NOEs between backbone amides and the Ha and Hb protons of histidine side chain were
measured using a 3D NOESY-15N-HSQC spectrum. Figure 2.3 shows (F2, F3) slices taken from the 3D NOESY-15N-HSQC and 3D HNHB spectra of ubiquitin 24H/28H at the 15N chemical shift frequencies of residues 24 and 28. Both strips show correlations to two β- protons, the intensities of which are proportional to the magnitude of the 3J(NHβ) couplings. The histidine residue at position 28 showed resolved cross-peaks for both Hbprotons in the
HNHB, indicating similar 3J(NHβ) couplings, and the Hb protons showed similar NOE
intensities with the HN of residue 28 and similar cross-peak intensities in the HNHB spectrum.” This suggests a c1 angle of 180o. The results for His24 were inconsistent, as the same Hbproton (Hb1 in Figure 2.3) appears to show a large 3J(NHβ) coupling, but at the same time also appears to be close to the HN atom of residues 24 and 25. This can be explained by chemical shift degeneracy of Hb1 and Hb2 of His24, i.e. the assignment of His24 Hb2 in Figure 2.3 may be wrong. This interpretation is supported by the absence of the expected Hb1–Hb2 cross-peak in a DQF-COSY spectrum. In this situation, the c1 angle cannot be determined from J-couplings.
51 Figure 2.3 Selected spectra regions from the HNHB (in red) and 3D NOESY-15N-HSQC (in blue) spectra of a 0.6 mM solution of uniformly 15N-labelled ubiquitin E24H/A28H taken at the (F2, F3) = (15N, 1HN) resonance frequencies of H24 and H28 and neighbouring residues.
To determine the side-chain orientation of His24, the E24H/A28H/S57C mutant was used to attach a paramagnetic C1-Tm3+ tag and measure PCSs of the Hd2 and He1 protons of the histidine imidazole rings. The ubiquitin S57C mutant labelled with the C1 tag has previously been shown to deliver relatively large PCSs and Dc tensors of good quality [14]. The attachment site was expected to deliver measurable PCSs for His24 without generating excessive line-broadening due to PREs. 15N-labelled protein samples were ligated with the C1 tag loaded with either Tm3+ or Y3+. A total of 42 PCSs were measured for the backbone amide protons from 2D [15N,1H]-HSQC spectra. The PCSs are reported in Table 1 and cross-peaks showing significant PCSs are assigned in Figure 2.4.
52 Figure 2.4 Superimpositions of [15N,1H]-HSQC spectra of a 0.3 mM solution of uniformly 15N-labelled ubiquitin E24H/A28H/S57C in NMR buffer (20 mM sodium phosphate, pH 6.5) at 25 oC. The protein was labelled with a C1 tag loaded with either Y3+ (diamagnetic reference, red cross-peaks) or Tm3+ (paramagnetic spectrum, blue cross-peaks). Assignments are shown for cross-peaks of backbone amides with significant PCSs.
Table 2.3 Experimental PCSs (in ppm) of the backbone amide protons of ubiquitin E24H/A28H/S57C with C1 tags.a
Res. no. Res. type PCS/ppm
3 Ile 0.000 4 Phe 0.084 5 Val 0.090 6 Lys 0.119 7 Thr 0.077 8 Leu 0.086 9 Thr 0.058 10 Gly 0.054 11 Lys 0.050 13 Ile 0.060 15 Leu 0.020 18 Glu 0.114 20 Ser 0.260 23 Ile 0.931 24 His 0.548 26 Val 0.392 27 Lys 0.306 29 Lys 0.178 33 Lys 0.072 34 Glu 0.072 35 Gly 0.068 36 Ile 0.083 39 Asp 0.126 40 Gln 0.124
53 41 Gln 0.157 42 Arg 0.186 43 Leu 0.305 44 Ile 0.282 45 Phe 0.408 46 Ala 0.267 47 Gly 0.237 49 Gln 0.342 50 Leu 0.460 51 Glu 0.762 53 Gly 0.591 64 Glu -0.156 65 Ser -0.060 66 Thr 0.092 67 Leu 0.177 68 His 0.235 70 Val 0.172 76 Gly 0.066
a PCSs measured as the difference in chemical shifts observed between samples tagged with C1-Tm3+ and C1-Y3+, respectively.
The PCSs measured from the resolved cross-peaks were used to fit the Dc tensor to the published coordinates of ubiquitin (PDB ID: 1D3Z [16]) using the program Numbat [17], which also allowed prediction of all remaining PCSs. The Dc-tensor parameters are listed in Table 2.4. PCS isosurfaces are depicted in Figure 2.5a and show that the Dc-tensor fit indeed located the metal ion at the expected position near residue 57. Figure 2.5b shows an excellent correlation between back-calculated and experimental PCSs.
54 Figure 2.5 (a) PCS isosurfaces representing the Dc tensor obtained with the C1-Tm3+ tag attached at position 57 of ubiquitin E24H/A28H/S57C. The isosurfaces correspond to PCSs of -1 ppm (dark blue), -0.1 ppm (light blue), 1 ppm (dark red) and 0.1 ppm (light red), and are plotted on the NMR structure of ubiquitin (PDB ID: 1D3Z [16]). The backbone of the protein is drawn in a ribbon representation (cyan) and the histidine side chains of the double-His motif are indicated by sticks. (b) Correlation between back-calculated and experimental PCSs of backbone amide protons.
Table 2.4Dc-tensor parameters of ubiquitin E24H/A28H/S57C fitted to the structure 1D3Z [16] using the PCSs measured with C1 tag loaded with Tm3+.a
Dcaxb Dcrhb Metal coordinatesc Euler angles (degrees)c Q- factord
x y z a b g
7.8 2.3 64.581 -81.567 14.087 133 145 35 0.03
a The program Numbat [17] was used to fit the Dc tensor. The fit used the first conformer of 1D3Z to determine the Dc tensor. The experimental PCSs are reported in Table 2.3.
b In units of 10-32 m3.
c Relative to the coordinates of the first conformer reported in the PDB file 1D3Z. For comparison, fitting the Dc tensor using amide proton PCSs reported for ubiquitin S57C with C1-Tb3+ tag [14] yielded the metal x, y and z coordinates as 66.367, -81.238 and 11.724, respectively.
d Calculated as the ratio of the root mean-square deviation between experimental and back- calculated PCSs.
55 To assign the resonances of the Hd2 and He1 protons of the histidine residues, a [13C,1H]- HSQC spectrum was recorded of a ubiquitin E24H/A28H/S57C sample selectively labelled with 15N/13C-histidine, which resulted in six cross-peaks with 13C-chemical shifts near 135 and 118 ppm, which were also observed in the [13C,1H]-HSQC spectrum of the corresponding uniformly 15N/13C-labelled sample (Figure 2.6a). Besides the histidine residues in positions 24 and 28, the samples also contained the natural residue His68. The Hd2 and He1 resonances of each histidine residue were linked by cross-peaks in the TOCSY-relayed-[13C,1H]-HSQC spectrum of Figure 2.6b and connected to corresponding backbone amide protons by cross- peaks observed in DQF-COSY and 2D-NOESY spectra.
PCSs of the histidine Hd2 and He1 protons were measured by recording [13C,1H]-HSQC spectra of uniformly 13C-labelled protein labelled with C1 tag at position 57, where the tag contained either Y3+ (diamagnetic reference) or Tm3+ (paramagnetic spectrum) (Figure 2.7). The PCS values are reported in Table 2.5.
Figure 2.6 [13C,1H]-HSQC and TOCSY-relayed [13C,1H]-HSQC spectra recorded of a 0.3 mM solution of uniformly 15N/13C-labelled ubiquitin E24H/A28H/S57C labelled with C1-Y3+ tag. The six cross-peaks corresponding to the Hd2 and He1 protons of the imidazole rings in the dHis motif and the natural histidine in position 68 are identified by circles.
56 Figure 2.7 PCS measurements of the histidine Hd2 and He1 protons. The figure shows superimpositions of [13C,1H]-HSQC spectra of a 0.3 mM solution of uniformly 13C-labelled ubiquitin E24H/A28H/S57C tagged with C1 loaded with either Y3+ (diamagnetic reference, red cross-peaks) or Tm3+ (paramagnetic spectrum, blue cross-peaks). The spectra were recorded in NMR buffer (20 mM sodium phosphate, pH 6.5) at 25 oC.
Table 2.5 PCS values of Hd2 and He1 measured for the histidine residues of ubiquitin E24H/A28H/S57C with C1-Tm3+ tag attached to Cys57.a
Residue no. Hd2 He1
24 0.241 0.166
28 0.141 0.096
68 0.145 0.099
a PCSs measured in ppm.
Using the Dc tensor determined from the PCSs of backbone amide protons (Tables 2.3 and 2.4), the PCSs of all other nuclear spins in the protein can be predicted, including the Hd2 and He1 protons of the histidine residues of the dHis motif for different side chain conformations. To this end, a series of PDB files was prepared, in which the c1 and c2 angles
57 of the histidine residues were changed systematically. Figure 2.8 shows the differences between predicted and experimental PCSs as a function of c1 and c2 angles for the histidine residues in positions 24 and 28.
For residue 24, varying the c1 angle while keeping c2 = -90o or 90o, the best match between predicted and experimental PCSs was obtained for c1 = 180o. For residue 28, c1 angles of 180o, -120o and 120o showed similarly good matches. Only c1 = 180o corresponds to a staggered conformation and this dihedral angle was also predicted from the coupling constant and NOE analysis (Figure 2.3). Predicting the PCSs for different c2 angles did not significantly improve the match (Figure 2.8c).
Figure 2.8 Sum of PCS deviations for the Hd2 and He1 protons of His24 and His28 in ubiquitin E24H/A28H/S57C. (a) PCS deviations for different c1 angles of His24, while keeping c2 = - 90o. (b) PCS deviations for different c1 angles of His28, while keeping c2 = -90o. (c) PCS deviations for different c2 angles of His28, with c1 set to 180o, -120o and 120o, respectively.
The modelled dHis-Co2+ motif metal position between histidines side chain that was achieved by modelling and is discussed in section 1 of this chapter, only suggested one conformation is possible when the position of the Co2+ ion is constrained to the planes of both imidazole rings with dihedral angles c1 near -180 and -60 for the histidine residues in positions i and i+4, respectively (Figure 2.9b).
In conclusion, histidines in absence of metal ion are not facing each other and immobilization of cobalt required conformational changes in histidine 28 to provide a binding site with µM affinity.
58 Figure 2.9 Side-chain conformations of the histidine residues in the a-helical dHis motif of ubiquitin E24H/A28H (a) before and (b) after binding to cobalt. The histidine residue in position His24 has a c1 angle of 180o irrespective of the presence or absence of cobalt, whereas His28 has to change from c1 = 180o (trans) to c1 = 60o (gauche-) in order to form the dHis- Co2+ motif.