Direct NMR detection of bifurcated hydrogen bonding in the α-helix N-caps of ankyrin repeat proteins.

Abstract

In biomolecules, bifurcated H-bonds typically involve the interaction of two donor protons with the two lone pairs of oxygen. Here, we present direct NMR evidence for a bifurcated H-bonding arrangement involving nitrogen as the acceptor atom. Specifically, the H-bond network comprises the Nδ1 atom of histidine and both the backbone N–H and side-chain Oγ-H of threonine within the conserved TXXH motif of ankyrin repeat (AR) proteins. Identification of the H-bonding partners is achieved via solution NMR H-bond scalar coupling (HBC) and H/D isotope shift experiments. Quantitative determination of 2hJNN HBCs supports that Thr N–H···Nδ1 His H-bonds within internal repeats are stronger (∼4 Hz) than in the solvent exposed C-terminal AR (∼2 Hz). In agreement, pKa values for the buried histidines bridging internal ARs are several units lower than those of the C-terminus. Quantum chemical calculations show that the relevant 2hJ and 1hJ couplings are dominated by the Fermi contact interaction. Finally, a Thr-to-Val replacement, which eliminates the Thr Oγ-H···Nδ1 His H-bond and decreases protein stability, results in a 25% increase in 2hJNN, attributed to optimization of the Val N–H···Nδ1 His H-bond. Overall, the results provide new insights into the H-bonding properties of histidine, a refined structural rationalization for the folding cooperativity of AR proteins, and a challenging benchmark for the calculation of HBCs.