Search

The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.

Potassium-sensitive hypokalaemic and normokalaemic periodic paralysis are inherited skeletal muscle diseases characterized by episodes of flaccid muscle weakness.sup.1,2. They are caused by single mutations in positively charged residues ('gating charges') in the S4 transmembrane segment of the voltage sensor of the voltage-gated sodium channel Na.sub.v1.4 or the calcium channel Ca.sub.v1.1.sup.1,2. Mutations of the outermost gating charges (R1 and R2) cause hypokalaemic periodic paralysis.sup.1,2 by creating a pathogenic gating pore in the voltage sensor through which cations leak in the resting state.sup.3,4. Mutations of the third gating charge (R3) cause normokalaemic periodic paralysis.sup.5 owing to cation leak in both activated and inactivated states.sup.6. Here we present high-resolution structures of the model bacterial sodium channel Na.sub.vAb with the analogous gating-charge mutations.sup.7,8, which have similar functional effects as in the human channels. The R2G and R3G mutations have no effect on the backbone structures of the voltage sensor, but they create an aqueous cavity near the hydrophobic constriction site that controls gating charge movement through the voltage sensor. The R3G mutation extends the extracellular aqueous cleft through the entire length of the activated voltage sensor, creating an aqueous path through the membrane. Conversely, molecular modelling shows that the R2G mutation creates a continuous aqueous path through the membrane only in the resting state. Crystal structures of Na.sub.vAb(R2G) in complex with guanidinium define a potential drug target site. Molecular dynamics simulations illustrate the mechanism of Na.sup.+ permeation through the mutant gating pore in concert with conformational fluctuations of the gating charge R4. Our results reveal pathogenic mechanisms of periodic paralysis at the atomic level and suggest designs of drugs that may prevent ionic leak and provide symptomatic relief from hypokalaemic and normokalaemic periodic paralysis.Crystal structures and molecular dynamics simulations of voltage-gated sodium channels containing mutations that cause hypokalaemic and normokalaemic periodic paralysis indicate the pathogenic mechanisms of these conditions and suggest a target for the design of potential therapeutic and symptomatic drugs., Author(s): Daohua Jiang [sup.1] , Tamer M. Gamal El-Din [sup.1] , Christopher Ing [sup.2] [sup.3] , Peilong Lu [sup.1] [sup.4] , Régis Pomès [sup.2] [sup.3] , Ning Zheng [sup.1] [sup.5] [...]

Dysfunction of the intramembrane protease [gamma]-secretase is thought to cause Alzheimer's disease, with most mutations derived from Alzheimer's disease mapping to the catalytic subunit presenilin 1 (PS1). Here we report an atomic structure of human [gamma]-secretase at 3.4 Å resolution, determined by single-particle cryo-electron microscopy. Mutations derived from Alzheimer's disease affect residues at two hotspots in PS1, each located at the centre of a distinct four transmembrane segment (TM) bundle. TM2 and, to a lesser extent, TM6 exhibit considerable flexibility, yielding a plastic active site and adaptable surrounding elements. The active site of PS1 is accessible from the convex side of the TM horseshoe, suggesting considerable conformational changes in nicastrin extracellular domain after substrate recruitment. Component protein APH-1 serves as a scaffold, anchoring the lone transmembrane helix from nicastrin and supporting the flexible conformation of PS1. Ordered phospholipids stabilize the complex inside the membrane. Our structure serves as a molecular basis for mechanistic understanding of [gamma]-secretase function. The atomic structure of human [gamma]-secretase at 3.4 Å resolution, determined by single-particle cryo-electron microscopy. Human [gamma]-secretase structure The human [gamma]-secretase complex, comprising presenilin 1 (PS1), PEN-2, APH-1, and nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Dysfunction of the enzyme is thought to cause Alzheimer's disease. This paper reports the first atomic structure of an intact human [gamma]-secretase complex, determined at 3.4 Å resolution by cryo-electron microscopy. The structure illustrates how a remarkably plastic active site is positioned inside the membrane through specific interactions of four components of [gamma]-secretase. Alzheimer's disease-derived mutations affect residues that cluster at two hotspots, each located at the center of a distinct four-transmembrane segment bundle in PS1., Author(s): Xiao-chen Bai [sup.1] , Chuangye Yan [sup.2] , Guanghui Yang [sup.2] , Peilong Lu [sup.2] , Dan Ma [sup.2] , Linfeng Sun [sup.2] , Rui Zhou [sup.2] , Sjors [...]

The γ-secretase complex, comprising presenilin 1 (PS1), PEN-2, APH-1 and nicastrin, is a membrane-embedded protease that controls a number of important cellular functions through substrate cleavage. Aberrant cleavage of the amyloid precursor protein (APP) results in aggregation of amyloid-β, which accumulates in the brain and consequently causes Alzheimer's disease. Here we report the three-dimensional structure of an intact human γ-secretase complex at 4.5 Å resolution, determined by cryo-electron-microscopy single-particle analysis. The γ-secretase complex comprises a horseshoe-shaped transmembrane domain, which contains 19 transmembrane segments (TMs), and a large extracellular domain (ECD) from nicastrin, which sits immediately above the hollow space formed by the TM horseshoe. Intriguingly, nicastrin ECD is structurally similar to a large family of peptidases exemplified by the glutamate carboxypeptidase PSMA. This structure serves as an important basis for understanding the functional mechanisms of the γ-secretase complex., γ-Secretase is a membrane-embedded aspartyl protease that cleaves a large number of transmembrane substrate proteins within their membrane-spanning regions, with the cleavage products serving as signalling molecules (1,2). This process [...]

Food-borne hemorrhagic Escherichia coli, exemplified by the strains O157:H7 and O104:H4 (refs 1, 2), require elaborate acid-resistance systems (ARs) (3) to survive the extremely acidic environment such as the stomach (pH ≅ 2). AR2 expels intracellular protons through the decarboxylation of L-glutamate (Glu) in the cytoplasm and exchange of the reaction product γ-aminobutyric acid (GABA) with extracellular Glu. The latter process is mediated by the Glu-GABA antiporter GadC (4,5), a representative member of the amino-acid-polyamine-organocation superfamily of membrane transporters. The functional mechanism of GadC remains largely unknown. Here we show, with the use of an in vitro proteoliposome-based assay, that GadC transports GABA/Glu only under acidic conditions, with no detectable activity at pH values higher than 6.5. We determined the crystal structure of E. coli GadC at 3.1 Å resolution under basic conditions. GadC, comprising 12 transmembrane segments (TMs), exists in a closed state, with its carboxy-terminal domain serving as a plug to block an otherwise inward-open conformation. Structural and biochemical analyses reveal the essential transport residues, identify the transport path and suggest a conserved transport mechanism involving the rigid-body rotation of a helical bundle for GadC and other amino acid antiporters., Other homologous amino-acid-polyamine-organocation (APC) family members include the key AR3 component arginine-agmatine (Arg-Agm) antiporter AdiC (6,7) (Fig. 1a), the lysine-cadaverine antiporter CadB, and the putrescine-ornithine antiporter PotE. Structural analysis of [...]

FocA is a representative member of the formate-nitrite transporter family, which transports short-chain acids in bacteria, archaea, fungi, algae and parasites. The structure and transport mechanism of the formate-nitrite transporter family remain unknown. Here we report the crystal structure of Escherichia coli FocA at 2.25 Å resolution. FocA forms a symmetric pentamer, with each protomer consisting of six transmembrane segments. Despite a lack of sequence homology, the overall structure of the FocA protomer closely resembles that of aquaporin and strongly argues that FocA is a channel, rather than a transporter. Structural analysis identifies potentially important channel residues, defines the channel path and reveals two constriction sites. Unlike aquaporin, FocA is impermeable to water but allows the passage of formate. A structural and biochemical investigation provides mechanistic insights into the channel activity of FocA. doi: 10.1038/nature08610, Formate is a major carbon source for methanogenic archaea such as Methanobacterium formicicum (1,2). It is also a signature metabolite of enteric bacteria under anaerobic conditions, during which pyruvate is [...]