Seventy-six percent of the g
positions in the leucine zipper of human B-ZIP proteins contain one of four long-side-chain amino acids: the acidic glutamic acid (E), the basic arginine (R) or lysine (K), or the polar glutamine (Q) (2
). Changing the g
positions in two heptads of the PAR B-ZIP domain from charged amino acids to alanine resulted in the formation of tetramers instead of dimers (48
). Thus, charged amino acids in the g
positions inhibit the higher-order oligomerization that would occur if the g
positions formed a continuous hydrophobic surface with the a
Charged amino acids in the g
positions contribute to leucine zipper stability. The most common g↔e′
pair has glutamic acid (E) in the g
position and an oppositely charged arginine (R) or lysine (K) in the following e′
position (E↔R and E↔K). These oppositely charged amino acids produce a g↔e′
pair that contributes −1.3 (E↔R) or −0.9 (E↔K) kcal/mol/pair more energy to dimer stability than a reference alanine pair (A↔A) (47
). The g↔e′
pairs R↔R and K↔K are stabilizing, contributing −0.1 and −0.3 kcal/mol/pair, respectively, relative to the A↔A pair. Presumably the repulsive electrostatic energies between the like charges in arginine or lysine pairs are overcome by the favorable Van der Waals interactions between the methylenes of arginine or lysine side chains and hydrophobic amino acids in the a
). E↔E is the only pair less stable than A↔A, contributing +0.4 kcal/mol/pair.
In addition to affecting stability, charged amino acids in the g
positions also regulate dimerization specificity (2
). In the analysis of stability presented above, we considered the stability of a charged g↔e′
pair relative to an A↔A pair without addressing any potential energetic interaction between the amino acids in the g↔e′
pair; the contribution of the individual E or R side chain to stability can be determined by examining the E↔A and A↔R pairs. Any excess stability conferred by the E↔R pair is due to the energetic interaction between E and R, termed the “coupling energy.” This can be calculated by using a double-mutant thermodynamic cycle (31
) that involves the analysis of four proteins. This idea is presented graphically in Fig. . For example, the coupling energy of the E↔R pair is derived from a comparison of a protein containing the E↔R pairs with three additional proteins mutated to contain the pairs A↔R, E↔A, and A↔A (47
). The levels of stability of the pairs relative to A↔A are as follows: E↔R, −1.3 kcal/mol; E↔A, −0.1 kcal/mol; and A↔R, −0.7 kcal/mol. The additional energy of the E↔R pair compared to the A↔R and E↔A pairs is −0.5 kcal/mol/salt-bridge [E↔R − (A↔R + E↔A) = coupling energy] [−1.3 − (−0.7 + −0.1) = −0.5] and represents the energy of interaction (coupling energy) between E and R. Table lists the stability of each g↔e′
pair relative to A↔A, and Table gives their coupling energy.
FIG. 5. Schematic describing the four proteins used for a double-mutant thermodynamic cycle. The top panel depicts two alanines in the g and e′ positions of a g↔e′. The second panel shows that an E↔A pair is 0.1 kcal/mol more stable (more ...)
Thermodynamic differences for g↔e′ pairs relative to A↔A (ΔΔGAA in kcal mol−1/pair) a
Coupling energy of interaction for g↔e′ pairs (ΔΔGint in kcal mol−1/pair) a
The larger coupling energy for the E↔R pair (−0.5 kcal/mol) than that of the E↔K pair (−0.3 kcal/mol) indicates that the E↔R pair contributes more to dimerization specificity than E↔K. The like-charged E↔E, K↔K, and R↔R pairs all have destabilizing coupling energies (+0.7, +0.6, and +0.8 kcal/mol, respectively) that are larger than the E↔R and E↔K attractive coupling energies (Table ). This suggests that preventing a repulsive g↔e′
pair is more important for driving dimerization specificity than forming an attractive pair. The energetic basis of the coupling energy for g↔e′
pairs is a subject of ongoing debate in the literature (47
), with some studies suggesting the measured coupling energy does not have a strong electrostatic component.
The suggestion that coupling energy may not be driven by charge interactions is highlighted by the polar glutamine that has a repulsive coupling energy in pairs with either acidic E or basic K (Table ). Because of the positive calculated coupling energies, we have color-coded E↔Q and Q↔E pairs in red as depicting repulsive acidic pairs and K↔Q, R↔Q, and Q↔K in blue as depicting repulsive basic pairs (Fig. ).
Many B-ZIP families, including JUN, CNC, and C/EBP, have only one charged amino acid in the g↔e′ pair. These charged amino acids contribute to the stability of the homodimer, as seen in the A↔R and E↔A pairs in the double-mutant thermodynamic analysis. Heterodimers, however, may be preferred if they form an attractive g↔e pair, because of the coupling energy. Thus, incomplete g↔e′ pairs can stabilize both homodimer and heterodimeric interactions.