Influence of a lysine 331 counterion on the pK(a) of aspartic acid 125: Evidence for a salt-bridge interaction and role in alpha(1b)-Adrenergic receptor activation

We have hypothesized previously that a salt-bridge constraint exists in the alpha(1b)-adrenergic receptor (AR). Docking of the agonist epinephrine can disrupt this constraint via competition of its protonated amine, leading t o an agonist-induced activation of second messengers. The amino acids, K331 and D125, which comprise this salt-bridge, should be closely associated wi th each other in the unbound form of the alpha(1b)-AR. This ionic associati on should stabilize the negative charge of D125, leading to an increase in its acid strength or a decreased pK(a). If the charged state of D125 is imp ortant for agonist binding, then changing the type of amino acid at positio n 331 should decrease the acid strength of D125, leading to epinephrine aff inity changes for the alpha(1b)-AR. To test this hypothesis, site-directed mutagenesis was performed at position 331 of the alpha(1b)-AR. The effect t hese substitutions had on D125 acid strength was quantitated via epinephrin e affinity changes calculated from competition binding experiments performe d at different pH values. For all mutations of the alpha(1b)-AR where the p ositive charge at position 331 was eliminated, there was a significant incr ease in the pK(a) (congruent to 0.73) of an acidic amino acid(s). In additi on, there was an increase in the binding affinity of epinephrine for these mutants that was associated with a gain in the basal production of inositol triphosphates. These results are consistent with an aspartic acid residue as the counterion for K331 of the salt-bridge constraint, which disrupted, is a part of the receptor activation process. Moreover, changes in the pK(a ) of D125 were not dependent on the type of amino acid substituted at posit ion 331. This suggests a mechanism in which K331 is no longer influencing D 125 after salt-bridge disruption in the wild-type alpha(1b)-AR, but may mov e to another stabilized position, analogous to what has been suggested for bacteriorhodopsin. Differences from the wild-type receptor in D125 pK(a) fo r the K331 mutations were used to estimate the free-energy potential of the constraining salt-bridge. This free energy (congruent to 1 kcal/mol) is si gnificant, but weak enough to be consistent with an activational mechanism where docking of the receptor agonist has sufficient free energy to cause d isruption of the salt-bridge.