Interhelical Ion Pairing in Coiled Coils: Solution Structure of a Heterodimeric Leucine Zipper and Determination of pKa Values of Glu Side Chains,

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Abstract
Residues of opposite charge often populate heptad positions g (heptad i on chain 1) and e‘ (heptad i + 1 on chain 2) in dimeric coiled coils and may stabilize the dimer by formation of interchain ion pairs. To investigate the contribution to stability of such electrostatic interactions we have designed a disulfide-linked heterodimeric zipper (AB zipper) consisting of the acidic chain Ac-E−VAQLEKE−VAQAEAE−NYQLEQE−VAQLEHE−CG-NH2 and the basic chain Ac-E−VQALKKR−VQALKAR−NYAAKQK−VQALRHK−CG-NH2 in which all e and g positions are occupied by either E or K/R to form a maximum of seven interhelical salt bridges. Temperature-induced denaturation experiments monitored by circular dichroism reveal a stable coiled coil conformation below 50 °C and in the pH range 1.2−8.0. Stability is highest at pH ∼ 4.0 [ΔGU (37 °C) = 5.18 ± 0.51 kcal mol-1]. The solution structure of the AB zipper at pH 5.65 has been elucidated on the basis of homonuclear 1H NMR data collected at 800 MHz [heavy atom rmsd's for the ensemble of 50 calculated structures are 0.47 ± 0.13 Å (backbone) and 0.95 ± 0.16 Å (all)]. Both chains of the AB zipper are almost entirely in α-helical conformation and form a superhelix with a left-handed twist. Overhauser connectivities reveal close contacts between g position residues (heptad i on chain 1) and residues d/f (heptad i on chain 1), residues a/d (heptad i + 1 on chain 1), and residue a‘ (heptad i + 1 on chain 2). Residues in position e (heptad i on chain 1) are in contact with residues a/b/d/f (heptad i on chain 1) and residue d‘ (heptad i on chain 2). These connectivities hint at a relatively defined alignment of the side chains across the helix interface. Partial H-bond formation between the functional groups of residues g and e‘(+1) is observed in the calculated structures. NMR pH titration experiments disclose pKa values for Glu δ-carboxylate groups: 4.14 ± 0.02 (E1), 4.82 ± 0.07 (E6), 4.52 ± 0.01 (E8), 4.37 ± 0.03 (E13), 4.11 ± 0.02 (E15), 4.41 ± 0.07 (E20), 4.82 ± 0.03 (E22), 4.65 ± 0.04 (E27), 4.63 ± 0.03 (E29), 4.22 ± 0.02 (E1‘). By comparison with pKa of Glu in unfolded peptides (∼4.3 ± 0.1), our pKa data suggest marginal or even unfavorable contribution of charged Glu to the stability of the AB zipper. The electrostatic energy gained from interhelical ion pairs is likely to be surpassed by hydrophobic energy terms upon protonation of Glu, due to increased hydrophobicity of uncharged Glu and, thus, better packing against apolar residues at the chain interface.