Earlier studies carried out by our group employing photoaffinity labeling with [benzoylphenylalanine13
]α-factor and oxidative cross linking experiments involving DOPA13
α-factor suggested that the Tyr13
position of α-factor interacts with the TM1 segment of Ste2p at residues R58 and Cys59, respectively (12
), and the presence of an aromatic ring at position 13 of α-factor was found to be necessary for efficient binding (11
). Arg58 on TM1 and His94 on TM2 in Ste2p are believed to be in the hydrophobic core of the membrane. Other members of the fungal pheromone GPCR subfamily conserve polar residues at similar positions (9
). The conservation of positively charged residues in the first and second TM domains of these fungal GPCRs, and the critical nature of Tyr13
of α-factor and of other fungal α-factors suggested to us that ligand recognition by Ste2p might involve a cation-π type of interaction.
Fluorine substitution has been utilized as an important tool to track cation-π interactions as fluorine is sterically similar to hydrogen but due to its extremely high electronegativity has a remarkable effect on the electronic structure of an aromatic ring. Progressive fluorination of the aromatic ring decreases the π-electron density and thereby decreases the ability of the π-face to interact with cations. Previously, we studied [Phe13
(3-F)]α-factor and [Phe13
(4-F)]α-factor using a radioactive binding competition assay and found that these analogs competed strongly with α-factor binding and were good agonists (29
). In the present investigation, using a direct fluorescent saturation binding assay and multiple fluorinated peptides we studied α-factor analogs with varying predicted cation-π binding energies and revealed that there is significant influence of the cation-π binding energies of the phenylalanine ring on the equilibrium binding constant Kd
of the receptor. Phenylalanine has a similar cation-π binding energy as that of tyrosine (27
) () and ideally phenylalanine replacement should not adversely affect the binding affinity. In our studies, the Kd
obtained for [Phe13
]α-factor is similar to that of native [Tyr13
]α-factor which appears to validate this contention.
Phe(3-F) (entry 3, ) and Phe(4-F) (entry 4, ) have similar calculated cation-π binding energies and α-factor analogs with these substitutions were found to have similar Kd
values in our binding studies. This indicates that the position of the fluorine atom does not have a significant effect on the binding affinity and the trend that we begin to see is mainly due to the decrease in cation-π binding energy. Progressive fluorination of the phenylalanine ring resulted in an increase in Kd
and the data fits well onto a linear correlation plot (). Phe(2,3,4,5,6-F5
) has the lowest cation-π binding energy among the fluorinated α-factor derivatives that were studied and this analog exhibited approximately a 10-fold decrease in binding affinity compared to α-factor. The associated decrease in binding affinity due to fluorination is not of the magnitude that was reported for ligand gated channels where up to a two log change in binding constants was observed. This suggests that the associated cation-π contribution to the α-factor–Ste2p binding may be weaker than the contribution to the ligand interactions in ion channels. In fact, we have recently found that position 13 of α-factor also interacts with Cys59 of Ste2p (13
) so that contributions of H-bonding between the hydroxyl group of Tyr13
to Cys59 and a cation-π interaction between Tyr13
and Arg58 both contribute to the binding of α-factor to Ste2p.
An additional way to test for the cation-π interaction would be to carry out a similar binding analysis on ste2 mutants where Arg58 is replaced by other residues. We have generated 3 such mutants ((R58A); (R58D) and (R58E)). All of these are seriously deficient in binding, with Kd’s for α-factor from 10 to 50-fold higher. As shown in the present study the fluorescent fluorinated α-factor analogs had Kd’s for the wildtype Ste2p ranging from 26.2 nM to 177.3 nM (). To determine the latter value we needed to use 4μM peptide, saturation was barely achieved, and the non-specific fluorescence was quite high. Since the Ste2p containing the substitution R58A binds α-factor with nearly 10-fold lower affinity saturation binding experiments would require very high concentrations of [Lys7(NBD),Phe13(2,3,4,5,6-F5)]α-factor (~40 μM). As stated above this peptide precipitates at 25 μM and the experiment, therefore, could not be done. However, the marked decrease in affinity noted with the three ste2 mutants that we generated supports our conclusion that the Arg58 side chain contributes to the binding energy and would be consistent with the cation-π interaction.
The use of NBD derivatives of α-factor might lead to a change in the interaction of position-13 of the pheromone and its receptor. However all of the analogs that we tested were agonists indicating productive binding to the receptor, and the potencies of the fluorinated α-factor analogs were within a factor of 3 to 4 of that of α-factor with the exception of the pentafluorinated analog. Although the trends in the binding affinities were not directly parallel to the trends in the agonist potencies, we have observed a similar lack of correlation in our previous study with α-factor analogs (29
). Moreover, the weakest binding fluorinated analog ([Phe13
)]α-factor) was by far the weakest agonist. Finally, α-factor efficiently competed with the binding of the Lys7
(NBD) analogs to Ste2p. Based on these observations and previous studies on α-factor derivatized at the Lys7
side chain (30
) we conclude that the fluorescent labeling does not result in a significant perturbation of the interactions of the position-13 side chain and the receptor. Interestingly, two non-fluorinated analogs, [Phe13
(4-Me)]α-factor and [Phe13
(4-OMe)]α-factor each showed a marked lack of correlation between the Kd
values and cation-π binding energies (). This finding contrasts with the correlation observed with fluorinated phenylalanine analogs () and indicates that while the insertion of fluorine in place of a hydrogen results in primarily an electronic effect on the benzene ring, other groups can have both electronic and steric effects on binding. These analogs have marginally higher cation-π binding energies as compared to phenylalanine and tyrosine () and would be predicted to result in stronger binding. We observed a 3-fold decrease in the binding ability for [Phe13
(4-Me)]α-factor and the difference reduces to 2-fold upon re-introduction of phenolic oxygen which should be a good electron donor. It is possible that the effect of re-introduction of the donor-oxygen atom is at least partially nullified by the steric bulk of the methoxy group. Despite the fact that it cannot participate in a cation-π interaction, the [Lys7
]α-factor does bind weakly to Ste2p. Thus, it is evident that additional effects involving steric, electrostatic and perhaps Van der Waals forces may also play a role in α-factor-Ste2p recognition at position-13 of the pheromone.
The results of this paper support the existence of a cation-π interaction between the carboxyl terminal Tyr13
residue of the α-factor peptide and the Arg58 residue in the first transmembrane domain of Ste2p. This interaction contributes to the favorable energetics of binding of ligand to receptor, since removal of Tyr from the carboxyl terminus drastically reduces the peptide affinity and leads to an inactive pheromone (34
). However this interaction involving the C-terminal of α-factor is not, by itself, sufficient for receptor activation because N-terminally truncated analogs of α-factor bind strongly to the receptor but do not lead to signal transduction (34
Detection of the interaction of Arg58 of the receptor with Tyr13
of α-factor raises the question of how the positively charged Arg58 is accommodated when there is no ligand bound to receptor, since the existence of an isolated arginine sidechain within the hydrophobic transmembrane region of Ste2p is expected to be energetically unfavorable. One possibility is that, in the absence of ligand, Arg58 participates in an intramolecular cation-π interaction with an aromatic residue elsewhere in the receptor. Each of the seven phenylalanine and two tryptophan residues within the predicted transmembrane regions of Ste2p can be mutated to non-aromatic residues without causing loss of function (35
). Similarly, aromatic residues do not appear to be required at the positions of two of the three tyrosine residues in these regions. The remaining tyrosine, Tyr266 can only be mutated to Trp, Phe or His (35
). However current models of Ste2p (9
), place Tyr266 too far from Arg58 for any direct interaction.
This suggests that the any aromatic side chain involved in an intramolecular cation-π interaction with Arg58 in the absence of ligand would reside in an extracellular tail or loop. The most likely site for the interacting side chains would be the first extracellular loop EL1, based on its expected proximity to Arg58 and on the following: 1) The results of cysteine scanning accessibility measurements on Ste2p indicate that burial of this loop in the transmembrane core of the receptor is involved in stabilizing the inactive state (37
); 2) Mutation of Phe119, also in EL1, has been reported to lead to constitutive activity, as would be expected if EL1 is involved in stabilizing the inactive state (38
). 3) Removal of residues 114 to 129 from EL1 of Ste2p yields a receptor that signals poorly but is constitutively active (Bhuiyan, Cohen and Hauser unpublished results); and 4) The substitution Tyr111C in EL1 of Ste2p results in a receptor that is incapable of signaling while retaining high affinity α-factor binding, suggesting that Tyr111 is involved in the activation pathway.
Thus, we hypothesize that either Tyr111 or F119 in EL1 may interact with Arg58 in the ligand-free receptor via a π-cation interaction, stabilizing the ground state (see for a model of the Tyr111-Arg58 interaction in Ste2p). Upon ligand binding, the intramolecular interaction is replaced by the cation-π interaction with Tyr13
of α-factor, anchoring the carboxyl terminal of the ligand to the receptor and allowing the amino terminal of the pheromone to establish additional interactions that induce a conformational change that is propagated through other transmembrane domains, perhaps through the sequential actions of a series of “micro-switches” (39
), to sites interacting with the G protein. A role for EL1 in signal transduction by other GPCRs has been proposed based on studies of rhodopsin, the dopamine receptor and the C5a receptor (40
). In addition, recent solid state and solution NMR studies have provided evidence for significant conformational changes in other extracellular loops of Class A GPCRs (44
Figure 6 Model for change in EL1 loop receptor interactions in presence and absence of α-factor. The transmembrane helices (TM) of Ste2p are shown in different colors and labeled. The structure of EL1 with a short 310-helix (106YSSVTYALT114) predicted (more ...)