Structural basis of calmodulin modulation of the rod cyclic nucleotide-gated channel.

Calmodulin (CaM) regulates many ion channels to control calcium entry into cells, and mutations that alter this interaction are linked to fatal diseases. The structural basis of CaM regulation remains largely unexplored. In retinal photoreceptors, CaM binds to the CNGB subunit of cyclic nucleotide-gated (CNG) channels and, thereby, adjusts the channel's Cyclic guanosine monophosphate (cGMP) sensitivity in response to changes in ambient light conditions. Here, we provide the structural characterization for CaM regulation of a CNG channel by using a combination of single-particle cryo-electron microscopy and structural proteomics. CaM connects the CNGA and CNGB subunits, resulting in structural changes both in the cytosolic and transmembrane regions of the channel. Cross-linking and limited proteolysis-coupled mass spectrometry mapped the conformational changes induced by CaM in vitro and in the native membrane. We propose that CaM is a constitutive subunit of the rod channel to ensure high sensitivity in dim light. Our mass spectrometry-based approach is generally relevant for studying the effect of CaM on ion channels in tissues of medical interest, where only minute quantities are available.

Continuous population-level monitoring of SARS-CoV-2 seroprevalence in a large European metropolitan region.

Effective public health measures against SARS-CoV-2 require granular knowledge of population-level immune responses. We developed a Tripartite Automated Blood Immunoassay (TRABI) to assess the IgG response against three SARS-CoV-2 proteins. We used TRABI for continuous seromonitoring of hospital patients and blood donors (n = 72'250) in the canton of Zurich from December 2019 to December 2020 (pre-vaccine period). We found that antibodies waned with a half-life of 75 days, whereas the cumulative incidence rose from 2.3% in June 2020 to 12.2% in mid-December 2020. A follow-up health survey indicated that about 10% of patients infected with wildtype SARS-CoV-2 sustained some symptoms at least twelve months post COVID-19. Crucially, we found no evidence of a difference in long-term complications between those whose infection was symptomatic and those with asymptomatic acute infection. The cohort of asymptomatic SARS-CoV-2-infected subjects represents a resource for the study of chronic and possibly unexpected sequelae.

The structure of cyclic nucleotide-gated channels in rod and cone photoreceptors.

Cyclic nucleotide-gated (CNG) channels play a central role in rod and cone photoreceptors of the vertebrate retina. In photoreceptors, light triggers a series of biochemical reactions that ultimately close CNG channels and evoke a brief voltage pulse, a signal that is later passed on to the brain. Malfunction of CNG channels can lead to loss of vision. Thus, understanding their function in atomic and mechanistic detail is important. Because of the complex subunit stoichiometry of these channels, elucidation of their structure has proved challenging. Recently, several cryoelectron microscopy (EM) structures of rod and cone CNG channels revealed unexpected structural features. We compare these structures side by side and highlight similarities and differences in key structural elements. We discuss the implications of the channels' structure for questions about their gating, ion permeation, and modulation. These results inform new strategies to further characterize the structural basis of CNG channels functioning in rods and cones.

Structural basis of the partially open central gate in the human CNGA1/CNGB1 channel explained by additional density for calmodulin in cryo-EM map.

The recently reported structure of the human CNGA1/CNGB1 CNG channel in the open state (Xue et al., 2021a) shows that one CNGA1 and one CNGB1 subunit do not open the central hydrophobic gate completely upon cGMP binding. This is different from what has been reported for CNGA homomeric channels (Xue et al., 2021b; Zheng et al., 2020). In seeking to understand how this difference is due to the presence of the CNGB1 subunit, we find that the deposited density map (Xue et al., 2021a) (EMDB 24465) contains an additional density not reported in the images of the original publication. This additional density fits well the structure of calmodulin (CaM), and it unambiguously connects the newly identified D-helix of CNGB1 to one of the CNGA1 helices (A1R) participating in the coiled-coil region. Interestingly, the CNGA1 subunit that engages in the interaction with this additional density is the one that, together with CNGB1, does not open completely the central gate. The sequence of the D-helix of CNGB1 contains a known CaM-binding site of exquisitely high affinity - named CaM2 (Weitz et al., 1998) -, and thus the presence of CaM in that region is not surprising. The mechanism through which CaM reduces currents across the membrane by acting on the native channel (Bauer, 1996; Hsu and Molday, 1993; Weitz et al., 1998) remains unclear. We suggest that the presence of CaM may explain the partially open central gate reported by Xue et al. (2021a). The structure of the open and closed states of the CNGA1/CNGB1 channel may be different with and without CaM present.

The structure of the native CNGA1/CNGB1 CNG channel from bovine retinal rods.

In rod photoreceptors of the retina, the cyclic nucleotide-gated (CNG) channel is composed of three CNGA and one CNGB subunits, and it closes in response to light activation to generate an electrical signal that is conveyed to the brain. Here we report the cryo-EM structure of the closed state of the native rod CNG channel isolated from bovine retina. The structure reveals differences between CNGA1 and CNGB1 subunits. Three CNGA1 subunits are tethered at their C terminus by a coiled-coil region. The C-helix in the cyclic nucleotide-binding domain of CNGB1 features a different orientation from that in the three CNGA1 subunits. The arginine residue R994 of CNGB1 reaches into the ionic pathway and blocks the pore, thus introducing an additional gate, which is different from the central hydrophobic gate known from homomeric CNGA channels. These results address the long-standing question of how CNGB1 subunits contribute to the function of CNG channels in visual and olfactory neurons.

A set of common movements within GPCR-G-protein complexes from variability analysis of cryo-EM datasets.

G-protein coupled receptors (GPCRs) are among the most versatile signal transducers in the cell. Once activated, GPCRs sample a large conformational space and couple to G-proteins to initiate distinct signaling pathways. The dynamical behavior of GPCR-G-protein complexes is difficult characterize structurally, and it might hinder obtaining routine high-resolution density maps in single-particle reconstructions. Here, we used variability analysis on the rhodopsin-Gi-Fab16 complex cryo-EM dataset, and the results provide insights into the dynamic nature of the receptor-complex interaction. We compare the outcome of this analysis with recent results obtained on the cannabinoid-Gi- and secretin-Gs-receptor complexes. Despite differences related to the biochemical compositions of the three samples, a set of consensus movements emerges. We anticipate that systematic variability analysis on GPCR-G-protein complexes may provide useful information not only at the biological level, but also for improving the preparation of more stable samples for cryo-EM single-particle analysis.

Cryo-EM structure of the rhodopsin-Gαi-ßγ complex reveals binding of the rhodopsin C-terminal tail to the gß subunit.

One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gß subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gß as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.

Charge Interactions Can Dominate Coupled Folding and Binding on the Ribosome.

Interactions between emerging nascent polypeptide chains and the ribosome can modulate cotranslational protein folding. However, it has remained unclear how such interactions can affect the binding of nascent chains to their cellular targets. We thus investigated on the ribosome the interaction between two intrinsically disordered proteins of opposite charge, ACTR and NCBD, which form a high-affinity complex in a coupled folding-and-binding reaction. Using fluorescence correlation spectroscopy and arrest-peptide-mediated force measurements in vitro and in vivo, we find that the ACTR-NCBD complex can form cotranslationally but only with ACTR as the nascent chain and NCBD free in solution, not vice versa. We show that this surprising asymmetry in behavior is caused by pronounced charge interactions: attraction of the positively charged nascent chain of NCBD to the negatively charged ribosomal surface competes with complex formation and prevents ACTR binding. In contrast, the negatively charged nascent ACTR is repelled by the ribosomal surface and thus remains available for productively binding its partner. Electrostatic interactions may thus be more important for cotranslational folding and binding than previously thought.

Understanding GPCR Recognition and Folding from NMR Studies of Fragments.

Cotranslational protein folding is a vectorial process, and for membrane proteins, N-terminal helical segments are the first that become available for membrane insertion. While structures of many G-protein coupled receptors (GPCRs) in various states have been determined, the details of their folding pathways are largely unknown. The seven transmembrane (TM) helices of GPCRs often contain polar residues within the hydrophobic core, and some of the helices in isolation are predicted to be only marginally stable in a membrane environment. Here we review our efforts to describe how marginally hydrophobic TM helices of GPCRs integrate into the membrane in absence of all compensating interhelical contacts, ideally capturing early biogenesis events. To this end, we use truncated GPCRs, here referred to as fragments. We present data from the human Y4 and the yeast Ste2p receptors in detergent micelles derived from solution NMR techniques. We find that secondary structure in the fragments is similar to corresponding parts of the entire receptors. However, uncompensated polar or charged residues destabilize the helices, and prevent proper integration into the lipid bilayer, in agreement with the biophysical scales from Wimley and White for the partitioning of amino acids into the membrane-interior. We observe that the stability and integration of single TM helices is improved by adding neighboring helices. We describe a topology study, in which all possible forms of the Y4 receptor were made so that the entire receptor is truncated from the N-terminus by one TM helix at a time. We discover that proteins with an increasing number of helices assume a more defined topology. In a parallel study, we focused on the role of extracellular loops in ligand recognition. We demonstrate that transferring all loops of the human Y1 receptor onto the E. coli outer membrane protein OmpA in a suitable topology results in a chimeric receptor that displays, albeit reduced, affinity and specificity for the cognate ligand. Our data indicate that not all TM helices will spontaneously insert into the helix, and we suggest that at least for some GPCRs, N-terminal segments might remain associated with the translocon until their interacting partners are biosynthesized.

Efficient Screening and Optimization of Membrane Protein Production in Escherichia coli.

Escherichia coli is one of the most widely used expression hosts for membrane proteins. However, establishing conditions for its recombinant production of membrane proteins remains difficult. Attempts to produce membrane proteins frequently result in either no expression or expression as misfolded aggregates. We developed an efficient pipeline for improving membrane protein overexpression in E. coli that is based on two approaches. The first involves transcriptional fusions, small additional RNA sequences upstream of the target open reading frame, to overcome no or poor overall expression levels. The other is based on a tunable promoter in combination with a fusion to green fluorescent protein serving as a reporter for the folding state of the target membrane protein. The latter combination allows adjusting the membrane protein expression rate to the downstream folding capacity, in order to decrease the formation of protein aggregates. This pipeline has proven successful for the efficient and parallel optimization of a diverse set of membrane proteins.

Mutational analysis of protein folding inside the ribosome exit tunnel.

Recent work has demonstrated that cotranslational folding of proteins or protein domains in, or in the immediate vicinity of, the ribosome exit tunnel generates a pulling force on the nascent polypeptide chain that can be detected using a so-called translational arrest peptide (AP) engineered into the nascent chain as a force sensor. Here, we show that AP-based force measurements combined with systematic Ala and Trp scans of a zinc-finger domain that folds in the exit tunnel can be used to identify the residues that are critical for intraribosomal folding. Our results suggest a general approach to characterize the folded state(s) that may form as a protein domain moves progressively down the ribosome exit tunnel.

Small protein domains fold inside the ribosome exit tunnel.

Cotranslational folding of small protein domains within the ribosome exit tunnel may be an important cellular strategy to avoid protein misfolding. However, the pathway of cotranslational folding has so far been described only for a few proteins, and therefore, it is unclear whether folding in the ribosome exit tunnel is a common feature for small protein domains. Here, we have analyzed nine small protein domains and determined at which point during translation their folding generates sufficient force on the nascent chain to release translational arrest by the SecM arrest peptide, both in vitro and in live E. coli cells. We find that all nine protein domains initiate folding while still located well within the ribosome exit tunnel.

Cotranslational Protein Folding inside the Ribosome Exit Tunnel.

At what point during translation do proteins fold? It is well established that proteins can fold cotranslationally outside the ribosome exit tunnel, whereas studies of folding inside the exit tunnel have so far detected only the formation of helical secondary structure and collapsed or partially structured folding intermediates. Here, using a combination of cotranslational nascent chain force measurements, inter-subunit fluorescence resonance energy transfer studies on single translating ribosomes, molecular dynamics simulations, and cryoelectron microscopy, we show that a small zinc-finger domain protein can fold deep inside the vestibule of the ribosome exit tunnel. Thus, for small protein domains, the ribosome itself can provide the kind of sheltered folding environment that chaperones provide for larger proteins.

Bicistronic mRNAs to enhance membrane protein overexpression.

Functional overexpression of membrane proteins is essential for their structural and functional characterization. However, functional overexpression is often difficult to achieve, and frequently either no expression or expression as misfolded aggregates is observed. We present an approach for improving the functional overexpression of membrane proteins in Escherichia coli using transcriptional fusions. The method involves the use of a small additional RNA sequence upstream to the RNA sequence of the target membrane protein and results in the production of a bicistronic mRNA. In contrast to the common approach of translational fusions to enhance protein expression, transcriptional fusions do not require protease treatment and subsequent removal of the fusion protein. Using this strategy, we observed improvements in the quantity and/or the quality of the produced material for several membrane proteins to levels compatible with structural studies. Our analysis revealed that translation of the upstream RNA sequence was not essential for increased expression. Rather, the sequence itself had a large impact on protein yields, suggesting that alternative folding of the transcript was responsible for the observed effect.

Mistic's membrane association and its assistance in overexpression of a human GPCR are independent processes.

The interaction of the Bacillus subtilis protein Mistic with the bacterial membrane and its role in promoting the overexpression of other membrane proteins are still matters of debate. In this study, we aimed to determine whether individual helical fragments of Mistic are sufficient for its interaction with membranes in vivo and in vitro. To this end, fragments encompassing each of Mistic's helical segments and combinations of them were produced as GFP-fusions, and their cellular localization was studied in Escherichia coli. Furthermore, peptides corresponding to the four helical fragments were synthesized by solid-phase peptide synthesis, and their ability to acquire secondary structure in a variety of lipids and detergents was studied by circular dichroism spectroscopy. Both types of experiments demonstrate that the third helical fragment of Mistic interacts only with LDAO micelles but does not partition into lipid bilayers. Interestingly, the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full-length Mistic as N-terminal fusions to achieve overexpression of a human G-protein-coupled receptor in E. coli, although with different effects on quantity and quality of the protein produced. A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. This information allowed us to discover a highly conserved Shine-Dalgarno sequence in the operon mstX-yugO that is important for downstream translation of the potassium ion channel yugO.

Topogenesis of heterologously expressed fragments of the human Y4 GPCR.

Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.