Nuclear Magnetic Resonance Structures of GCN4p Are Largely Conserved When Ion Pairs Are Disrupted at Acidic pH but Show a Relaxation of the Coiled Coil Superhelix

Abstract

To understand the roles ion pairs play in stabilizing coiled coils, we determined nuclear magnetic resonance structures of GCN4p at three pH values. At pH 6.6, all acidic residues are fully charged; at pH 4.4, they are half-charged, and at pH 1.5, they are protonated and uncharged. The α-helix monomer and coiled coil structures of GCN4p are largely conserved, except for a loosening of the coiled coil quaternary structure with a decrease in pH. Differences going from neutral to acidic pH include (i) an unwinding of the coiled coil superhelix caused by the loss of interchain ion pair contacts, (ii) a small increase in the separation of the monomers in the dimer, (iii) a loosening of the knobs-into-holes packing motifs, and (iv) an increased separation between oppositely charged residues that participate in ion pairs at neutral pH. Chemical shifts (HN, N, C', Cα, and Cβ) of GCN4p display a seven-residue periodicity that is consistent with α-helical structure and is invariant with pH. By contrast, periodicity in hydrogen exchange rates at neutral pH is lost at acidic pH as the exchange mechanism moves into the EX1 regime. On the basis of H-N nuclear Overhauser effect relaxation measurements, the α-helix monomers experience only small increases in picosecond to nanosecond backbone dynamics at acidic pH. By contrast, C rotating frame T relaxation (T) data evince an increase in picosecond to nanosecond side-chain dynamics at lower pH, particularly for residues that stabilize the coiled coil dimerization interface through ion pairs. The results on the structure and dynamics of GCNp4 over a range of pH values help rationalize why a single structure at neutral pH poorly predicts the pH dependence of the unfolding stability of the coiled coil.

Conformational Dynamics of Asparagine at Coiled-Coil Interfaces

“…Indeed, the aforementioned pH titration of GCN4-p1 reveals a range of Asn side-chain conformations. 44 Related to this, the native dimeric state of GCN4-p1, which has a central Asn-at- a , forms trimers under certain conditions. 45 In this state, the Asn side chains point out from the hydrophobic core and water fills the space left behind.…”

“… 42 , 43 Nonetheless, pH titrations removing any formal electrostatic interactions result in subtle changes in the overall CC structure and supercoil. 44 …”

Structural characterization of the self‐association domain of swallow

“…(c) Wireframe representation of the lowest‐energy structure in the Swa DIMER ensemble with black lines indicating the location of the intermolecular NOEs used in the structure calculation to constrain the dimer structure. (d) Overlay of the lowest‐energy structure from the Swa DIMER structural ensemble (in green) with the leucine zipper region of GCN4 (in blue, PDB 5IEW 15 ) illustrating the “bulge” in the center of the Swa DIMER coiled‐coil…”

“…We were able to completely assign the 1 H, 13 C, and 15 N chemical shifts of all backbone and sidechain resonances for Swa DIMER with the exception of amino, hydroxyl, carboxyl, and guanidinium functional groups (due to fast exchange with the solvent), and phenylalanine/tyrosine γ-carbons and tyrosine ζ-carbons. In doing so, we corrected F I G U R E 1 Schematic domain structure of Swallow, model of its interaction with LC8, and helical wheel diagram for the selfassociation domain.…”

“…Previous examples of coiled‐coils studied using nuclear magnetic resonance (NMR) spectroscopy include monomers that form hairpins (Protein Data Bank 9 entries 2K48 10 and 2KE4 11 ), parallel homodimers (1JUN, 12,13 2GD7, 14 5IEW, 15 6N2M 16 ), antiparallel homodimers (1HF9, 17 2LW9, 18 2N9B, 19 and 6E4H 20 ), heterodimers (2A93 21 ), homodimers that form four‐helix bundles (2MAJ 22 ), homotrimers (2FXP, 23 2KP8, 24 and 2N64 25 ), and even computationally designed heterodimeric four‐helix bundles (6DMP 26 ). Swa DIMER is, to our knowledge, the longest coiled‐coil structure determined by NMR spectroscopy to date, albeit only slightly longer than the coiled‐coil structures in some previous studies 14,25 …”