To perform their physiological functions, amino methyl propionic acid receptors (AMPARs) cycle through active, resting, and desensitized states, and dysfunction in AMPAR activity is associated with various neurological disorders. Transitions among AMPAR functional states, however, are largely uncharacterized at atomic resolution and are difficult to examine experimentally. Here, we report long-timescale molecular dynamics simulations of dimerized AMPAR ligand-binding domains (LBDs), whose conformational changes are tightly coupled to changes in AMPAR functional states, in which we observed LBD dimer activation and deactivation upon ligand binding and unbinding at atomic resolution. Importantly, we observed the ligand-bound LBD dimer transition from the active conformation to several other conformations, which may correspond with distinct desensitized conformations. We also identified a linker region whose structural rearrangements heavily affected the transitions to and among these putative desensitized conformations, and confirmed, using electrophysiology experiments, the importance of the linker region in these functional transitions.

Huntington's disease neurodegeneration occurs when the number of consecutive glutamines in the huntingtin exon-1 (HTTExon1) exceeds a pathological threshold of 35. The sequence homogeneity of HTTExon1 reduces the signal dispersion in NMR spectra, hampering its structural characterization. By simultaneously introducing three isotopically labeled glutamines in a site-specific manner in multiple concatenated samples, 18 glutamines of a pathogenic HTTExon1 with 36 glutamines were unambiguously assigned. Chemical shift analyses indicate the α-helical persistence in the homorepeat and the absence of an emerging toxic conformation around the pathological threshold. Using the same type of samples, the recognition mechanism of Hsc70 molecular chaperone has been investigated, indicating that it binds to the N17 region of HTTExon1, inducing the partial unfolding of the poly-Q. The proposed strategy facilitates high-resolution structural and functional studies in low-complexity regions.

The parathyroid hormone (PTH) 1 receptor (PTH1R) is a G protein-coupled receptor (GPCR) that regulates skeletal development and calcium homeostasis. Here, we describe cryo-EM structures of the PTH1R in complex with fragments of the two hormones, PTH and PTH-related protein, the drug abaloparatide, as well as the engineered tool compounds, long-acting PTH (LA-PTH) and the truncated peptide, M-PTH(1-14). We found that the critical N terminus of each agonist engages the transmembrane bundle in a topologically similar fashion, reflecting similarities in measures of Gαs activation. The full-length peptides induce subtly different extracellular domain (ECD) orientations relative to the transmembrane domain. In the structure bound to M-PTH, the ECD is unresolved, demonstrating that the ECD is highly dynamic when unconstrained by a peptide. High resolutions enabled identification of water molecules near peptide and G protein binding sites. Our results illuminate the action of orthosteric agonists of the PTH1R.

The genotoxin colibactin produced by Escherichia coli is involved in the development of colorectal cancers. This secondary metabolite is synthesized by a multi-protein machinery, mainly composed of non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) enzymes. In order to decipher the function of a PKS-NRPS hybrid enzyme implicated in a key step of colibactin biosynthesis, we conducted an extensive structural characterization of the ClbK megaenzyme. Here we present the crystal structure of the complete trans-AT PKS module of ClbK showing structural specificities of hybrid enzymes. In addition, we report the SAXS solution structure of the full-length ClbK hybrid that reveals a dimeric organization as well as several catalytic chambers. These results provide a structural framework for the transfer of a colibactin precursor through a PKS-NRPS hybrid enzyme and can pave the way for re-engineering PKS-NRPS hybrid megaenzymes to generate diverse metabolites with many applications.

γ-Hemolysin (γ-HL) is a hemolytic and leukotoxic bicomponent β-pore-forming toxin (β-PFT), a potent virulence factor from the Staphylococcus aureus Newman strain. In this study, we performed single-particle cryoelectron microscopy (cryo-EM) of γ-HL in a lipid environment. We observed clustering and square lattice packing of octameric HlgAB pores on the membrane bilayer and an octahedral superassembly of octameric pore complexes that we resolved at resolution of 3.5 Å. Our atomic model further demonstrated the key residues involved in hydrophobic zipping between the rim domains of adjacent octameric complexes, providing additional structural stability in PFTs post oligomerization. We also observed extra densities at the octahedral and octameric interfaces, providing insights into the plausible lipid-binding residues involved for HlgA and HlgB components. Furthermore, the hitherto elusive N-terminal region of HlgA was also resolved in our cryo-EM map, and an overall mechanism of pore formation for bicomponent β-PFTs is proposed.

Tryptophan hydroxylase 2 (TPH2) catalyzes the rate-limiting step in serotonin biosynthesis in the brain. Consequently, regulation of TPH2 is relevant for serotonin-related diseases, yet the regulatory mechanism of TPH2 is poorly understood and structural and dynamical insights are missing. We use NMR spectroscopy to determine the structure of a 47 N-terminally truncated variant of the regulatory domain (RD) dimer of human TPH2 in complex with L-Phe, and show that L-Phe is the superior RD ligand compared with the natural substrate, L-Trp. Using cryo-EM, we obtain a low-resolution structure of a similarly truncated variant of the complete tetrameric enzyme with dimerized RDs. The cryo-EM two-dimensional (2D) class averages additionally indicate that the RDs are dynamic in the tetramer and likely exist in a monomer-dimer equilibrium. Our results provide structural information on the RD as an isolated domain and in the TPH2 tetramer, which will facilitate future elucidation of TPH2's regulatory mechanism.

Structures and dynamics of transmembrane (TM) receptor regions are key to understanding their signaling mechanism across membranes. Here we examine configurations of TM region dimers, assembled using the recent Martini 3 force field for coarse-grain (CG) molecular dynamics simulations. At first glance, our results show only a reasonable agreement with ab initio predictions using PREDDIMER and AlphaFold2 Multimer and with nuclear magnetic resonance (NMR)-derived structures. 5 of 11 CG TM structures are similar to the NMR structures (within <3.5 Å root-mean-square deviation [RMSD]) compared with 10 and 9 using PREDDIMER and AlphaFold2, respectively (with 8 structures of the later within 1.5 Å). Surprisingly, AlphaFold2 predictions are closer to NMR structures when the 2001 instead of 2020 database is used for training. The CG simulations reveal that alternative configurations of TM dimers readily interconvert with a predominant population. The implications for transmembrane signaling are discussed, including for the development of peptide-based pharmaceuticals.