Orientation, positional, additivity, and oligomerization-state effects of interhelical ion pairs in alpha-helical coiled-coils

J Mol Biol. 1998 Nov 13;283(5):993-1012. doi: 10.1006/jmbi.1998.2125.

Abstract

The role of interhelical g-e' ion pairs in the dimerization specificity and stability of alpha-helical coiled-coils is highly controversial. Synthetic 35-residue coiled-coils based on the heptad repeat QgVaGbAcLdQeK f were used to investigate the effect of orientation of interhelical ion pairs between lysine and glutamic acid residues on coiled-coil stability. Stability was estimated from urea denaturation at 20 degreesC, monitoring unfolding with circular-dichroism spectroscopy. Double mutant cycles were employed to estimate the net interaction energy, Delta DeltaGuint, for the two orientations of the ion pair; Ee-Kg and Ke-Eg. Delta DeltaGuint was found to be about 1.4-fold higher for the Ee-Kg orientation in a coiled-coil containing an N-terminal disulfide bridge. The Delta DeltaGuint value was similar whether obtained from the middle heptad or averaged over all five heptads of the coiled-coil, suggesting that ion pairs contribute additively to coiled-coil stability. The effect of uncompensated charges was also illustrated by single substitutions of Gln with either Lys or Glu, resulting in Lys-Gln or Glu-Gln g-e' pairs. These substitutions were found to be twice as destabilizing at position g as at position e, and Lys was about twice as destabilizing as Glu at both positions e and g. In the absence of an interhelical disulfide bridge, Glu and Lys substitutions in the middle heptad were equally destabilizing at positions e and g (Lys continued to be more destabilizing than Glu) and the Delta DeltaGuint value for Lys-Glu ion pairs was not orientation dependent. These and previous results suggest the non-covalently-linked synthetic coiled-coils behave as molten globules, whereas a disulfide-bridge may "lock in" the structural differences between positions of the heptad repeat. Interhelical Lys-Glu ion pairs in either orientation promoted the formation of trimeric coiled-coils (in the absence of a disulfide bridge) while Gln-Gln g-e' interactions led to dimer formation. The results support a role for g-e' ionic attractions in controlling coiled-coil specificity, stability and oligomerization state, possibly through effects on the side-chain packing at the subunit interface.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Drug Stability
  • Glutamic Acid / chemistry
  • Hydrogen-Ion Concentration
  • Ions
  • Lysine / chemistry
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Osmolar Concentration
  • Peptides / chemistry
  • Peptides / genetics
  • Protein Conformation
  • Protein Denaturation
  • Protein Structure, Secondary*
  • Protein Structure, Tertiary
  • Proteins / chemistry*
  • Proteins / genetics
  • Static Electricity
  • Thermodynamics

Substances

  • Ions
  • Peptides
  • Proteins
  • Glutamic Acid
  • Lysine