. †

Sample preparation
To a protein sample, 1.1 eq. of ligand dissolved in deuterated dimethyl sulphoxide (DMSO-d 6 ) were added and the sample was stirred well. The excess of ligand was removed by ultraltration using 4 ml Amicon ultraltration tubes (MW cut-off 10 kDa). Triuoroacetate as internal standard for uorine chemical shi  calibration as well as 5% v/v D 2 O were added and the pH was adjusted to 6.80.

NMR experiments
A 1 H-15 N-HSQC experiment reported by Grzesiek et al. 40 with a pulse sequence including a water suppression scheme using selective water ip-back pulses and gradients to suppress radiation damping was used for the determination of the PCS, since it does not cause the small additional shi due to dipolar coupling (rdc) introduced in TROSY-1 H 15 N-HSQC spectra. For backbone assignment of uniformly 2 H 13 C 15 N labelled hCA II S50C Lu-DOTA-M8 and all spectra recorded for protein characterization, TROSY 41 variants of HSQC, HNCO, HNCA, HN(CO) CA, HN(CA)CO and HNCACB experiments incorporating a WATERGATE scheme were used. 42-44 NMR spectra were recorded on Bruker Avance III or Avance III HD spectrometers operating at 600.13 MHz proton frequency, equipped with 5 mm BBFO, 1 H/ 13 C/ 15 N TXI or 1 H/ 19 F-13 C/ 15 N-D QCI-F probe heads, all tted with z-axis pulsed eld gradients.

Results and discussion
In order to ensure a rigid and stable attachment point for the Ln-DOTA-M8-SSPy and Ln-M7PyThiazole-DOTA complexes without compromising the global fold of hCA II, ve solvent exposed serine residues in regions of the protein with rigid secondary structure were selected and mutated to cysteine (Fig. 3). The native cysteine residue 206 was replaced by a serine for all constructs to avoid any interference. Double mutants containing this modication, e.g. hCA II S50C C206S, are abbreviated in the following, e.g. as hCA II S50C.
Although an NMR assignment for the wild type protein is available, 45 a backbone assignment of uniformly 2 H 13 C 15 N labelled hCA II S50C Lu-DOTA-M8 was performed to have an unambiguous assignment including the shied residues close to the tagging site. In order to provide a convenient starting point for the PCS assignment, selectively 15 N leucine labelled hCA II mutants were expressed. Having selectively 15 N leucine labelled protein and an assignment of uniformly labelled hCA II tagged with Lu-DOTA-M8 for our purpose in hand, more than eight shied peaks were readily assigned for each mutant from an overlay of the 1 H-15 N HSQC spectra of diamagnetic Lu-DOTA-M8-SSPy and paramagnetic Tm-DOTA-M8-SSPy attached to selectively 15 N leucine labelled hCA II (Fig. 4). Based on the assigned PCS for the leucine residues, the remaining PCS can be back-calculated using the initially tted anisotropy tensor. Although it is possible to start the analysis of the PCS from the fully labelled protein (in the less crowded regions of the spectrum), this method simplies considerably the analysis of the spectra. The mutant hCA II S173C was excluded from further analysis due to the appearance of two sets of signals in the HSQC spectrum of the thulium tagged construct. These two sets arise most likely from two equally populated positions of the tag molecule when attached to the protein, since residue 173 is located on the edge of a beta sheet ( Fig. 9 and 10 in ESI †).
Parameters describing the magnetic susceptibility tensor of the lanthanide metal were then determined from this initial set of PCS using the program Numbat and the X-ray structure of hCA II (PDB code: 3KS3). 46,47 The initially obtained tensor set allowed to back-calculate the expected PCS for the remaining leucine residues. Upon unambiguous assignment of all remaining shied peaks apart from those leucine residues in a distance smaller than 10.9Å from the tag, where the signals were either broadened beyond the limit of detection due to PRE or shied outside of the spectral window, rened tensor parameters were then calculated from this larger set of PCS.
Subsequently, a complete assignment and evaluation of the magnetic susceptibility tensors using all detectable resonances was performed (Fig. 5). The rened tensors for the uniformly labelled hCA II coincided within 20% with the initial tensors of the 15 N selectively leucine labelled protein constructs. Table 1 lists the rened tensor parameters obtained for the different protein mutants and used Monte-Carlo methods. All metal coordinates were located in a distance range of 6-8Å to the g-oxygen of the serine residue at the position where the  corresponding sulphur atom of the protein mutant is expected and the obtained Q-factors for the ts of 3.5-10.6% were excellent. For the mutants S50C, S217C and S220C similar values for Dc ax were found, whereas this value was almost twice as large for the S166C mutant indicating a lower exibility of the tag when attached to the protein. 48 This was also reected in the obtained pseudocontact shis, where signicantly larger shis were found for the S166C mutant. An extensive protocol of the PCS assignment and tting procedure applied is given in the ESI. † In order to have a further anisotropy tensor at hand, Luand Tm-M7PyThiazole-DOTA were attached to selectively 15 N leucine labelled hCA II S166C and the anisotropy tensor of the magnetic susceptibility was determined ( Table 1). The additional lanthanide chelating tag was introduced, since it was expected to be orthogonal to the already investigated constructs and we wanted to demonstrate that successful localization of the ligand is possible with a combination of tensors from uniformly and selectively leucine labelled constructs.
In order to test the newly developed approach for the localization of ligands within a protein of interest, three sulphonamide ligands were added to the protein as a dimethyl sulphoxide (DMSO) solution in small excess of 1.1 eq. to ensure saturation of the protein with ligand. Complete loading of the protein with ligand was then conrmed by 1 H-15 N HSQC spectra. Due to the high affinity of phenyl-sulphonamide based ligands, 38,39 the excess ligand could then be removed by ultra-ltration. Chemical shi changes upon ligand binding were only observed for the residues in the binding pocket of the protein, which indicates that the overall structure of the protein did not change upon binding to the ligand. 19 F PCS in the range of À0.078 to À0.409 could be observed for the ve investigated hCA II constructs (Fig. 6, 6 and 7 in   50 it can be concluded that the aromatic ring of the ligands used in this study pointing out of the enzyme's pocket still exhibits residual mobility. The largest PCS using Tm-DOTA-M8-SSPy was observed for the hCA II S166C mutant, caused by the lower mobility of the tag with respect to the protein. 48 The observed PCSs of a given value are found on an isosurface dened as follows: Dc rh sin 2 q cos 2f ! whereas d PCS is an experimentally determined PCS, Dc ax and Dc rh are the axial and rhombic components of the magnetic susceptibility tensor. The solutions for a given PCS and Dc ax and Dc rh of this equation for the polar coordinates r, q and 4, deliver the mentioned isosurface. The position and orientation of this isosurface within a reference coordinate system is determined by three Euclidean coordinates of the corresponding lanthanide metal and three Euler angles (a, b, g) given in the ZYZ 0 convention. 46 According to these considerations, when four backcalculated PCSs of different tensors are available, the position of a specic nucleus can be determined by the method of least squares. A sum of square residuals was dened as follows: where i is the index of a corresponding tensor and n is the total number of individual tensors available. d PCS i (experiment) is the experimental PCS for the analyzed tensors and d PCS i ðx 0 i ; y 0 i ; z 0 i Þ is dened as follows: i Þ are the coordinates x, y, z transformed into the according tensor frame. This transformation is carried out by translation of the reference coordinate system to the position of the corresponding metal center and rotation of the coordinate system by the three Euler angles (a, b, g) which are given for each tensor. Dc ax,i , Dc rh,i and r i correspond to the axial and rhombic tensor components and the distance between lanthanide metal and the observed position (x, y, z) respectively.
The optimized position of the uorine atoms of the used ligands was then determined by minimization of the target function s(x, y, z) dened above using the SciPy library. 51 Error analysis of the determined uorine position was performed applying a Monte-Carlo protocol where the tensor parameters were varied for every iteration according to the uncertainties determined in Numbat. For each uorine position, 10 000 iterations were carried out with a random seed in order to ensure comparability. The resulting values and uncertainties were the average and standard deviation of these 10 000 iterations.
Interestingly, upon analysis of the obtained pseudocontact shis and calculation of the position of the ligand, we unambiguously and successfully localized all three inhibitors within the protein using uorine pseudocontact shi restraints over a distance range of 22-38Å. Since the achieved distances for the localization of a ligand within a protein using lanthanide Fig. 6 One-dimensional 19 F NMR spectra (565 MHz, 298 K) of F2-Inh in the presence of different hCA II constructs. Spectra of diamagnetic hCA II constructs with F2-Inh did not show any differences, therefore only one diamagnetic reference spectrum is displayed. 19 F chemical shift was calibrated to an internal signal of trifluoroacetate at À79.0 ppm. The back-calculated positions for the uorine atoms of FM-519 and FM-520 show a deviation of 3.3 and 0.8Å when compared to the closely related X-ray structure of the penta-uoro derivate (PDB 1G54 (ref. 50)). Both the graphical analysis using the tensor isosurfaces for a given PCS value as well as the performed Monte-Carlo simulations yield similar positions for the uorine atoms ( Fig. 7 and 8, Table 2).
The results obtained for FM-519 and FM-520 show that the method is applicable for the localization of ligands of a protein with a reasonable precision by only acquiring one-dimensional 19 F spectra and analysis of the obtained pseudocontact shis.
Compared to the X-ray structure (PDB 1G52 (ref. 50)), the determined uorine positions for F2-Inh differed by 1.6Å for the uorine atom in meta position of the diuorophenyl substituent and by 2.6Å for the uorine atom in ortho position (Fig. 9). Interestingly, this result is only obtained when the diuorophenyl substituent of the ligand is rotated by 157 in a way that it aligns in a nearly perpendicular fashion with the neighbouring phenylalanine ring of the residue F131. Supported by MP2 Pos. dev. obtained using combinations with angle score < 30 (Å) calculations that investigated the inuence of the uorine substitution pattern of the diuorophenyl substituent on the interactions with a benzene ring and were published in a previous study 52 (ref. 52, page 2, motif 1b), we propose that in the solution state structure of F2-Inh the diuorophenyl substituent adopts the described position (Fig. 9).
Notably, the intramolecular uorine-uorine distance in F2-Inh was determined as 3.5Å, reproducing the distance obtained in the X-ray structure (2.8Å) with an accuracy of 0.7Å. This result corroborates the high accuracy of the method presented in this work. Upon primary localization of the uorine-containing ligand within the protein, further optimization of the position could be performed using protein-ligand docking soware.
In order to investigate the precision and accuracy of the graphical analysis of the presented method by using three isosurfaces, we iterated through all possible combinations of three isosurfaces for the measured PCS for FM-520. Using a Python script, the normal vectors at the intersection point were determined and their intersection angle was extracted.
The closer the obtained angle matches 90 , the more precisely the position of a uorine atom is dened at the intersection point. We then added up the obtained normalized differences to 90 of the different pairs of the normal vectors to get an angle score. An angle score of 0 means perfectly orthogonal intersections, while 90 results from parallel isosurfaces. The positions determined from three isosurfaces with an angle score below 30 closely matches the determined position using Monte-Carlo protocols with 4 tensors (orange spheres in Fig. 10). For three isosurfaces with an unfavourable angle score of 40 we found a signicant deviation of 10Å from the above position (red sphere in Fig. 10). However, the achieved accuracy would still be high enough to discriminate e.g. two distant binding sites in a protein.
In summary, the results for FM-519, FM-520 and F2-Inh show that it is possible to unambiguously localize a ligand approaching an accuracy of 0.8Å based solely on the uorine pseudocontact shi caused by lanthanide chelating tags by measuring 4-5 data points per ligand obtained from onedimensional 19 F spectra, which provide one unique solution for the position the ligand. Interestingly, for F2-Inh the results obtained with our method suggest differences of the solution state and crystal state structure. The graphical analysis using only three tensors shows a precision that is sufficient to discriminate two binding sites within a protein. When only combinations are taken into account showing an angle score below 30 , the obtained accuracy allows a clear localization of the uorine atom and the position matches the one determined in Monte-Carlo protocols using 4 tensors. By omitting the need for solvent suppression, chemical modication of the ligand and extensive measurement times during the screening process, the method constitutes a fast, reliable and convenient approach to screen a high number of uorine-containing ligands for a specic protein of interest.

Conclusions
To conclude, it was demonstrated for the rst time that the position of ligands within a protein can be determined solely by measurement of one-dimensional 19 F NMR experiments and analysis of the obtained 19 F PCS over a distance range of 22-38Å in a protein of a molecular weight of 30 kDa. Human carbonic anhydrase II was expressed in different labelling schemes for ve different single-cysteine constructs and three ligands were tested. During the drug candidate screening process, the presented method drastically reduces NMR measurement times compared to NOE experiments and eliminates the need for isotope labelled protein as well as labour intensive chemical modication of the ligand. Due to the large PCS observed over a distance range of 22-38Å, the method could be extended to proteins signicantly larger than 30 kDa, allows for the investigation of deeply buried ligand binding sites, avoids interference of the tag with the ligand  interaction site and renders monitoring of ternary or quaternary protein ligand complexes feasible.

Conflicts of interest
There are no conicts to declare.