Abstract
G protein-coupled receptors (GPCRs) are integral membrane proteins that are pivotal in transmitting signals from outside to within the cell. More than 800 GPCRs exist in humans that, albeit sharing a common architecture of seven transmembrane helices, respond to very distinct extracellular stimuli, including hormones, neurotransmitters, odorants, and photons. The binding of a ligand, or the absorption of a photon in the case of rhodopsin, leads to alterations in the conformational landscape of the receptors, which ultimately results in GPCR activation. Activated GPCRs act, among other roles, as guanine nucleotide exchange factors that activate G proteins and thus initiate signaling cascades. The involvement of GPCRs in many biologically important processes makes them a major pharmaceutical target, with approximately a third of all marketed drugs targeting GPCRs.
While most studies focus on the elucidation of the activation mechanism, we were interested in studying the complex conformational landscape of inactive GPCRs, a deeper understanding of which is of pharmacological interest as it can, for example, improve subtype selectivity of drugs. Our studies focused on the α1B-adrenergic receptor (α1B-AR), which is involved in the regulation of smooth muscle contraction and vasoconstriction, and which is targeted by a number of marketed drugs. To study the α1B-AR with NMR, we relied on a thermostabilized construct of this receptor, termed α1B-AR-B1D1, which can be expressed in E. coli and purified in detergent micelles with relatively high protein yields.
Backbone amides in α1B-AR-B1D1 were investigated using [15N,1H]-TROSY spectra, which showed remarkable differences between inverse agonists. Whereas many differences in chemical shifts are found between the different ligands, there also exist similarities that suggest a division of the inverse agonists into three classes. Class I is formed by quinazoline derivatives (alfuzosin, cyclazosin, and prazosin), class II by other small ligands (carvedilol, HEAT, nicergoline, and tamsulosin), and class III by niguldipine and SNAP 5089, which are small-molecule ligands, and by ρ-TIA, which is an allosterically binding peptide. This classification was further supported by a quantitative approach that determines spectral similarities and subsequently by [13C,1H]-HSQC spectra probing Ile and Leu δ- methyl and Val γ-methyl groups that showed the same grouping of the ligands. The presence of three distinct spectra groups suggests that each of the investigated inverse agonists modulates the conformational landscape of α1B-AR-B1D1 in one out of three possible ways.
To find potential differences in how the three ligand classes affect the conformational landscape of α1B- AR-B1D1, we determined dynamics in the ps−ns range for Ile, Leu, and Val side chains in the form of order parameters (S2axis) for their methyl groups. Dynamics in this fast time regime are essential for protein function and are connected directly to entropy. We determined the side-chain dynamics of the prazosin-, tamsulosin-, and ρ-TIA-bound α1B-AR-B1D1. The resulting dynamic profiles were very similar between the different inverse agonists indicating that no major overall differences exist. Very rigid side chains were present that have not been found in other membrane proteins so far. The peptide ρ-TIA leads to an increase in the flexibility of I1764×56 and possibly of I2145×49, which is adjacent to the P2155×50 of the highly conserved PIF motif. Further, side-chain dynamics obtained with the ρ-TIA- bound α1B-AR-B1D1 match less well to the α1B-AR crystal structure than the dynamics obtained with prazosin and tamsulosin. These observations suggest that ρ-TIA, and thus possibly the other class III ligands, alter the conformational landscape of α1B-AR-B1D1 with respect to class I and II ligands.
Interestingly, several general trends were observed for the side-chain dynamics in α1B-AR-B1D1, which have not yet been described for other membrane proteins. Among them are the correlations between side-chain dynamics and structural properties of the α1B-AR: side chains are more dynamic when they are more protein-surface exposed and when they are less densely packed. Further, side-chain dynamics follow the z-axis of the protein, with flexible side chains being found at the extra- and intracellular ends of the receptor, and rigid ones at the center of the membrane bilayer. Another surprising observation was that Leu side chains were on average more dynamic than Ile side chains, despite that they are expected to give rise to identical δ-methyl order parameters when located in the same environment. The difference in average dynamics likely stems from that Leu is generally less densely packed and more protein-surface exposed than Ile in α1B-AR-B1D1, a feature that is a common among all class A GPCRs. One possible explanation for this asymmetric distribution of Leu and Ile is that Leu forms more beneficial contacts with lipids and is thus more frequently located on the protein surface than Ile. We further explored whether the asymmetry might stem from that Leu but not Ile tunes the hydrophobicity of class A GPCRs to facilitate their correct insertion into membranes and/or to stably anchor them within membranes. In particular, we investigated whether receptors compensate excess hydrophilicity by an increase in the number of Leu rather than Ile residues. We found evidence for this hypothesis in the frequencies with which Leu and Ile occur in the sequences of class A GPCRs.
Besides differences in side-chain dynamics, the ligand classes affect the binding of Na+ ions. Class A GPCRs share a conserved Na+ binding pocket in the center of the helical bundle, with the binding of a Na+ ion stabilizing inactive conformational states. The binding of Na+ is crucial for receptor function and it has been theorized that the egress of the Na+ ion into the cytosol provides a small amount of energy that promotes receptor activation. The binding of a Na+ ion by prazosin-bound α1B-AR-B1D1 leads to the population of an additional conformational state. The binding affinity for Na+ was determined based on the δ-methyl groups of I2195×54 (Kd = 42 mM) and I1784×58 (Kd = 74 mM). These affinities report on the response of the α1B-AR-B1D1 to the binding of Na+ rather than on the binding itself. Therefore, the higher affinity of I2195×54, which is in closer proximity to the Na+ binding pocket than I1784×58, indicates loose allosteric coupling between the two residues and thus incomplete transmission of the conformational information. Interestingly, while the Na+ affinity remains similar with tamsulosin- and carvedilol-bound α1B-AR-B1D1, class III ligand-bound α1B-AR-B1D1 appears to be unable to bind Na+.
Metallothioneins (MTs) are small and Cys-rich proteins that coordinate large amounts of metal ions within metal-thiolate clusters. In gastropods, that is, in snails and slugs, MTs are often found as a means of Cd2+ detoxification. Over the course of millions of years, gastropods moved into new habitats with different environmental Cd2+ levels, which led to independent gains and losses of Cd2+-selectivity in their MTs. Two additional MT isoforms exist in some gastropods. The first of these isoforms is involved in Cu+ metabolism whereas the second one has an unknown function. The evolution of Cd2+ and Cu+ binding properties in all three isoforms was investigated by studying extant and reconstructed ancestral proteins. NMR was used to measure the extent of conformational dynamics based on general spectra properties and 15N T2 relaxation. MTs that evolved to bind a specific metal ion (generally) form conformationally stable complexes, whereas complexes with non-cognate metals display conformational exchange. We introduced the term conformity to describe this property in order to differentiate it from selectivity, which relates to the capability of MTs to form homometallic complexes with fixed stoichiometries. The ancestor to all three MT isoforms was Cd2+-conform. The Cd2+- conformity was subsequently lost in the new MT isoform that emerged through a gene duplication of this ancestral MT. Cu+-conformity evolved independently in two isoforms, after these isoforms split from one another in a second gene duplication event.
Whereas most gastropod MTs share a two-domain architecture, there are notable exceptions, one of them being the three-domain MT of the common periwinkle Littorina littorea (LlMT), in which the N- terminal domain underwent a partial sequence duplication. The highly cooperatively bound metal ions can be transferred between two metalated proteins and between metalated and apo proteins through direct contact between proteins. Up to eight signals per residue are present in [15N,1H]-HSQC spectra of mixed Cd2+/Zn2+ metalated domains, indicating that the chemical shifts are sensitive to the precise arrangement of metal ions within a cluster and that the metal ions rearrange on very slow timescales.
C- and N-terminal domains in gastropod MTs have distinct sequences and display different properties regarding metal binding. Whereas C-domains are more conserved in terms of their amino acid sequence, N-domains are more conserved in their structure, indicating that the N-domain fold is more resistant to mutations. The C-domain (at least in LlMT) has an approx. 5-fold higher affinity for Cd2+ ions than the N-domain, but the N-domain shows generally a higher conformity when binding the non-cognate metal. Thus, the N-domain can be described as the generalist and the C-domain as the specialist among the two domains.
Baumann, Christian