Anomalous amide proton chemical shifts as signatures of hydrogen bonding to aromatic sidechains

Abstract

Hydrogen bonding between an amide group and the p-$\pi$ cloud of an aromatic ring was first identified in a protein in the 1980s. Subsequent surveys of high-resolution X-ray crystal structures found multiple instances, but their preponderance was determined to be infrequent. Hydrogen atoms participating in a hydrogen bond to the p-$\pi$ cloud of an aromatic ring are expected to experience an upfield chemical shift arising from a shielding ring current shift. We surveyed the Biological Magnetic Resonance Data Bank for amide hydrogens exhibiting unusual shifts as well as corroborating nuclear Overhauser effects between the amide protons and ring protons. We found evidence that Trp residues are more likely to be involved in p-$\pi$ hydrogen bonds than other aromatic amino acids, whereas His residues are more likely to be involved in in-plane hydrogen bonds, with a ring nitrogen acting as the hydrogen acceptor. The p-$\pi$ hydrogen bonds may be more abundant than previously believed. The inclusion in NMR structure refinement protocols of shift effects in amide protons from aromatic sidechains, or explicit hydrogen bond restraints between amides and aromatic rings, could improve the local accuracy of sidechain orientations in solution NMR protein structures, but their impact on global accuracy is likely be limited.

Figure 1

Definition of the azimuthal angle ( $\mathit{\theta }$ ) and demarcation of regions of upfield and downfield ring current shifts. For protons above the plane of a Tyr or Phe ring the upfield shift can reach 1.5 ppm for distances from the ring center around 3 Å; for protons in the plane of the ring the downfield shift approaches 2 ppm at 3 Å. For Trp the effects can be significantly larger. Local mobility (e.g. fluctuations about the ${\mathit{\chi }}_{\mathrm{2}}$ sidechain dihedral angle of the aromatic residue) can substantially diminish ring current shifts.​​​​​​​

Figure 2

Manual federation of BMRB and PDB via a customized workflow.

Figure 3

The distribution of amide chemical shifts as a function of the distance of the amide proton from the center of the nearest aromatic ring.

Figure 4

Distribution of azimuth angles for outlier ( $>\mathrm{3}\mathit{\sigma }$ ) amide proton shifts. Upfield shifts are shown in the top row, downfield shifts in the bottom row.

Figure 5

Proportions of amide protons with at least one NOE restraint to an aromatic ring proton ( $y$ axis), as a function of the $Z$ score of the amide proton ( $x$ axis). Proportions are calculated with respect to the total number of amide hydrogens with chemical shifts reported in entries with at least one amide–aromatic restraint. The numbers over each point in panel (a) are the total number of such amides (including those lacking any NOE restraints to a nearby aromatic) with that $Z$ score. In panel (b), the restrained amide protons are further demarcated by the type of aromatic sidechain to which they are restrained.

Figure 6

Shown are the number of restrained amide–aromatic pairs (that is amide protons and aromatic rings with at least one defined restraint between them) for the four aromatic residue types and three $Z$ score classifications: upfield ( $Z\le -\mathrm{2}$ ), downfield ( $Z\ge \mathrm{2}$ ), and normal ( $-\mathrm{2}\le Z\le \mathrm{2}$ ). The colors of the bars correspond to the number of restraints between the pairs; bar heights are plotted using a logarithmic scale.

Figure 7

Examples of amide protons with extreme upfield shifts. (a, b) PDB:2MWH. The G93 amide proton is directly below the W23 aromatic ring ( $Z$   $=$   $-$ 7, ${\mathit{\delta }}_{\mathrm{GLY}}$   $=$  2.937 ppm, $d$   $=$  3.99 Å, $\mathit{\theta }$   $=$  43.9 ${}^{\circ }$ , ${\stackrel{\mathrm{‾}}{\mathit{\delta }}}_{\mathrm{GLY}}$   $=$  8.237 ppm, ${\mathit{\sigma }}_{\mathrm{GLY}}$   $=$  0.770 ppm). (c, d) PDB:2MWH. The G26 amide proton is directly below the W90 aromatic ring ( $Z$   $=$   $-$ 6.43, ${\mathit{\delta }}_{\mathrm{GLY}}$   $=$  3.38 ppm, $d$   $=$  2.98 Å, $\mathit{\theta }$   $=$  25.0 ${}^{\circ }$ , ${\stackrel{\mathrm{‾}}{\mathit{\delta }}}_{\mathrm{GLY}}$   $=$  8.327 ppm, ${\mathit{\sigma }}_{\mathrm{GLY}}$   $=$  0.770 ppm). The amide proton is represented as a yellow sphere, and the aromatic sidechain is shown in red.

Figure 8

Examples of amide protons with extreme downfield shifts. (a, b) PDB:2NCL. The D28 amide proton is near the plane of Y37 aromatic ring ( $Z$   $=$  5.21, ${\mathit{\delta }}_{\mathrm{ASP}}$   $=$  11.387 ppm, $d$   $=$  5.62 Å, $\mathit{\theta }$   $=$  72.0 ${}^{\circ }$ , ${\stackrel{\mathrm{‾}}{\mathit{\delta }}}_{\mathrm{ASP}}$   $=$  8.299 ppm, ${\mathit{\sigma }}_{\mathrm{ASP}}$   $=$  0.588 ppm). (c, d) PDB:2KKZ. The L61 amide proton forms a hydrogen bond with the sidechain nitrogen of H86 ( $Z$   $=$  6.66, ${\mathit{\delta }}_{\mathrm{LEU}}$   $=$  12.56 ppm, $d$   $=$  3.22 Å, $\mathit{\theta }$   $=$  69.7 ${}^{\circ }$ , ${\stackrel{\mathrm{‾}}{\mathit{\delta }}}_{\mathrm{LEU}}$   $=$  8.217 ppm, ${\mathit{\sigma }}_{\mathrm{LEU}}$   $=$  0.735 ppm). The amide proton is represented as a yellow sphere, and the aromatic sidechain is shown in red.

Figure 9

The van der Waals interaction energies for ALA approaching PHE with its amide N-H aligned with the ring normal. On the $x$ axis is the distance from the ALA nitrogen to the PHE ring center. VdW interaction energies for each distance were calculated by subtracting the VdW energies of ALA and PHE in isolation from the energies calculated at that distance from one another. All calculations were performed in MoSART using the AMBER99 force field.

Figure 10

Correlation of $Z$ scores with order parameters.

Figure 11

Trends in total BMRB structure depositions (blue), runs executed using the BMRB CS-Rosetta server (green), and depositions citing CS-Rosetta (red).

Figure 12

The distribution of amide chemical shifts for depositions citing CS-Rosetta as a function of distance from the center of the nearest ring (compare Fig. 3).