Person:

Liao, Maofu

Though many functional RNAs fold into intricate and precise 3D architectures, it is difficult to acquire their high-resolution structures. Herein, we present a nanoarchitectural strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy, termed RNA oligomerization-enabled cryo-EM via installing kissing-loops (ROCK), involves RNA construct engineering to install kissing-loop sequences onto functionally nonessential stems for the homomeric self-assembly into closed nanoarchitectures with multiplied molecular weights and mitigated structural flexibility. ROCK enables the cryo-EM reconstruction of the Tetrahymena group I intron at 2.98 Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA including the previously unknown peripheral domains. ROCK is also applied to two smaller RNAs to produce modest-resolution maps, revealing the conformational change of the Azoarcus group I intron and the bound ligand in the FMN riboswitch. Our work unleashes the largely unexplored potential of cryo-EM in RNA structural studies.

The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.

Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome—a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain. Hrd1 is the crucial membrane component for retro-translocation, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction—from the cytosol into or across membranes. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.

Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium, and/or sodium across the membrane. They play critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are the primary targets for some of the most commonly prescribed drugs in clinic. Here, we determined the cryo-EM structure of a Na-K-Cl cotransporter NKCC1, an extensively-studied member of the CCC transporters. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations, and computational studies uncover the ion translocation pathway, ion-binding sites, and key residues for transport activity. These results provide insights into ion selectivity, coupling, and translocation, and establish a framework for understanding the physiological functions of CCC transporters and interpreting disease-related mutations.

Triglycerides (triacylglycerols, TGs) store metabolic energy in organisms and have industrial uses for foods and fuels. Excessive accumulation of TGs in humans causes obesity and is associated with metabolic diseases1. TG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes2-4 whose structures and catalytic mechanisms are unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at 3.0-Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at 3.2-Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction center is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density, possibly an acyl-acceptor molecule, is located within the reaction center, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, give rise to a model of catalysis for DGAT’s generation of TGs.

HIV-1 envelope glycoprotein [Env; trimeric (gp160)3 cleaved to (gp120/gp41)3] interacts with primary receptor CD4 and coreceptor (e.g. chemokine receptor CCR5 or CXCR4) to allow viral entry by catalyzing fusion of viral and target cell membranes. Encounter of gp120 with the coreceptor was thought to be the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-EM structure, at 3.9Å resolution, of a full-length gp120 in complex with a soluble CD4 and an unmodified human CCR5. The V3 loop of gp120 inserts into the chemokine binding pocket formed by seven transmembrane helices of CCR5, which adopts an inactive conformation, while the N-terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41, but it brings the Env trimer close to the target membrane. The extended N-terminus of gp120, gripped by gp41 in the prefusion or CD4-bound Env trimer, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to promote fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry and may guide development of vaccines and therapeutics.

Lipopolysaccharide (LPS) in Gram-negative bacteria is essential for outer membrane formation and antibiotic resistance. The LPS transport proteins A-G (LptA-G) move LPS from the inner to outer membrane. The ATP-binding cassette (ABC) transporter LptB2FG, tightly associated with LptC, extracts LPS out of the inner membrane. The mechanism of the entire LptB2FGC complex and the role of LptC are poorly understood. Here, we used single-particle cryo-EM to characterize the structures of LptB2FG and LptB2FGC in the nucleotide-free and vanadate-trapped states. These cryo-EM structures resolve the bound LPS, reveal the transporter-LPS interactions with side-chain details, and uncover the basis of coupling LPS capture and extrusion to conformational rearrangements of LptB2FGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptB2FG, representing an unprecedented regulatory mechanism for ABC transporters. Our results suggest a role of LptC in coordinating LptB2FG action and the periplasmic Lpt protein interactions to achieve efficient LPS transport.