NMR Evidence for Unusual Bifurcated Hydrogen Bonding in the TXXH Alpha-Helix N-capping Motif

Abstract

Hydrogen bonds (H-bonds) are generally weak (< 20 kJ/mol) but ubiquitous interactions that determine in large part the cooperative folding and selfassembly of biological macromolecules in aqueous solution. Although important, most H-bonds are inferred indirectly and modeled using theoretical idealized geometry, therefore leaving instances of bifurcated or strained H-bonds undetected. Here, we present direct solution NMR evidence for an unusual bifurcated H-bonding arrangement comprising threonine Og-H and N-H donor protons and histidine acceptor Nd1 within the conserved TXXH alpha-helix N-capping motif of consensus ankyrin repeat (AR) proteins. The threonine N-H/Nd1 histidine H-bonds were detected in the TXXH motif of three and four-repeat AR proteins via 2h J NN hydrogen bond scalar coupling (HBC) NMR correlation experiments. Comparison of the 2h J NN couplings and histidine pK a values demonstrated that the N-H/Nd1 H-bonds of internal AR ( 2h J NN~4 Hz, pK a < 3) were stronger than those of the solvent-exposed C-terminal AR ( 2h J NN~2 Hz, pK a~5 .7). In addition to the N-H/Nd1 2h J NN couplings, we also observed distinct 1h J HN HBCs connecting the threonine Og-H and histidine Nd1 within the TXXH motif of internal ARs. Together, the 2h J NN and 1h J HN HBCs supported that a single histidine Nd1 acceptor had two H-bond donors. Accordingly, two-bond reciprocal isotope shifts were detected between the threonine N-H and Og-H protons in solvents with mixed H 2 O/D 2 O composition. Finally, a threonine to valine replacement, which eliminated the Og-H/Nd1 H-bond and resulted in a destabilized protein, lead to a relaxed valine N-H/Nd1 histidine interaction with enhanced 2h J NN (~5.2 Hz) likely owing to an optimized bonding geometry. Overall, our results provide new insight into the H-bonding ability of histidine and a challenging benchmark for the calculation of HBCs and their relation to H-bond energetics. Oxidation of methionine disrupts the structure and function of a range of proteins, but little is understood about the chemistry that underlies these perturbations. We show that methionine oxidation dramatically increases the strength of the methionine-aromatic interaction motif, a driving force for protein folding and protein-protein interaction that has emerged in recent years. We have performed a structural bioinformatics analysis of the PDB and CSD and found that interactions between DMSO (a small molecule analog of methionine sulfoxide (MetOx)) and aromatic groups are prevalent, even compared to methioninearomatic interactions. We then performed quantum mechanical calculations and found that DMSO-aromatic interactions are substantially strengthened compared to DMS-aromatic, especially in the cases of tyrosine and tryptophan which are able to donate a hydrogen bond to the sulfonyl oxygen. The relative bond energies correlate roughly with the relative abundance of phenylalaninetyrosine-and tryptophan-DMSO interactions found in the PDB. Through protein-stability experiments we show that oxidation of methionine w...