Structural basis of the acyl-transfer mechanism of human GPAT1.

Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.

Structural basis for chemokine receptor CCR6 activation by the endogenous protein ligand CCL20.

Chemokines are important protein-signaling molecules that regulate various immune responses by activating chemokine receptors which belong to the G protein-coupled receptor (GPCR) superfamily. Despite the substantial progression of our structural understanding of GPCR activation by small molecule and peptide agonists, the molecular mechanism of GPCR activation by protein agonists remains unclear. Here, we present a 3.3-Å cryo-electron microscopy structure of the human chemokine receptor CCR6 bound to its endogenous ligand CCL20 and an engineered Go. CCL20 binds in a shallow extracellular pocket, making limited contact with the core 7-transmembrane (TM) bundle. The structure suggests that this mode of binding induces allosterically a rearrangement of a noncanonical toggle switch and the opening of the intracellular crevice for G protein coupling. Our results demonstrate that GPCR activation by a protein agonist does not always require substantial interactions between ligand and the 7TM core region.

Design and performance of a superconducting neutron resonance spin flipper.

Despite the challenges, neutron resonance spin echo still holds the promise to improve upon neutron spin echo for the measurement of slow dynamics in materials. We present a bootstrap, radio frequency neutron spin flipper using high temperature superconducting technology capable of flipping neutron spin with either nonadiabatic or adiabatic modes. A frequency of 2 MHz has been achieved, which would achieve an effective field integral of 0.35 T m for a meter of separation in a neutron resonance spin echo spectrometer at the current device specifications. In bootstrap mode, the self-cancellation of Larmor phase aberrations can be achieved with the appropriate selection of the polarity of the gradient coils.

Insights into the ubiquitin transfer cascade catalyzed by the Legionella effector SidC.

The causative agent of Legionnaires' disease, Legionella pneumophila, delivers more than 330 virulent effectors to its host to establish an intracellular membrane-bound organelle called the Legionella containing vacuole. Among the army of Legionella effectors, SidC and its paralog SdcA have been identified as novel bacterial ubiquitin (Ub) E3 ligases. To gain insight into the molecular mechanism of SidC/SdcA as Ub ligases, we determined the crystal structures of a binary complex of the N-terminal catalytic SNL domain of SdcA with its cognate E2 UbcH5C and a ternary complex consisting of the SNL domain of SidC with the Ub-linked E2 UbcH7. These two structures reveal the molecular determinants governing the Ub transfer cascade catalyzed by SidC. Together, our data support a common mechanism in the Ub transfer cascade in which the donor Ub is immobilized with its C-terminal tail locked in an extended conformation, priming the donor Ub for catalysis.

Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.

Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes1-4. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain1,5. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond6-8. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity, and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD 9 in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.

Exploiting the ubiquitin and phosphoinositide pathways by the Legionella pneumophila effector, SidC.

Intracellular bacterial pathogens use secreted effector proteins to alter host cellular processes, with the goal of subverting host defenses and allowing the infection to progress. One such pathogen, Legionella pneumophila, secretes ~300 proteins into its host to alter a number of pathways including intracellular trafficking, phosphoinositide metabolism, and cell signaling. The Legionella effector SidC was previously found to bind to PI(4)P and was responsible for the enrichment of ER proteins and ubiquitinated species on the Legionella-containing vacuoles. Through our recent work, we have discovered that SidC contains a unique N-terminal E3 ubiquitin ligase domain and a C-terminal novel PI(4)P-binding domain. Our results demonstrate that SidC serves to link two distinct cellular pathways, ubiquitin and phosphoinositide. However, how the ubiquitin ligase activity regulates host membrane trafficking events remains to be investigated.

Structure of the Legionella Virulence Factor, SidC Reveals a Unique PI(4)P-Specific Binding Domain Essential for Its Targeting to the Bacterial Phagosome.

The opportunistic intracellular pathogen Legionella pneumophila is the causative agent of Legionnaires' disease. L. pneumophila delivers nearly 300 effector proteins into host cells for the establishment of a replication-permissive compartment known as the Legionella-containing vacuole (LCV). SidC and its paralog SdcA are two effectors that have been shown to anchor on the LCV via binding to phosphatidylinositol-4-phosphate [PI(4)P] to facilitate the recruitment of ER proteins to the LCV. We recently reported that the N-terminal SNL (SidC N-terminal E3 Ligase) domain of SidC is a ubiquitin E3 ligase, and its activity is required for the recruitment of ER proteins to the LCV. Here we report the crystal structure of SidC (1-871). The structure reveals that SidC contains four domains that are packed into an arch-like shape. The P4C domain (PI(4)P binding of SidC) comprises a four α-helix bundle and covers the ubiquitin ligase catalytic site of the SNL domain. Strikingly, a pocket with characteristic positive electrostatic potentials is formed at one end of this bundle. Liposome binding assays of the P4C domain further identified the determinants of phosphoinositide recognition and membrane interaction. Interestingly, we also found that binding with PI(4)P stimulates the E3 ligase activity, presumably due to a conformational switch induced by PI(4)P from a closed form to an open active form. Mutations of key residues involved in PI(4)P binding significantly reduced the association of SidC with the LCV and abolished its activity in the recruitment of ER proteins and ubiquitin signals, highlighting that PI(4)P-mediated targeting of SidC is critical to its function in the remodeling of the bacterial phagosome membrane. Finally, a GFP-fusion with the P4C domain was demonstrated to be specifically localized to PI(4)P-enriched compartments in mammalian cells. This domain shows the potential to be developed into a sensitive and accurate PI(4)P probe in living cells.

The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus.

Compounds capable of stimulating soluble guanylate cyclase (sGC) activity might become important new tools to treat hypertension. While rational design of these drugs would be aided by elucidation of the sGC three-dimensional structure and molecular mechanism of activation, such efforts also require quantities of high quality enzyme that are challenging to produce. We implemented the titerless infected-cells preservation and scale-up (TIPS) methodology to express the heterodimeric sGC. In the TIPS method, small-scale insect cell cultures were first incubated with a recombinant baculovirus which replicated in the cells. The baculovirus-infected insect cells (BIIC) were harvested and frozen prior to cell lysis and the subsequent escape of the newly replicated virus into the culture supernatant. Thawed BIIC stocks were ultimately used for subsequent scale up. As little as 1 mL of BIIC was needed to infect a 100-L insect cell culture, in contrast to the usual 1L of high-titer, virus stock supernatants. The TIPS method eliminates the need and protracted time for titering virus supernatants, and provides stable, concentrated storage of recombinant baculovirus in the form of infected cells. The latter is particularly advantageous for virus stocks which are unstable, such as those for sGC, and provides a highly efficient alternative for baculovirus storage and expression. The TIPS process enabled efficient scale up to 100-L batches, each producing about 200mg of active sGC. Careful adjustment of expression culture conditions over the course of several 100-L runs provided uniform starting titers, specific activity, and composition of contaminating proteins that facilitated development of a process that reproducibly yielded highly active, purified sGC.

X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase.

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.