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.

A collection of programs for one-dimensional Ising analysis of linear repeat proteins with point substitutions

“…Comparing DGX with DGR reveals that intrinsic stability is decreased by 1.6 kcal mol -1 . This is consistent with the loss of an internal hydrogen bond between the threonine (Og)H and the i+3 histidine sidechain Nd1 (Preimesberger et al, 2015). This hydrogen bond is part of a hydrogen bond network that includes a second, bifurcated hydrogen bond from the i+3 histidine Nd1 to the threonine main-chain (N)H, and from the i+3 histidine Ne2 (N)H to the main-chain C=O from the residue preceding the threonine in the next repeat (asparagine i+32; Figure 5A).…”

“…To illustrate how the Ising fitting programs perform with a more complex heteropolymeric model and to examine the effects of a mutation on intrinsic and interfacial stabilities, we performed Ising fitting and bootstrap analysis on folding transitions of a series of capped consensus ankyrin repeats in which a conserved threonine from one or two R repeats is substituted with a valine at position 7 in the ankyrin TPLH motif (referred to here as T7V). The T7V substitution has previously been shown to destabilize a fourrepeat NXRC construct (where repeat X is an R-type repeat that harbors the point substitution) by 2.6 kcal mol -1 using a simple two-state model (Preimesberger et al, 2015). By combining the X-substitution series ( Figure 1B) with the homopolymeric capped constructs analyzed with model 1 above, 11 we were able to fit a model for heteropolymeric capped repeats containing ten thermodynamic parameters (model 2).…”

“…Rather, at least in the examples shown here, high parameter uncertainty appears to mask correlation, whereas low parameter uncertainty reveals correlation. Mosavi et al, 2002); note that the histidine ring has been flipped by 180° around c2 to match the bonding pattern determined by Preimesberger et al, 2015). Threonine at position 7 (yellow) forms a bifurcated hydrogen bond to the Nd1 of histidine 10 via its main-chain NH and side-chain OH within the same repeat.…”

“…Comparison of DGR-1,X and DGX-1,R with DGR-1,R shows that the N-terminal interface is destabilized by valine substitution by +1.0 kcal mol -1 , but that this stabilization is partly compensated by a stabilization of the C-terminal interface by -0.4 kcal mol -1 ( Figure 5A). The modest stabilization of the C-terminal interface may reflect a re-orientation of the i+3 histidine side-chain in the substituted repeat to optimize the two remaining hydrogen bonds (Preimesberger et al, 2015).…”

The Heteronuclear Multiple-Quantum Correlation Experiment: Perspective from Classical Vectors, Nonclassical Vectors, and Product Operators

“…Nuclear magnetic resonance (NMR) has become an indispensable technique in diverse fields such as chemistry [1][2][3][4], biochemistry [5][6][7][8], structural biology [9][10][11][12], materials science [13][14][15], and biomedicine [16][17][18]. However, the richness and complexity of NMR, joined to a vast literature, appear intimidating to novice users.…”