Fig 1.
Phylogenetic analysis of the GPCR crystal structure set.
Maximum likelihood tree (A) analysis, maximum likelihood distance-based NeighborNet (B), and Evolutionary Trace sequence similarity-based tree (C) of the 7TM domain of crystallized receptors [3–28]. Naming according to the Uniprot database [53]. Receptors with hydrophobic residues highlighted in green, adenosine receptors in orange, biogenic amine receptors in red, non-rhodopsin-like receptors in purple, peptide-binding receptors in cyan, purine receptors in yellow. Numbers in (A) denote how often the respective node was found in bootstrap replicas (1000 samples in total). Numbers in (C) denote the ratio of sequence similarity between both sequence groups forming the respective node. The two tree analyses show a separation of rhodopsin-like GPCRs into small molecule receptors, i.e. amine receptors, on one side, and peptide/purine receptors on the other side, with rhodopsin (OPSD) being in between both subgroups. The connection with the non-rhodopsin-like GPCRs is formed in the middle between both branches, too, and is positioned close to rhodopsin and neurotensin receptor 1 (NTR1). The network and both tree analyses all show a good topological agreement, indicating a general tree-like evolution signal. However, the network exhibits large meshes around the connection point of non-rhodopsin-like and rhodopsin-like GPCRs, indicating a certain ambiguity about their precise connection.
Fig 2.
Phylogenetic analysis of the full rhodopsin-like GPCR crystal structure set.
Phylogenetic analysis of known human rhodopsin-like GPCR protein 7TM sequences after assignment of the transmembrane helices by alignment of the full scope of human GPCR protein sequences with the respective 7TM sequences from receptors with available x-ray structures. A: classification according to known ligand binding. The tree shows three major branches: olfactory receptors (in orange), small molecule binders (red), peptide binders (cyan). Orphan receptors in black. Olfactory receptors form a subclass of their own within the rhodopsin-like class. Small molecule binding GPCRs form a clear cluster. Peptide receptors cluster at the left end of the tree, starting with the neuropeptides subbranch. A 2nd set of small ligand receptors, which are the purine, leukotriene, and the free fatty acids receptors, mixes with protease-activated receptors. B: classification according to ligand properties as found in X-ray crystallography structures. Color scheme like in Fig 1. Known crystallized ligands can be subdivided into hydrophobic ligands (green), biogenic amines (red), hydrophilic ligands (orange), peptides (cyan), and purines (yellow). The small molecule binding receptor families closest to the common node with non-rhodopsin GPCRs are hydrophobic ligand GPCRs. The overall picture is in good agreement with the one found for the crystallized GPCRs from Fig 1.
Fig 3.
Consensus binding volume of peptide and small molecule ligands.
A: Volume accessed by peptide ligands [9,18] and small organic ligands [7,8,11–13,21,22,36,37] as observed in rhodopsin-like GPCR crystal structures after superposition of their 7TM helices. Left and right images are views rotated by 90 degrees. Peptide ligand volume as orange surface, small organic ligand volume as blue mesh. Helices from rhodopsin (PDB ID 1U19) [36] displayed in yellow as optical reference. Both types of ligands bind to the targeting receptors between helices II-VII. While peptide ligands bind around the height of the extracellular end of the helical bundle, small molecule ligands bind in a volume within the extracellular half of the helical bundle. Both volumes share a common region. B, C: Overlay of small ligand accessed volume (blue mesh) and peptide ligand accessed volume (orange surface) with N termini and el2 from rhodopsin (B) and S1PR1 (C). In rhodopsin, the N terminus and el2 fill out the peptide binding volume, while el2 shows a good match with the region claimed by both peptide binding volume and small ligand binding volume. In S1PR1, N terminus and el2 still both are positioned within the peptide binding volume. However, el2 is shortened compared to rhodopsin, and only partially fills the volume claimed by both peptide and small ligand binding.
Fig 4.
Development of peptide and small molecule binding GPCRs by protein self-interaction with el2.
Peptide ligand binding volume in rhodopsin-like GPCRs (see Fig 3) as mesh, small organic ligands (see Fig 3) in grey sticks with polar oxygen / nitrogen atoms as spheres, el2 as yellow cartoon, N termini as red cartoon. In all known non-rhodopsin GPCR structures (purple circle) [23–25,28], el2 forms a β–hairpin structure, which surrounds the peptide binding volume. In rhodopsin-like peptide receptor structures with bound peptide ligand (blue circle) [9,18], el2 forms a β-hairpin, which flanks this domain, too. This common arrangement suggests that during evolution, both rhodopsin-like and non-rhodopsin peptide binders kept common ancestral peptide ligand binding features. Furthermore, both classes kept a common position and shape of el2. The length of the rhodopsin el2 is in good agreement with the one observed in peptide-binding GPCRs. In rhodopsin (green circle, top), el2 goes right through the common peptide / small molecule ligand volume, and is held there by steric constraints from the N terminus (see Fig 3B). Retinal binds underneath and outside of this contact domain at a position close to the protein center. In the sphingosine receptor (green circle, bottom), el2 is disordered and has contracted in comparison to rhodopsin, while still being in contact with the N terminus (compare Fig 3C). The ligand has advanced into the common ligand domain, performing contacts there. We therefore assume that an opsin precursor forms the link between peptide-binding GPCRs and small molecule-binding GPCRs. In small molecule-binding GPCRs (red / orange circle), el2 is disordered or helical and has retracted from the peptide-binding domain. Small molecule ligands bind in the peptide contact volume at the position of el2 in rhodopsin, and below it. It seems that the ancestral rhodopsin-like GPCRs initially possessed an el2 in the form of a β-hairpin. Peptide binders retained el2 in this form. Small molecule ligands for GPCRs seem to have then developed as allosteric binders via an opsin ancestor as key intermediate, with el2 substituting a bound peptide ligand. Along the proposed pathway, small molecule ligands then seem to have substituted el2, who retracted from the binding site and lost the β-hairpin conformation. Purine receptors seem to have undergone a similar development.
Fig 5.
NeighborNet analysis of GPCR ligand binding sites.
Top: uncorrected P distance based NeighborNet of residues forming the ligand binding sites in GPCR crystal structures. Ligands and characteristic residues in sticks, their characteristic contact-forming polar oxygen/nitrogen atoms as spheres and mesh. Trp6.48 in yellow displayed as visual point of reference. The network analysis reveals four dominant categories of ligand binding cavities: peptide ligand receptors (cyan), hydrophobic ligand receptors (green), containing opsins and sphingosine receptors, polar ligand receptors (orange), containing adenosine receptors; and ammonium ligand receptors (red), containing muscarinic acetylcholine receptors, biogenic amine receptors, and opioid receptors. Hydrophobic ligand receptors share a glutamate at the similar positions 3.28 or 3.29. Surprisingly, opioid receptors are found in a group together with biogenic amine receptors, but not with peptide ligand receptors. They share the major contact residue Asp3.32 with acetylcholine and biogenic amine receptors. Bottom: Sequence alignment of amino acids forming small molecule binding sites in analyzed GPCR crystal structures.
Fig 6.
NeighborNet analysis of GPCR sodium binding sites.
Uncorrected P distance based NeighborNet analysis of residues forming the central sodium / water binding site in GPCR crystal structures. Left: NeighborNet analysis. The network analysis reveals four dominant categories of ligand binding cavities: peptide ligand receptors (cyan), hydrophobic ligand receptors (green), containing opsins and sphingosine receptors, polar ligand receptors (orange), containing adenosine receptors; and ammonium ligand receptors (red), containing muscarinic acetylcholine receptors, biogenic amine receptors, and opioid receptors. The overall topology of the network is in good agreement with the one of the ligand binding site presented in Fig 5, which suggests an evolutionary connection between both sites. Top right: overview of residues forming the central sodium/water binding site (in PDB ID 4N6H [35]). Sodium in purple sphere, water oxygen atoms in red spheres, binding site forming amino acids as sticks. Bottom right: sequence comparison of analyzed amino acid positions.