Design of heterotetrameric coiled coils: evidence for increased stabilization by Glu(-)-Lys(+) ion pair interactions.
Electrostatic interactions between charged amino acids often affect heterospecificity in coiled coils as evidenced by the interaction between the oncoproteins, fos and jun. Such interactions have been successfully exploited in the design of heteromeric coiled coils in a number of laboratories. It has been suggested that heterospecificity in these dimeric coiled-coil systems is driven not by specific electrostatic interactions in the heterodimers but rather by electrostatic repulsion acting to destabilize the homodimer state relative to the heterodimer state. We show that it is possible to design ion pair interactions that directly stabilize the heterotetrameric coiled-coil state. Synthetic peptides were used whose sequences are based on the C-terminal tetramerization domain of Lac repressor, as a model system for four-chain coiled coils (Fairman et al., 1995). These Lac-based peptides, containing either glutamic acid (Lac21E) or lysine (Lac21K) at all b and c heptad positions, only weakly self-associate but, when mixed, afford a highly stable heterotetramer. This study represents the first experimental evidence for the importance of the b and c heptad positions to the stability of coiled coils. Finally, pH dependence and NaCl dependence studies show that heterotetramer stability is driven by ion pair interactions between glutamate and lysine; these interactions contribute about 0.6 kcal/mol of stabilizing free energy for each potential glutamate-lysine pair.
Fairman
R
R
Division of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Chao
H G
HG
Lavoie
T B
TB
Villafranca
J J
JJ
Matsueda
G R
GR
Novotny
J
J
D000595:Amino Acid Sequence; D002151:Calorimetry; D003196:Computer Graphics; D004563:Electrochemistry; D018698:Glutamic Acid; D006358:Hot Temperature; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016759:Oncogene Protein p65(gag-jun) / Q000737:chemistry; D016761:Oncogene Proteins v-fos / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D046911:Macromolecular Substances; D016759:Oncogene Protein p65(gag-jun); D016761:Oncogene Proteins v-fos; D010455:Peptides; D018698:Glutamic Acid; D008239:Lysine