The aim of this work was to better map the amino acid residues surrounding the binding pocket of the MC4R for one of its agonists, NDP-MSH, using a covalent attachment approach, coupled with a more accurate modeling of the receptor in its activated state. In order to identify specific interactions between the MC4R and NDP-MSH, we cross-linked cysteine-containing peptide analogues to the endogenous and mutated Cys residues on the receptor. The peptide ligands were labeled with biotin at their N-terminus and cross-linked to the receptor, and the complex was detected using streptavidin polyperoxidase.
In this study, we have demonstrated direct interactions between Cys-containing ligand homologues and both the WT receptor and versions containing substituted cysteines, which resulted in receptors to which the peptide ligand was covalently attached. The results revealed the proximity of residues at positions 5–9 of NDP-MSH to residues Leu106, Asp122, Ile125, Asp126, His264, and Met292 of the MC4R. Each position on the core peptide was shown to interact to varying degrees with at least two of the aforementioned residues on TM3, TM6, and TM7. However, a certain amount of caution should be exercised when analyzing the cross-linking data. There is the possibility of interpreting interactions that are not “real” based on a low level of cross-linking due to the potential of trapping serendipitous interactions (e.g., as the peptide enters the binding cleft) or flexibility (as the receptor and ligand “accommodate” the interaction). Furthermore, when charged residues either on the ligand or on the peptide are substituted with cysteines, important electrostatic interactions between the ligand and the receptor may be affected.
To aid the interpretation of these binding data, we constructed a theoretical model whereby the core tetrapeptide was docked initially, followed in a sequential manner by the N-terminus and the C-terminus of the peptide, akin to the proposed binding mode of adrenaline to the β2
A number of research groups have developed R
-state models for the MC4R, including Schiöth et al.
, who utilized an MC4R molecular model to suggest that a 76° counterclockwise turn of TM3 may be important for MC4R activation.41
Pogozheva et al.33
employed two different structural templates, namely, a model of the inactive conformation of the hMC4R29
and a model of the active conformation of the μ-opioid receptor. They then used distance constraints from the inactive conformation of rhodopsin, together with experimental constraints compatible with active states in several GPCRs, to develop an R
model of the MC4R into which they positioned NDP-MSH into the binding pocket using distance geometry calculations. Cho et al.20
to position an NMR structure of NDP-MSH in the binding pocket of their MC4R model, followed by refinement with a short molecular dynamics simulation. They determined that NDP-MSH formed a β-turn conformation around the d
sequence, with the hydrophobic side chain of the d
residue located away from the negatively charged side chains of the acidic residues. In their model, the Arg8
residue is involved in charge–charge interactions with the acidic residues. Finally, Hogan et al.23
used the observation that a switch in the orientation of Trp258 (from perpendicular to the plane of the membrane to parallel with the plane of the membrane)49
and a change in the conformation of the aromatic cluster of residues in TM6 are both involved in receptor activation.67
In developing their MC4R R
model, Hogan et al.
first changed the conformation of Phe254, Trp258, and Phe262 in their bovine-rhodopsin-based MC4R model, and then reduced the kink induced by Pro260 in TM6 from an initial 30° to a final lower kink of 11°.23
Here, we utilized the recently elucidated crystal structures to generate R
-state models of the MC4R, from which the structure of a complex with NDP-MSH was developed in a ligand-based modeling approach. This approach allowed us to examine the proposed interactions and the roles of the various mutations studied here and reported earlier in the literature. Previous NMR studies on isolated NDP-MSH have resolved two different structures for the peptide ligand. Cho et al.
indicated that the central residues forming the turn conformation in NDP-MSH are the His6
In contrast, Hogan et al.
later studied cyclic decapeptides with the consensus tetrapeptide sequence His-d
-Trp locked in a type II′ β-turn.23
With the use of our MC4R R
-state model based on the opsin template, the d
turn structure fitted the biochemical data best. On the other hand, some of the biochemical data did fit both models; however, it may be that different conformational forms of the ligand interact preferentially with different conformational states of the receptor. Our model is, however, limited by focusing on the predicted active state based on the active state of opsin.51
It appears that agonist binding may occur in at least two stages. Firstly, the core tetrapeptide is predicted to neutralize the negative repulsion between TM2 and TM3. The documented interaction between Arg8
on the peptide and Asp122 on the receptor34
was in agreement with our data, which also indicated that Asp126 is within interaction distance.62
In this study, as supported by both cross-linking studies and modeling analysis, both Glu5
are shown to be in close proximity to TM3, including the residues Asp122, Asp126, and Ile125 (see Supplementary Material
in the core tetrapeptide is not predicted to form direct interactions with the acidic bundle on TM3 of the MC4R, but maintains the “bioactive” conformation of the peptide ligand.32,68–70
Some data, however, have implicated residues Asp122, Asp126, His264, and Phe21832,34
in forming direct interactions with His6
). Here, our cross-linking studies and model suggest that His6
lies in close proximity to Asp122 and Asp126, but does not interact with the receptor. Rather, His6
forms interactions with residues of the peptide ligand itself, including d
, and Trp9
, thus supporting the theory of the importance of His6
in stabilizing the structure of the peptide ligand.22,32,68–70
Previous data have highlighted the importance of burying the hydrophobic residues (d
) in the hydrophobic cleft on TM6. Interestingly, one of the strongest cross-links detected was that between Ile125 (TM3) and d
on the peptide. This interaction was pivotal in our decision to use the d
β-turn of NDP-MSH over the His6
turn for the selection of our model complex. Interactions between TM3 and d
have only recently been suggested,23
implicating a third interaction in addition to the ionic TM3 interaction and TM6 hydrophobic binding pocket. The ionic interactions may allow rotation of TM3 towards TM2, which unlocks the putative activation motif DRY. Interestingly, one might expect the positively charged residue at position 8 (Arg8
) to be responsible for activation. However, Arg8
has been implicated mainly in potency and affinity, while the peptide ligand position 7 (Phe/Nal7
) is mainly responsible for agonist/antagonist characteristics.71
For example, nonpeptide agonists for the MC4R have been developed excluding any involvement of the Arg8
has been shown to increase the potency of the ligand; some even argue that it interacts more strongly with the receptor than with l
In the antagonist, d
-Nal may exert steric hindrance, preventing the movement of TM3. The lack of interaction between the l
peptide analogue and any of the key predicted residues of the receptor may be of some significance, and it may suggest that there is an alternative binding cavity sampled by l
-Phe that has not been exploited in our cross-linking study (e.g., with Phe284 and Tyr287 of TM6).
Experimentally, Trp9 on the peptide ligand was shown to be in close proximity to both TM6 and TM7, but the modeling data also indicated that residues on TM3, including Asp126, Ile129, Cys130, and Leu133, lie in the proximity of this residue. However, the predicted proximity of Cys130 to Trp9 at 5.2 Å in the model is not supported by the cross-linking studies. Perhaps the environment is not conducive to oxidation or the residues are not appropriately positioned.
In contrast, Met292 of TM7 was newly identified to interact with Trp9
. This interaction is interesting, as the equivalent residue in bovine rhodopsin Lys296 is involved in covalently attaching the chromophore to the receptor. For the first time, this residue has been shown to be located in the NDP-MSH pocket, as demonstrated by cross-linking () and functional studies (). In this study, NDP-MSH exhibited the characteristics of the binding of an antagonist to the mutant M292C-MC4R. Furthermore, previous studies demonstrated that M292C-MC4R exerted a dominant-negative effect on activation when in complex with another form of the receptor (unpublished data). Also, a cysteine at the equivalent position in the neurokinin NK2 receptor was shown to be cross-linked to the C-terminal residues of that ligand.72
All these reinforce the significance of this residue in the ligand binding cavity of many GPCRs.
The residues His6
, and Trp9
on the peptide ligand all showed a level of interaction with TM6 at residue His264. This residue in our model does not appear to be directly involved in the active binding pocket, but previous data have implicated it as important for ligand binding.33,34,40,73
Nickolls et al.
calculated a nonsignificant change in affinity for the peptide agonist NDP-MSH with H264A-MC4R.37
Here, we show that activity is dramatically reduced when His264 is mutated to Cys. This evidence points toward His264 being involved in the active-state structure of the MC4R. Leu106 is also not predicted to be directly involved in the agonist binding pocket, but cross-linking has shown interactions with residues His6
on the peptide. This residue is located at the top of TM2 on ECL1 in the model and is predicted to interact with the backbone of nearby Ile102 and Leu107. The side chain is exposed to the solvent, however, and, given its loop position, is likely to be flexible and thus could adopt alternative conformations. The residue at this position in the melanocortin-1 receptor (MC1R), when mutated, caused constitutive activity.36
This mutation L106C may alter the proposed ionic arrangement between TM2 and TM3, as suggested from studies of the homologous residue in MC1R, thereby resulting in constitutive activity.36
A large body of evidence suggests that Arg8
is involved in stabilizing the equilibrium between Asp122 (TM3) and Glu100 (TM2).43
Thus, if the equilibrium is altered by the mutation L106C, the function of Arg8
becomes redundant. This finding is supported by our binding data () where peptide 8, which contains the replacement of Arg8
when bound to L106C-MC4R, behaves similarly to the interaction of NDP-MSH with WT MC4R.
After the core tetrapeptide of NDP-MSH had been docked, its N-terminal and C-terminal regions were mapped into the model, and a series of potential hydrogen bonds between TM4, ECL2, and TM5 was apparent, consistent with the biochemical data. Previous data have suggested that substitution at positions Ser1
, and Ser3
of NDP-MSH, with less hydrophilic residues, slightly reduces affinity and potency.63,64
From our model, Ser1
of the peptide ligand is predicted to form a hydrogen bond with Val179 on TM4 of the receptor; thus, a hydrophobic residue at this peptide position can be expected to reduce affinity. Our data and model also validate a novel interaction between Cys196 (TM5) and Asp189 (ECL2) with the N-terminal region of the peptide (see Supplementary Material
). Investigation into the region on the MC4R (TM5) where the amino-terminus of the peptide agonist docks raised the possibility of a hydrogen-bond interaction between Ser188 on the receptor and Ser3
on the peptide. It is perhaps relevant that mutation of Asp189 to Ala dramatically decreased both the affinity and the potency of NDP-MSH.
It is important to note that all attempts to mutate or extend ECL2 destroyed the functionality of the MC4R (Cox et al.
, unpublished data). The modeling work suggests that the small ECL2 forms important interactions with the peptide ligand and is clearly very critical.32
The binding of the short agonist MTII, which lacks the N-terminal Ser1
motif of NDP-MSH activity, is not affected in the D189A mutant. Therefore, the hydrophilic nature of TM4 and TM5 may be important in forming a binding pocket for the amino-terminus of the peptide ligand. Although the N-terminal region of the peptide, when absent, is not required, it may exert an effect on the conformation of the “bioactive” sequence when present. The residue Cys196(5.42) in TM5 of the MC4R is equivalent to Ser204(5.42) in TM5 of the β2
-adrenergic receptor. This residue is one of a cluster of Ser residues at the TM5/ECL2 interface that are mainly involved in hydrogen bonding to the catecholamine agonist.74
These data begin to shed light on the critical nature of ECL2 in WT MC4R.
For the first time, direct interactions of the carboxyl-terminus of NDP-MSH with the MC4R were observed. The residue at position Val13
was shown to be in close proximity to Cys257(6.47) located on TM6. Cys257(6.47) is at an interesting position, as the equivalent residue in the β2
-adrenergic receptor is accessible, but only in the active conformation.55
Furthermore, recent studies that engineered metal ion binding sites in the β2
-adrenergic receptor demonstrated that this region moves towards TM3, in particular towards Asp(2.29), upon activation by an agonist.75
It is suggested that this mechanism of action is common to all GPCRs.76
The amino-terminus and carboxyl-terminus of the NDP-MSH ligand have previously been implicated in potency.28
The docking of the carboxyl-terminus produced here provides tentative support for the suggestion that the MC4R is activated by sequential interactions of the agonist with the receptor.65
Subsequent interactions may include possible hydrogen bonds between TM5 and the amino-terminus of the peptide. Once the receptor is activated, the N-terminal and C-terminal regions of the peptide are able to dock at the TM4/TM5 and TM6/TM7 interfaces, respectively, potentiating the signal. If the peptide ligand docks by a series of steps, each exposing more and more interfaces, this would complement experiments performed on the β2
-adrenergic receptor that led to the proposal of multiple binding states.65