Like other photoreceptor proteins, the archaerhodopsin-3 (AR3) protein has a desensitized, inactive state which is formed in the prolonged absence of light. This dark-adapted (DA) state must be converted to the light-adapted (LA) or resting state, before the protein can generate a proton motive force. In general, receptor desensitization is commonly achieved through reversible covalent or non-covalent modifications, which typically modulate intramolecular bonding networks to stabilize a conformation that is distinct from the active resting or ground state.

Here, we present high-resolution crystal structures of the LA and DA states of AR3, solved to 1.1 Å and 1.3 Å resolution respectively [1]. We observe significant differences between the two states in the dynamics of internal water molecules that are coupled via H-bonds to the retinal Schiff base. These changes modulate the polarity of the environment surrounding the chromophore, influence the relative stability of 13-cis and all-trans retinal isomers and facilitate the conversion between the two forms. These crystal structures also allow us to gain a better understanding of the extent to which the conformation of the chromophore is coupled to the networks of internal water molecules, see Fig. 1. They highlight how minimal displacements of charged and hydrophilic groups within the low dielectric environment of the membrane can induce changes in ligand conformation and vice versa. Finally, these structures also provide high-resolution structural information that increases our understanding of the mechanism of H+ translocation by AR3, and will facilitate the design of further, more efficient AR3 mutants for applications in optogenetics.

Membrane proteins with multiple transmembrane domains play critical roles in cell physiology, but little is known about the machinery coordinating their biogenesis at the endoplasmic reticulum. Here we describe a ~ 360 kDa ribosome-associated complex comprising the core Sec61 channel and five accessory factors: TMCO1, CCDC47 and the Nicalin-TMEM147-NOMO complex. Cryo-electron microscopy reveals a large assembly at the ribosome exit tunnel organized around a central membrane cavity. Similar to protein-conducting channels that facilitate movement of transmembrane segments, cytosolic and luminal funnels in TMCO1 and TMEM147, respectively, suggest routes into the central membrane cavity. High-throughput mRNA sequencing shows selective translocon engagement with hundreds of different multi-pass membrane proteins. Consistent with a role in multi-pass membrane protein biogenesis, cells lacking different accessory components show reduced levels of one such client, the glutamate transporter EAAT1. These results identify a new human translocon and provide a molecular framework for understanding its role in multi-pass membrane protein biogenesis.

Obesity is a global epidemic causing increased morbidity and impaired quality of life. The melanocortin receptor 4 (MC4R) is a G protein-coupled receptor that plays a key role in regulation of food consumption and energy expenditure in the central nervous system, thus becoming a prime target for anti-obesity drugs. We present the cryo-EM structure of the human MC4R-G_s signaling complex bound to the agonist setmelanotide, a cyclic peptide recently approved for the treatment of obesity. The work reveals the mechanism of MC4R activation, highlighting a molecular switch that initiates satiation signaling. Coupled to signaling assays and molecular dynamics simulations, the structure demonstrates the role calcium plays in receptor activation, but not inhibition. Altogether, these results fill a gap in understanding MC4R activation and provide guidelines for a structure-based design of novel and more efficient weight management drugs.

The Commander complex is a conserved regulator of intracellular trafficking. This ancient complex consists of 15 core components as well as a number of associated proteins that can be sub-divided into 3 categories: the COMMD/Coiled coil domain containing protein (CCDC) 22/CCDC93 (CCC) complex, the Retriever complex (a distant relative of the Retromer complex) and a number of associated proteins. Functionally Commander couples to Sorting Nexin 17 (SNX17) to facilitate the recycling of over 100 cell surface proteins including key receptors such as, LDLR, LRP1, p-Selectin and the amyloid precursor protein. In addition, Commander dysfunction has been linked to various disease pathologies including X-linked intellectual disability. It is therefore crucial to understand the structure, mechanism and function of this complex, an understanding that has remain largely elusive to date.

In the work presented here I have followed on from our previously published work on the structure of individual COMMD proteins [1] by reconstituting the core COMMD protein complex in vitro. The successful reconstitution of this complex using a polycistronic E. coli vector has allowed for the identification of two distinct subcomplexes of the COMMD family, a result supported by in vivo experiments conducted using quantitative mass spectrometry and a panel of COMMD knockout eHAP cell lines.

Identification of these distinct subcomplexes also allowed for the resolution of a ~3.3 Å crystal structure of COMMD subcomplex B. Revealing an intimately assembled tetramer, with interfaces along the β-strands of the highly conserved COMM domain and more subunit specific interactions between the loops on the COMM domain and the more variable helical N-terminal domain (See Figure 1). Intriguingly despite the formation of distinct subcomplexes in in vitro expression there is sufficient evidence to suggest COMMDs exist as a decameric assembly in the endogenous cellular environment. With this in mind we have used the aforementioned structure to model this assembly, revealing a geometrically perfect star assembly (See Figure 2).

[1] Healy, M. D., Hospenthal, M. K., Hall, R. J., Chandra, M., Chilton, M., Tillu, V., Chen, K., Celligoi, D. J., McDonald, F. J., Cullen, P. J., Lott, J. S., Collins, B. M., Ghai, R. (2018). eLIFE. 7, e35895.

Cholesterol-dependent cytolysins (CDCs) are bacterial pore-forming toxins that are secreted as soluble monomers and oligomerise into large circular pre-pores on the surface of cholesterol-rich membranes. Various structural changes and transitions results in insertion of β-hairpins into the lipid bilayer, forming a large β-barrel pore that results in cell lysis. We have identified a widely distributed family of bacterial proteins that share substantial structural similarity with CDCs. We have the named these proteins the “CDC-like” (CDCL) proteins, which derive from predominantly Gram-negative bacterial phyla. Many of these CDLS exist as homologous pairs. One partner of the CDCL pair, termed CDCL long, consists of four domains: three similar to CDCs and a unique fourth domain. The other partner, CDCL short, possesses three domains, all similar to CDCs. One CDCL pair, referred to as ALY long (ALY^L) and ALY short (ALY^S), originate from the species Elizabethkingia anophelis; an emerging and opportunistic pathogen of unknown virulence and transmission. We have solved the crystal structure of ALY^L, which consists of characteristic CDC domain 1 – 3 structure; however, domain 4 differs from that of CDCs significantly. In the presence of lipids, ALY^S forms a circular oligomer, while ALY^S in combination with ALY^L forms a functional pore capable of inserting into membranes. Unlike CDCs, formation of these pores is not cholesterol dependent. To determine the atomic structure of ALY pores, cryo-EM single-particle analysis is currently being pursued. Further studies include HDX-MS and lipid binding analysis to study the conformational changes and lipid binding details of pore formation. An understanding of pore formation by ALY may yield new knowledge of Elizabethkingia anophelis virulence, in addition to providing a system that could be applied to biotechnological applications.

Humans are chronically exposed to mixtures of xenobiotics referred to as endocrine-disrupting chemicals (EDCs). A vast body of literature links exposure to these chemicals with increased incidences of reproductive, metabolic, or neurological disorders. Moreover, recent data demonstrate that, when used in combination, chemicals have outcomes that cannot be predicted from their individual behavior. In its heterodimeric form with the retinoid X receptor (RXR), the pregnane X receptor (PXR) plays an essential role in controlling the mammalian xenobiotic response and mediates both beneficial and detrimental effects. Our previous work shed light on a mechanism by which a binary mixture of xenobiotics activates PXR in a synergistic fashion. Structural analysis revealed that mutual stabilization of the compounds within the ligand-binding pocket of PXR accounts for the enhancement of their binding affinity. In order to identify and characterize additional active mixtures, we combined a set of cell-based, biophysical, structural, and in vivo approaches. Our study reveals features that confirm the binding promiscuity of this receptor and its ability to accommodate bipartite ligands. We reveal previously unidentified binding mechanisms involving dynamic structural transitions and covalent coupling and report four binary mixtures eliciting graded synergistic activities. Last, we demonstrate that the robust activity obtained with two synergizing PXR ligands can be enhanced further in the presence of RXR environmental ligands. Our study reveals insights as to how low-dose EDC mixtures
may alter physiology through interaction with RXR–PXR and potentially several other nuclear receptor heterodimers.