Crystal structure of the potassium-importing KdpFABC membrane complex.

Cellular potassium import systems play a fundamental role in osmoregulation, pH homeostasis and membrane potential in all domains of life. In bacteria, the kdp operon encodes a four-subunit potassium pump that maintains intracellular homeostasis, cell shape and turgor under conditions in which potassium is limiting. This membrane complex, called KdpFABC, has one channel-like subunit (KdpA) belonging to the superfamily of potassium transporters and another pump-like subunit (KdpB) belonging to the superfamily of P-type ATPases. Although there is considerable structural and functional information about members of both superfamilies, the mechanism by which uphill potassium transport through KdpA is coupled with ATP hydrolysis by KdpB remains poorly understood. Here we report the 2.9 Å X-ray structure of the complete Escherichia coli KdpFABC complex with a potassium ion within the selectivity filter of KdpA and a water molecule at a canonical cation site in the transmembrane domain of KdpB. The structure also reveals two structural elements that appear to mediate the coupling between these two subunits. Specifically, a protein-embedded tunnel runs between these potassium and water sites and a helix controlling the cytoplasmic gate of KdpA is linked to the phosphorylation domain of KdpB. On the basis of these observations, we propose a mechanism that repurposes protein channel architecture for active transport across biomembranes.

Cryo-EM structure of ABCG5/G8 in complex with modulating antibodies.

The heterodimer of ATP-binding cassette transporter ABCG5 and ABCG8 mediates the excretion of sterols from liver and intestine, playing a critical role in cholesterol homeostasis. Here, we present the cryo-EM structure of ABCG5/G8 in complex with the Fab fragments from two monoclonal antibodies at 3.3Å resolution. The high-resolution structure reveals a unique dimer interface between the nucleotide-binding domains (NBD) of opposing transporters, consisting of an ordered network of salt bridges between the conserved NPXDFXXD motif and serving as a pivot point that may be important for the transport cycle. While mAb 11F4 increases the ATPase activity potentially by stabilization of the NBD dimer formation, mAb 2E10 inhibits ATP hydrolysis, likely by restricting the relative movement between the RecA and helical domain of ABCG8 NBD. Our study not only provides insights into the structural elements important for the transport cycle but also reveals novel epitopes for potential therapeutic interventions.

A novel six-rhodopsin system in a single archaeon.

Microbial rhodopsins, a diverse group of photoactive proteins found in Archaea, Bacteria, and Eukarya, function in photosensing and photoenergy harvesting and may have been present in the resource-limited early global environment. Four different physiological functions have been identified and characterized for nearly 5,000 retinal-binding photoreceptors, these being ion transporters that transport proton or chloride and sensory rhodopsins that mediate light-attractant and/or -repellent responses. The greatest number of rhodopsins previously observed in a single archaeon had been four. Here, we report a newly discovered six-rhodopsin system in a single archaeon, Haloarcula marismortui, which shows a more diverse absorbance spectral distribution than any previously known rhodopsin system, and, for the first time, two light-driven proton transporters that respond to the same wavelength. All six rhodopsins, the greatest number ever identified in a single archaeon, were first shown to be expressed in H. marismortui, and these were then overexpressed in Escherichia coli. The proteins were purified for absorption spectra and photocycle determination, followed by measurement of ion transportation and phototaxis. The results clearly indicate the existence of a proton transporter system with two isochromatic rhodopsins and a new type of sensory rhodopsin-like transducer in H. marismortui.

Molecular Insight into Recognition of the CGRPR Complex by Migraine Prevention Therapy Aimovig (Erenumab).

Calcitonin-gene-related peptide (CGRP) plays a key role in migraine pathophysiology. Aimovig (erenumab; erenumab-aooe in the United States) is the only US Food and Drug Administration (FDA)-approved monoclonal antibody (mAb) therapy against the CGRP receptor (CGRPR) for the prevention of migraine. Aimovig is also the first FDA-approved mAb against a G-protein-coupled receptor (GPCR). Here, we report the architecture and functional attributes of erenumab critical for its potent antagonism against CGRPR. The crystal structure of erenumab in complex with CGRPR reveals a direct ligand-blocking mechanism, enabled by a remarkable 21-residue-long complementary determining region (CDR)-H3 loop, which adopts a tyrosine-rich helix-turn tip and projects into the deep interface of the calcitonin receptor-like receptor (CLR) and RAMP1 subunits of CGRPR. Furthermore, erenumab engages with residues specific to CLR and RAMP1, providing the molecular basis for its exquisite selectivity. Such structural insights reveal the drug action mechanism of erenumab and shed light on developing antibody therapeutics targeting GPCRs.

Cryo-EM structures of NPC1L1 reveal mechanisms of cholesterol transport and ezetimibe inhibition.

The intestinal absorption of cholesterol is mediated by a multipass membrane protein, Niemann-Pick C1-Like 1 (NPC1L1), the molecular target of a cholesterol lowering therapy ezetimibe. While ezetimibe gained Food and Drug Administration approval in 2002, its mechanism of action has remained unclear. Here, we present two cryo-electron microscopy structures of NPC1L1, one in its apo form and the other complexed with ezetimibe. The apo form represents an open state in which the N-terminal domain (NTD) interacts loosely with the rest of NPC1L1, leaving the NTD central cavity accessible for cholesterol loading. The ezetimibe-bound form signifies a closed state in which the NTD rotates ~60°, creating a continuous tunnel enabling cholesterol movement into the plasma membrane. Ezetimibe blocks cholesterol transport by occluding the tunnel instead of competing with cholesterol binding. These findings provide insight into the molecular mechanisms of NPC1L1-mediated cholesterol transport and ezetimibe inhibition, paving the way for more effective therapeutic development.

Molecular mechanism of an antagonistic antibody against glucose-dependent insulinotropic polypeptide receptor.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone involved in regulating glucose and lipid metabolism. GIP receptor (GIPR) antagonism is believed to offer therapeutic potential for various metabolic diseases. Pharmacological intervention of GIPR, however, has limited success due to lack of effective antagonistic reagents. Previously we reported the discovery of two mouse anti-murine GIPR monoclonal antibodies (mAbs) with distinctive properties in rodent models. Here, we report the detailed structural and biochemical characterization of these two antibodies, mAb1 and mAb2. In vitro and in vivo characterizations demonstrated mAb2 is a full GIPR antagonistic antibody and mAb1 is a non-neutralizing GIPR binder. To understand the molecular basis of these two antibodies, we determined the co-crystal structures of GIPR extracellular domain in complex with mAb1 and with mAb2 at resolutions of 2.1 and 2.6 Å, respectively. While the non-neutralizing mAb1 binds to GIPR without competing with the ligand peptide, mAb2 not only partially occludes the ligand peptide binding, but also recognizes the GIPR C-terminal stalk region in a helical conformation that acts as a molecular mimic of the ligand peptide and locks GIPR in a novel auto-inhibited state. Furthermore, administration of mAb2 in diet-induced obesity mice for 7 weeks leads to both reduction in body weight gain and improvement of metabolic profiles. In contrast, mAb1 has no effect on body weight or other metabolic improvement. Together, our studies reveal the unique molecular mechanism of action underlying the superior antagonistic activity of mAb2 and signify the promising therapeutic potential of effective GIPR antagonism for the treatment of metabolic disorders.

Nephrogenic systemic fibrosis in advanced chronic kidney disease: a single hospital's experience in Taiwan.

Nephrogenic systemic fibrosis (NSF) or nephrogenic fibrosing dermopathy (NFD) clinically resembles scleromyxedema which develops in the setting of advanced chronic kidney diseases. Limited data exist about its epidemiology in Asian countries. A total of 153 magnetic resonance imaging (MRI) examinations, including 81 contrast-enhancement, were identified in 127 patients with advanced chronic kidney disease at stage five undergoing MRI or angiography examination between January 2005 and July 2007, in our hospital. The diagnosis of NFD/NSF was established based on clinical manifestation and histopathology. NFD/NSF was diagnosed in none of the 105 patients on haemodialysis but in one of the 22 patients on peritoneal dialysis. This 24-year-old woman was a case of systemic lupus erythematosus since age 15 and who developed skin lesions two months before the initiation of peritoneal dialysis but nine months after four exposures to gadodiamide during MRI study. The skin condition had significantly improved within three months under a combination regimen of systemic pentoxifylline and topical clobetasol propionate ointment, with further amelioration during subsequent treatment with colchicine. Our results lend support to the predisposition of gadolinium-containing contrast agents to the development of NFD/NSF in patients with advanced renal failure, even before the initiation of dialysis. The cause of a lower incidence rate in our series remains to be determined.