Structure and regulation of the BsYetJ calcium channel in lipid nanodiscs

BsYetJ is a bacterial homolog of transmembrane BAX inhibitor-1 motif-containing 6 (TMBIM6) membrane protein that plays a key role in the control of calcium homeostasis. However, the BsYetJ (or TMBIM6) structure embedded in a lipid bilayer is uncharacterized, let alone the molecular mechanism of the calcium transport activity. Herein, we report structures of BsYetJ in lipid nanodiscs identified by double electron–electron resonance spectroscopy. Our results reveal that BsYetJ in lipid nanodiscs is structurally different from those crystallized in detergents. We show that BsYetJ conformation is pH-sensitive in apo state (lacking calcium), whereas in a calcium-containing solution it is stuck in an intermediate, inert to pH changes. Only when the transmembrane calcium gradient is established can the calcium-release activity of holo-BsYetJ occur and be mediated by pH-dependent conformational changes, suggesting a dual gating mechanism. Conformational substates involved in the process and a key residue D171 relevant to the gating of calcium are identified. Our study suggests that BsYetJ/TMBIM6 is a pH-dependent, voltage-gated calcium channel.

A rod conformation of the Pyrococcus furiosus Rad50 coiled coil

The Rad50-Mre11 nuclease complex plays a vital role in DNA repair in all domains of life. It recognizes and processes DNA double-strand breaks. Rad50 proteins fold into an extended structure with a ~20-60 nm long coiled coil connecting a globular ABC ATPase domain with a zinc hook dimerization domain. A published structure of an archaeal Rad50 zinc hook shows coiled coils pointing away from each other. Here we present the crystal structure of an alternate conformation displaying co-aligned coiled coils. Archaeal Rad50 may thus switch between rod-shaped and ring-like conformations as recently proposed for a bacterial homolog.

Cryo-EM structures of a lipid-sensitive pentameric ligand-gated ion channel embedded in a phosphatidylcholine-only bilayer

The lipid dependence of the nicotinic acetylcholine receptor from the Torpedo electric organ has long been recognized, and one of the most consistent experimental observations is that, when reconstituted in membranes formed by zwitterionic phospholipids alone, exposure to agonist fails to elicit ion-flux activity. More recently, it has been suggested that the bacterial homolog ELIC (Erwinia chrysanthemi ligand-gated ion channel) has a similar lipid sensitivity. As a first step toward the elucidation of the structural basis of this phenomenon, we solved the structures of ELIC embedded in palmitoyl-oleoyl-phosphatidylcholine- (POPC-) only nanodiscs in both the unliganded (4.1-Å resolution) and agonist-bound (3.3 Å) states using single-particle cryoelectron microscopy. Comparison of the two structural models revealed that the largest differences occur at the level of loop C—at the agonist-binding sites—and the loops at the interface between the extracellular and transmembrane domains (ECD and TMD, respectively). On the other hand, the transmembrane pore is occluded in a remarkably similar manner in both structures. A straightforward interpretation of these findings is that POPC-only membranes frustrate the ECD–TMD coupling in such a way that the “conformational wave” of liganded-receptor gating takes place in the ECD and the interfacial M2–M3 linker but fails to penetrate the membrane and propagate into the TMD. Furthermore, analysis of the structural models and molecular simulations suggested that the higher affinity for agonists characteristic of the open- and desensitized-channel conformations results, at least in part, from the tighter confinement of the ligand to its binding site; this limits the ligand’s fluctuations, and thus delays its escape into bulk solvent.

The Amino Terminus of LeuT Changes Conformation in an Environment Sensitive Manner

Neurotransmitter:sodium symporters are highly expressed in the human brain and catalyze the uptake of substrate through the plasma membrane by using the electrochemical gradient of sodium as the energy source. The bacterial homolog LeuT, a small amino acid transporter isolated from the bacteria Aquifex aeolicus, is the founding member of the family and has been crystallized in three conformations. The N-terminus is structurally well defined and strongly interacts with the transporter core in the outward-facing conformations. However, it could not be resolved in the inward-facing conformation, which indicates enhanced mobility. Here we investigate conformations and dynamics of the N-terminus, by combining molecular dynamics simulations with experimental verification using distance measurements and accessibility studies. We found strongly increased dynamics of the N-terminus, but also that helix TM1A is subject to enhanced mobility. TM1A moves towards the transporter core in the membrane environment, reaching a conformation that is closer to the structure of LeuT with wild type sequence, indicating that the mutation introduced to create the inward-facing structure might have altered the position of helix TM1A. The mobile N-terminus avoids entering the open vestibule of the inward-facing state, as accessibility studies do not show any reduction of quenching by iodide of a fluorophore attached to the N-terminus.

SLC6 neurotransmitter transporter family (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Members of the solute carrier family 6 (SLC6) of sodium- and (sometimes chloride-) dependent neurotransmitter transporters [29, 22, 70] are primarily plasma membrane located and may be divided into four subfamilies that transport monoamines, GABA, glycine and neutral amino acids, plus the related bacterial NSS transporters [99]. The members of this superfamily share a structural motif of 10 TM segments that has been observed in crystal structures of the NSS bacterial homolog LeuTAa, a Na+-dependent amino acid transporter from Aquiflex aeolicus [126] and in several other transporter families structurally related to LeuT [45].

Pseudomonas aeruginosaesterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization

The human membrane-bound α/β-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein fromPseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence ofn-octyl β-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified fromEscherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space groupI4122 and diffracted to a resolution of 2.54 Å. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.