Corey W. Liu

The dynamic process of β2-adrenergic receptor activation

G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β(2)-adrenergic receptor (β(2)AR), a prototypical GPCR. We labeled β(2)AR with (13)CH(3)ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not observed in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound preparations. They also show that for β(2)AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The observed heterogeneity may be important for β(2)ARs ability to engage multiple signaling and regulatory proteins.

Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the β2 adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

Small-molecule displacement of a cryptic degron causes conditional protein degradation

The ability to rapidly regulate the functions of specific proteins in living cells is a valuable tool for biological research. Here we describe a novel technique by which the degradation of a specific protein is induced by a small molecule. A protein of interest is fused to a Ligand-Induced Degradation (LID) domain resulting in the expression of a stable and functional fusion protein. The LID domain is comprised of the FK506- and rapamycin-binding protein (FKBP) and a 19-amino acid degron fused to the C-terminus of FKBP. In the absence of the small molecule Shield-1, the degron binds to the FKBP protein and the fusion protein is stable. Shield-1 binds tightly to FKBP thereby displacing the degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. Structure-function studies of the 19-residue peptide showed that a four-amino acid sequence within the peptide is responsible for degradation.

Solution structure and proposed domain domain recognition interface of an acyl carrier protein domain from a modular polyketide synthase.

Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide‐producing assemblies. However, impaired domain–domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain–domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6‐deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10‐kD domain is a three‐helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14–94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 ± 0.09 Å for the backbone atoms (1.21 ± 0.13 Å for all non‐hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 ± 0.10 Å (0.99 ± 0.11 Å for non‐hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein–protein recognition of ACP domains, consistent with the previously observed specificity. Site‐directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.

Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

A longstanding proposal in enzymology is that enzymes are electrostatically and geometrically complementary to the transition states of the reactions they catalyze and that this complementarity contributes to catalysis. Experimental evaluation of this contribution, however, has been difficult. We have systematically dissected the potential contribution to catalysis from electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of the transition state and reaction intermediate, bind and accept two hydrogen bonds in an active site oxyanion hole. The binding of substituted phenolates of constant molecular shape but increasing p K a models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen bonds increase by 0.50–0.76 ppm/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K a unit). The small effect of increased charge localization on affinity occurs despite the shortening of the hydrogen bonds and a large favorable change in binding enthalpy (ΔΔH = −2.0 kcal/mol/p K a unit). This shallow dependence of binding affinity suggests that electrostatic complementarity in the oxyanion hole makes at most a modest contribution to catalysis of ˜300-fold. We propose that geometrical complementarity between the oxyanion hole hydrogen-bond donors and the transition state oxyanion provides a significant catalytic contribution, and suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from a single dominant contribution.

Molecular Framework for the Activation of RNA-dependent Protein Kinase

The RNA-dependent protein kinase (PKR) plays an integral role in the antiviral response to cellular infection. PKR contains three distinct domains consisting of two conserved N-terminal double-stranded RNA (dsRNA)-binding domains, a C-terminal Ser-Thr kinase domain, and a central 80-residue linker. Despite rich structural and biochemical data, a detailed mechanistic explanation of PKR activation remains unclear. Here we provide a framework for understanding dsRNA-dependent activation of PKR using nuclear magnetic resonance spectroscopy, dynamic light scattering, gel filtration, and autophosphorylation kinetics. In the latent state, PKR exists as an extended monomer, with an increase in self-affinity upon dsRNA association. Subsequent phosphorylation leads to efficient release of dsRNA followed by a greater increase in self-affinity. Activated PKR displays extensive conformational perturbations within the kinase domain. We propose an updated model for PKR activation in which the communication between RNA binding, central linker, and kinase domains is critical in the propagation of the activation signal and for PKR dimerization.

Solution mapping of t cell receptor docking footprints on peptide-MHC

T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR–pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a “recognition code” between conserved elements of the TCR and MHC genes. Although ≈17 cocrystal structures of unique TCR–pMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H–2Ld–QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a high-affinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many Ld-specific TCRs.

Complementary Phosphorus Speciation in Agricultural Soils by Sequential Fractionation, Solution P Nuclear Magnetic Resonance, and Phosphorus K-edge X-ray Absorption Near-Edge Structure Spectroscopy.

Ultisols in China need phosphorus (P) fertilization to sustain crop production but are prone to P loss in runoff. Balancing P inputs and loss requires detailed information about soil P forms because P speciation influences P cycling. Analytical methods vary in the information they provide on P speciation; thus, we used sequential fractionation (SF), solution P nuclear magnetic resonance (P-NMR), and P K-edge X-ray absorption near-edge structure (XANES) spectroscopy to investigate organic P (P) and inorganic P (P) species in Chinese Ultisols managed for different crops and with different fertilizer inputs in the first study to combine these techniques to characterize soil P. Sequential fractionation showed that moderately labile NaOH-P was the largest P pool in these soils, P varied from 20 to 47%, and residual P ranged from 9 to 31%. Deoxyribonucleic acid (1-5%) and -inositol hexakisphosphate (-IHP, 4-10%) were the major P forms from P-NMR. Orthophosphate diesters determined by NMR were significantly correlated with labile NaHCO-P in SF ( > 0.981; < 0.001). Soil P was shown to be predominantly associated with iron and soluble calcium (Ca) by XANES. Furthermore, XANES identified hydroxyapatite in the soil receiving the highest rates of Ca-phosphate fertilizer, which had the highest HCl-P pool by SF, and also identified IHP (7%) in the soil with the highest proportion of -IHP from P-NMR. These results strongly suggest that a combined use of SF, solution P-NMR, and P K-edge XANES spectroscopy will provide the comprehensive information about soil P species needed for effective soil P management.

Substrate‐driven conformational changes in ClC‐ec1 observed by fluorine NMR

The CLC ‘Cl− channel’ family consists of both Cl−/H+ antiporters and Cl− channels. Although CLC channels can undergo large, conformational changes involving cooperativity between the two protein subunits, it has been hypothesized that conformational changes in the antiporters may be limited to small movements localized near the Cl− permeation pathway. However, to date few studies have directly addressed this issue, and therefore little is known about the molecular movements that underlie CLC‐mediated antiport. The crystal structure of the Escherichia coli antiporter ClC‐ec1 provides an invaluable molecular framework, but this static picture alone cannot depict the protein movements that must occur during ion transport. In this study we use fluorine nuclear magnetic resonance (NMR) to monitor substrate‐induced conformational changes in ClC‐ec1. Using mutational analysis, we show that substrate‐dependent 19F spectral changes reflect functionally relevant protein movement occurring at the ClC‐ec1 dimer interface. Our results show that conformational change in CLC antiporters is not restricted to the Cl− permeation pathway and show the usefulness of 19F NMR for studying conformational changes in membrane proteins of known structure.

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF3‐S‐ACP, was developed as a 19F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.