Gaojie Song

Conformational states of the full-length glucagon receptor.

Class B G protein-coupled receptors are composed of an extracellular domain (ECD) and a seven-transmembrane (7TM) domain, and their signalling is regulated by peptide hormones. Using a hybrid structural biology approach together with the ECD and 7TM domain crystal structures of the glucagon receptor (GCGR), we examine the relationship between full-length receptor conformation and peptide ligand binding. Molecular dynamics (MD) and disulfide crosslinking studies suggest that apo-GCGR can adopt both an open and closed conformation associated with extensive contacts between the ECD and 7TM domain. The electron microscopy (EM) map of the full-length GCGR shows how a monoclonal antibody stabilizes the ECD and 7TM domain in an elongated conformation. Hydrogen/deuterium exchange (HDX) studies and MD simulations indicate that an open conformation is also stabilized by peptide ligand binding. The combined studies reveal the open/closed states of GCGR and suggest that glucagon binds to GCGR by a conformational selection mechanism.

Unexpected fold in the circumsporozoite protein target of malaria vaccines

Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an “αTSR” domain. The αTSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but αTSR does not. Interestingly, polymorphic T-cell epitopes map to specialized αTSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

Structure of the full-length glucagon class B G-protein-coupled receptor.

The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and transmembrane domain in an inactive conformation. The two domains are connected by a 12-residue segment termed the stalk, which adopts a β-strand conformation, instead of forming an α-helix as observed in the previously solved structure of the GCGR transmembrane domain. The first extracellular loop exhibits a β-hairpin conformation and interacts with the stalk to form a compact β-sheet structure. Hydrogen–deuterium exchange, disulfide crosslinking and molecular dynamics studies suggest that the stalk and the first extracellular loop have critical roles in modulating peptide ligand binding and receptor activation. These insights into the full-length GCGR structure deepen our understanding of the signalling mechanisms of class B G-protein-coupled receptors.

Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators

The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling.

Shape change in the receptor for gliding motility in Plasmodium sporozoites.

Sporozoite gliding motility and invasion of mosquito and vertebrate host cells in malaria is mediated by thrombospondin repeat anonymous protein (TRAP). Tandem von Willebrand factor A (VWA) and thrombospondin type I repeat (TSR) domains in TRAP connect through proline-rich stalk, transmembrane, and cytoplasmic domains to the parasite actin-dependent motility apparatus. We crystallized fragments containing the VWA and TSR domains from Plasmodium vivax and Plasmodium falciparum in different crystal lattices. TRAP VWA domains adopt closed and open conformations, and bind a Mg2+ ion at a metal ion–dependent adhesion site implicated in ligand binding. Metal ion coordination in the open state is identical to that seen in the open high-affinity state of integrin I domains. The closed VWA conformation associates with a disordered TSR domain. In contrast, the open VWA conformation crystallizes with an extensible β ribbon and ordered TSR domain. The extensible β ribbon is composed of disulfide-bonded segments N- and C-terminal to the VWA domain that are largely drawn out of the closed VWA domain in a 15 Å movement to the open conformation. The extensible β ribbon and TSR domain overlap at a conserved interface. The VWA, extensible β ribbon, and TSR domains adopt a highly elongated overall orientation that would be stabilized by tensile force exerted across a ligand-receptor complex by the actin motility apparatus of the sporozoite. Our results provide insights into regulation of “stick-and-slip” parasite motility and for development of sporozoite subunit vaccines.

Structural insight into acute intermittent porphyria.

Acute intermittent porphyria (AIP), an inherited disease of heme biosynthesis, is one of the most common types of porphyria. Reduced activity of the enzyme porphobilinogen deaminase (PBGD), which catalyzes the sequential condensation of 4 molecules of porphobilinogen to yield preuroporphyrinogen, has been linked to the symptoms of AIP. We have determined the 3‐dimensional structure of human PBGD at 2.2 Å resolution. Analysis of the structure revealed a dipyrromethane cofactor molecule covalently linked to C261, sitting in a positively charged cleft region. In addition to the critical catalytic D99, a number of other residues are seen hydrogen bonded to the cofactor and play a role in catalysis. Sequential entry of 4 pyrrole molecules into the active site is accomplished by movement of the domains around the hinges. H120P mutation resulted in an inactive enzyme, supporting the role of H120 as a hinge residue. Interestingly, some of the mutations of the human PBGD documented in patients suffering from AIP are located far away from the active site. The structure provides insights into the mechanism of action of PBGD at the molecular level and could aid the development of potential drugs for the up‐regulation of PBGD activity in AIP.— Song, G., Li, Y., Cheng, C, Zhao, Y., Gao, A., Zhang, R., Joachimiak, A., Shaw, N., Liu, Z.‐J. Structural insight into acute intermittent porphyria. FASEB J. 23, 396–404 (2009)

Structural Determinants of Binding the Seven-transmembrane Domain of the Glucagon-like Peptide-1 Receptor (GLP-1R)

The glucagon-like peptide-1 receptor (GLP-1R) belongs to the secretin-like (class B) family of G protein-coupled receptors. Members of the class B family are distinguished by their large extracellular domain, which works cooperatively with the canonical seven-transmembrane (7TM) helical domain to signal in response to binding of various peptide hormones. We have combined structure-based site-specific mutational studies with molecular dynamics simulations of a full-length model of GLP-1R bound to multiple peptide ligand variants. Despite the high sequence similarity between GLP-1R and its closest structural homologue, the glucagon receptor (GCGR), nearly half of the 62 stably expressed mutants affected GLP-1R in a different manner than the corresponding mutants in GCGR. The molecular dynamics simulations of wild-type and mutant GLP-1R·ligand complexes provided molecular insights into GLP-1R-specific recognition mechanisms for the N terminus of GLP-1 by residues in the 7TM pocket and explained how glucagon-mimicking GLP-1 mutants restored binding affinity for (GCGR-mimicking) GLP-1R mutants. Structural analysis of the simulations suggested that peptide ligand binding mode variations in the 7TM binding pocket are facilitated by movement of the extracellular domain relative to the 7TM bundle. These differences in binding modes may account for the pharmacological differences between GLP-1 peptide variants.

Mechanism of the Rpn13-induced activation of Uch37

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other’s ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37ΔHb,Hc,KEKE, a truncation removal of the C-terminal extension region (residues 256–329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270–407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.

Structures of the Toxoplasma gliding motility adhesin

Significance Structures of the major adhesin in Toxoplasma show how its ligand-binding domain is displayed above the cell surface at the tip of a stalk with six elongated domains. A prodomain inhibits conformational change from closed to open. An associating protein binds to the most membrane-proximal domain. Comparison with orthologues in Plasmodium reveals remarkable specializations as well as similarities between diverse apicomplexans. Micronemal protein 2 (MIC2) is the key adhesin that supports gliding motility and host cell invasion by Toxoplasma gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1–6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2–M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal structures of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual 1C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state.

Extending the Structural View of Class B GPCRs

The secretin-like class B family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis. Recent structures of various receptors in complex with a variety of orthosteric and allosteric ligands provide fundamental new insights into the function and mechanism of class B GPCRs, including: (i) ligand-induced changes in the relative orientation of the extracellular and transmembrane receptor domains; (ii) intramolecular interaction networks that stabilize conformational changes to accommodate intracellular G protein binding; and (iii) allosteric modulation of receptor activation. This review provides a comprehensive analysis of the structural, biochemical, and pharmacological data on class B GPCRs for understanding ligand-receptor interaction and modulation mechanisms and assessing the potential implications for drug discovery for the secretin-like GPCR family.