Ligand-bound Conformation Research Articles

The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains

In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested.

Metabolite Recognition Principles and Molecular Mechanisms Underlying Riboswitch Function

Riboswitches are mRNA elements capable of modulating gene expression in response to specific binding by cellular metabolites. Riboswitches exert their function through the interplay of alternative ligand-free and ligand-bound conformations of the metabolite-sensing domain, which in turn modulate the formation of adjacent gene expression controlling elements. X-ray crystallography and NMR spectroscopy have determined three-dimensional structures of virtually all the major riboswitch classes in the ligand-bound state and, for several riboswitches, in the ligand-free state. The resulting spatial topologies have demonstrated the wide diversity of riboswitch folds and revealed structural principles for specific recognition by cognate metabolites. The available three-dimensional information, supplemented by structure-guided biophysical and biochemical experimentation, has led to an improved understanding of how riboswitches fold, what RNA conformations are required for ligand recognition, and how ligand binding can be transduced into gene expression modulation. These studies have greatly facilitated the dissection of molecular mechanisms underlying riboswitch action and should in turn guide the anticipated development of tools for manipulating gene regulatory circuits.

How opioid drugs bind to receptors

The search for safe, non-addictive versions of morphine and other opioid drugs has just received a boost with the solving of the crystal structures of the receptors to which the drugs bind. See Articles p.321 & p.327 , Letters p.395 & p.400 Four papers in this issue of Nature present the long-awaited high-resolution crystal structures of the four known opioid receptors in ligand-bound conformations. These G-protein-coupled receptors are the targets of a broad range of drugs, including painkillers, antidepressants, anti-anxiety agents and anti-addiction medications. Brian Kobilka’s group reports the crystal structure of the µ-opioid receptor bound to a morphinan antagonist and the δ-opioid receptor bound to naltrindole. Raymond Stevens’ group reports on the κ-opioid receptor bound to the selective antagonist JDTic, and the nociceptin/orphanin FQ receptor bound to a peptide mimetic. In an associated News and Views, Marta Filizola and Lakshmi Devi discuss the implications of these landmark papers for research on the mechanisms underlying receptor function and drug development.

Guanine nucleotides differentially modulate backbone dynamics of the STAS domain of the SulP/SLC26 transport protein Rv1739c of Mycobacterium tuberculosis

Enzymatic catalysis and protein signaling are dynamic processes that involve local and/or global conformational changes occurring across a broad range of time scales. (1) H-(15) N relaxation NMR provides a comprehensive understanding of protein backbone dynamics both in the apo (unliganded) and ligand-bound conformations, enabling both fast and slow internal motions of individual amino acid residues to be observed. We recently reported the structure and nucleotide binding properties of the sulfate transporter and anti-sigma factor antagonist (STAS) domain of Rv1739c, a SulP anion transporter protein of Mycobacterium tuberculosis. In the present study, we report (1) H-(15) N NMR backbone dynamics measurements [longitudinal (T(1) ), transverse (T(2) ) and steady-state ({(1) H}-(15) N) heteronuclear NOE] of the Rv1739c STAS domain, in the absence and presence of saturating concentrations of GTP and GDP. Analysis of measured relaxation data and estimated dynamic parameters indicated distinct features differentiating the binding of GTP and GDP to Rv1739c STAS. The 9.55 ns overall rotational correlation time of Rv1739c STAS increased to 10.48 ns in the presence of GTP, and to 13.25 ns in the presence of GDP, indicating significant nucleotide-induced conformational changes. These conformational changes were accompanied by slow time scale (μs to ms) motions in discrete regions of the protein, as reflected by guanine nucleotide-induced changes in relaxation parameters. The observed nucleotide-specific alterations in the relaxation properties of individual STAS residues reflect an increased molecular anisotropy and/or the emergence of conformational equilibria governing functional properties of the STAS domain.

Potent Inhibitors of 5‐Lipoxygenase Identified using Pseudoreceptors

From pseudoreceptors to inhibitors: With limited knowledge about a ligand-bound receptor conformation, pseudoreceptor models can be helpful for the identification of novel inhibitors, as demonstrated here for 5-lipoxygenase, a validated target for the treatment of inflammation, cardiovascular diseases, and cancer.

Allosteric control of ligand-binding affinity using engineered conformation-specific effector proteins

We describe a phage display methodology to engineer synthetic antigen binders (sABs) that recognize either the apo- or the ligand-bound conformation of maltose binding protein (MBP). sABs that preferentially recognize the maltose-bound form of MBP act as positive allosteric effectors by significantly increasing the affinity for maltose. A crystal structure of a sAB bound to the closed form of MBP reveals the basis for the exhibited allosteric effect. We show that sABs which recognize the bound form of MBP can rescue the function of a binding-deficient mutant by restoring its natural affinity for maltose. Further, the sABs can enhance maltose binding in vivo by providing a growth advantage to bacteria under low maltose conditions. The results demonstrate that structure-specific sABs can be engineered to dynamically control ligand-binding affinities by modulating the transition between different conformations.

The Role of Oligomerization and Cooperative Regulation in Protein Function: The Case of Tryptophan Synthase

The oligomerization/co-localization of protein complexes and their cooperative regulation in protein function is a key feature in many biological systems. The synergistic regulation in different subunits often enhances the functional properties of the multi-enzyme complex. The present study used molecular dynamics and Brownian dynamics simulations to study the effects of allostery, oligomerization and intermediate channeling on enhancing the protein function of tryptophan synthase (TRPS). TRPS uses a set of α/β–dimeric units to catalyze the last two steps of L-tryptophan biosynthesis, and the rate is remarkably slower in the isolated monomers. Our work shows that without their binding partner, the isolated monomers are stable and more rigid. The substrates can form fairly stable interactions with the protein in both forms when the protein reaches the final ligand–bound conformations. Our simulations also revealed that the α/β–dimeric unit stabilizes the substrate–protein conformation in the ligand binding process, which lowers the conformation transition barrier and helps the protein conformations shift from an open/inactive form to a closed/active form. Brownian dynamics simulations with a coarse-grained model illustrate how protein conformations affect substrate channeling. The results highlight the complex roles of protein oligomerization and the fine balance between rigidity and dynamics in protein function.

CD69 Suppresses Sphingosine 1-Phosophate Receptor-1 (S1P1) Function through Interaction with Membrane Helix 4

Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P1). The activation antigen CD69 associates with and inhibits the function of S1P1, inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P1-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp1A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S1P1 showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S1P1 in cell lysates, likely reflecting degradation. Unexpectedly, the S1P1-CD69 complex exhibited a much longer half-life for binding of S1P than S1P1 alone. In contrast to wild-type CD69, a non-S1P1 binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P1 and suggest that CD69 induces an S1P1 conformation that shares some properties of the ligand-bound state, thereby facilitating S1P1 internalization and degradation.

Two-dimensional NMR and All-atom Molecular Dynamics of Cytochrome P450 CYP119 Reveal Hidden Conformational Substates

Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [(13)C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.

Conformational transitions upon ligand binding: holo-structure prediction from apo conformations.

Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 Å backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 Å backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.

Conformational Transitions Upon Ligand Binding: Holo Structure Prediction from Apo Conformations

Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design.Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited.We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 A backbone RMSD upon ligand binding. In all cases, receptor models within 1.5 A backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for eight of ten cases in the top-ranked complex models.The developed protocol is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.

Ligand Binding Turns Moth Pheromone-binding Protein into a pH Sensor

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

Sphingosine Kinase-1 Associates with Integrin α Vβ 3 to Mediate Endothelial Cell Survival

Sphingosine Kinase-1 Associates with Integrin α Vβ 3 to Mediate Endothelial Cell Survival

Ligand Entry and Exit Pathways in the β2-Adrenergic Receptor

The recently determined crystal structure of the human β2-adrenergic (β2AR) G-protein-coupled receptor provides an excellent structural basis for exploring β2AR–ligand binding and dissociation process. Based on this crystal structure, we simulated ligand exit from the β2AR receptor by applying the random acceleration molecular dynamics (RAMD) simulation method. The simulation results showed that the extracellular opening on the receptor surface was the most frequently observed egress point (referred to as pathway A), and a few other pathways through interhelical clefts were also observed with significantly lower frequencies. In the egress trajectories along pathway A, the D192–K305 salt bridge between the extracellular loop 2 (ECL2) and the apex of the transmembrane helix 7 (TM7) was exclusively broken. The spatial occupancy maps of the ligand computed from the 100 RAMD simulation trajectories indicated that the receptor–ligand interactions that restrained the ligand in the binding pocket were the major resistance encountered by the ligand during exit and no second barrier was notable. We next performed RAMD simulations by using a putative ligand-free conformation of the receptor as input structure. This conformation was obtained in a standard molecular dynamics simulation in the absence of the ligand and it differed from the ligand-bound conformation in a hydrophobic patch bridging ECL2 and TM7 due to the rotation of F193 of ECL2. Results from the RAMD simulations with this putative ligand-free conformation suggest that the cleft formed by the hydrophobic bridge, TM2, TM3, and TM7 on the extracellular surface likely serves as a more specific ligand-entry site and the ECL2–TM7 hydrophobic junction can be partially interrupted upon the entry of ligand that pushes F193 to rotate, resulting in a conformation as observed in the ligand-bound crystal structure. These results may help in the design of β2AR-targeting drugs with improved efficacy, as well as in understanding the receptor subtype selectivity of ligand binding in the β family of the adrenergic receptors that share almost identical ligand-binding pockets, but show notable amino acid sequence divergence in the putative ligand-entry site, including ECL2 and the extracellular end of TM7.

Dynamic Linear Response Theory for Conformational Relaxation of Proteins

Dynamic linear response theory is adapted to the problem of computing the time evolution of the atomic coordinates of a protein in response to the unbinding of a ligand molecule from a binding pocket within the protein. When the ligand dissociates from the molecule, the protein molecule finds itself out of equilibrium and its configuration begins to change, ultimately coming to a new stable configuration corresponding to equilibrium in a force field that lacks the ligand-protein interaction terms. Dynamic linear response theory (LRT) relates the nonequilibrium motion of the protein atoms that ensues after the ligand molecule dissociates to equilibrium dynamics in the force field, or equivalently, on the potential energy surface (PES) relevant to the unliganded protein. In general, the connection implied by linear response theory holds only when the ligand-protein force field is small. However, in the case where the PES of the unliganded protein system is a quadratic (harmonic oscillator) function of the coordinates, and the force of the ligand upon the protein molecule in the ligand-bound conformation is constant (the force on each atom in the protein is independent of the location of the atom), dynamic LRT is exact for any ligand-protein force field strength. An analogous statement can be made for the case where the atoms in the protein are subjected to frictional and random noise forces in accord with the Langevin equation (to account for interaction of the protein with solvent, for example). We numerically illustrate the application of dynamic LRT for a simple harmonic oscillator model of the ferric binding protein, and for an analogous model of T4 lysozyme. Using a physically appropriate value of the viscosity of water to guide the choice of friction parameters, we find relaxation time scales of residue-residue distances on the order of several hundred ps. Comparison is made to relevant experimental measurements.

Exploring the Thermodynamics of Activation Pathways of Bovine Rhodopsin with Fast Molecular Dynamics Simulations

Recent progress made in the understanding of the structure and function of G-protein coupled receptors (GPCRs) points to compelling biophysical and crystallographic evidence for distinct conformational changes of the prototypical family member bovine rhodopsin/opsin upon illumination. To investigate possible activation pathways of bovine rhodopsin from an early inactive intermediate (lumirhodopsin) produced by the cis-trans photoisomerization of its retinal chromophore to an activated ligand-bound opsin-like conformation of the protein, we carried out several independent biased molecular dynamics simulations of the receptor in an explicit dipalmitoylphosphatidylcholine (DPPC) membrane bilayer. The simulations revealed significantly different activation pathways between inactive and opsin-like activated forms of bovine rhodopsin, whose thermodynamic behavior was evaluated by metadynamics using as reaction coordinates both the position along the identified representative pathways, and the distance from them. The results point to three common metastable states that are likely to correspond to a photoactivated deprotonated Meta I-380/Meta IIa, and two different Meta IIb-like intermediates of bovine rhodopsin. Inferences from these molecular models are expected to shed new light into mechanisms of allosteric modulation of rhodopsin function.

Nonlocal Helix Formation Is Key to Understanding S-Adenosylmethionine-1 Riboswitch Function

Riboswitches are noncoding RNAs that regulate gene expression in response to changing concentrations of specific metabolites. Switching activity is affected by the interplay between the aptamer domain and expression platform of the riboswitch. The aptamer domain binds the metabolite, locking the riboswitch in a ligand-bound conformation. In absence of the metabolite, the expression platform forms an alternative secondary structure by sequestering the 3′ end of a nonlocal helix called P1. We use all-atom structure-based simulations to characterize the folding, unfolding, and metabolite binding of the aptamer domain of the S-adenosylmethionine-1 (SAM-1) riboswitch. Our results suggest that folding of the nonlocal helix (P1) is rate-limiting in aptamer domain formation. Interestingly, SAM assists folding of the P1 helix by reducing the associated free energy barrier. Because the 3′ end of the P1 helix is sequestered by an alternative helix in the absence of metabolites, this observed ligand-control of P1 formation provides a mechanistic explanation of expression platform regulation.

Probing the Role of Parasite-Specific, Distant Structural Regions on Communication and Catalysis in the Bifunctional Thymidylate Synthase−Dihydrofolate Reductase from Plasmodium falciparum

Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or nonactive regions that might modulate catalysis: (1) an N-terminal tail and (2) a linker region tethering DHFR to TS, and encoding a crossover helix that forms critical electrostatic interactions with the DHFR active site. The role of these nonactive sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady-state kinetic characterization of the full-length, wild-type (WT) P. falciparum TS-DHFR enzyme and probe the role of distant, nonactive regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an activated (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s-1 to 130 s-1 in the WT enzyme. The TS rate is also reciprocally activated by approximately 1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the DHFR single rate and the bifunctional TS-DHFR rate by a factor of 2. The 2-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication between TS active site and DHFR ligands is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from approximately 14 s-1 to approximately 40 s-1. In summary, our results demonstrate a complex interplay of domain-domain communication and nonactive-site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their nonactive site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria.

Visualizing spatially correlated dynamics that directs RNA conformational transitions

RNAs fold into three-dimensional (3D) structures that subsequently undergo large, functionally important, conformational transitions in response to a variety of cellular signals. RNA structures are believed to encode spatially tuned flexibility that can direct transitions along specific conformational pathways. However, this hypothesis has proved difficult to examine directly because atomic movements in complex biomolecules cannot be visualized in 3D by using current experimental methods. Here we report the successful implementation of a strategy using NMR that has allowed us to visualize, with complete 3D rotational sensitivity, the dynamics between two RNA helices that are linked by a functionally important trinucleotide bulge over timescales extending up to milliseconds. The key to our approach is to anchor NMR frames of reference onto each helix and thereby directly measure their dynamics, one relative to the other, using 'relativistic' sets of residual dipolar couplings (RDCs). Using this approach, we uncovered super-large amplitude helix motions that trace out a surprisingly structured and spatially correlated 3D dynamic trajectory. The two helices twist around their individual axes by approximately 53 degrees and 110 degrees in a highly correlated manner (R = 0.97) while simultaneously (R = 0.81-0.92) bending by about 94 degrees. Remarkably, the 3D dynamic trajectory is dotted at various positions by seven distinct ligand-bound conformations of the RNA. Thus even partly unstructured RNAs can undergo structured dynamics that directs ligand-induced transitions along specific predefined conformational pathways.

Structural and functional characterization of a novel type of ligand-independent RXR-USP receptor.

Retinoid X receptor (RXR) and Ultraspiracle (USP) play a central role as ubiquitous heterodimerization partners of many nuclear receptors. While it has long been accepted that a wide range of ligands can activate vertebrate/mollusc RXRs, the existence and necessity of specific endogenous ligands activating RXR-USP in vivo is still matter of intense debate. Here we report the existence of a novel type of RXR-USP with a ligand-independent functional conformation. Our studies involved Tribolium USP (TcUSP) as representative of most arthropod RXR-USPs, with high sequence homology to vertebrate/mollusc RXRs. The crystal structure of the ligand-binding domain of TcUSP was solved in the context of the functional heterodimer with the ecdysone receptor (EcR). While EcR exhibits a canonical ligand-bound conformation, USP adopts an original apo structure. Our functional data demonstrate that TcUSP is a constitutively silent partner of EcR, and that none of the RXR ligands can bind and activate TcUSP. These findings together with a phylogenetic analysis suggest that RXR-USPs have undergone remarkable functional shifts during evolution and give insight into receptor-ligand binding evolution and dynamics.