Direct NMR Detection of Bifurcated Hydrogen Bonding in the α‑Helix N‑Caps of Ankyrin Repeat Proteins

• Published in: Journal of the American Chemical Society 2015-01, Vol.137 (3), p.1008-1011
• Main Authors: Preimesberger, Matthew R, Majumdar, Ananya, Aksel, Tural, Sforza, Kevin, Lectka, Thomas, Barrick, Doug, Lecomte, Juliette T. J
• Format: Article
• Language: English
• Subjects: Ankyrin Repeat; Ankyrins - chemistry; Communication; Hydrogen Bonding; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Quantum Theory
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja510784g

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 2h J NN 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, pK a 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 2h J and 1h J 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 2h J NN, 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.