A behavioral protocol to assess the relationship between three cognitive biases and future depression severity.

According to the cognitive model of depression, memory bias, interpretation bias, and attention bias are associated with the development and maintenance of depression. Here, we present a protocol for investigating whether and how the novel coronavirus disease 2019 (COVID-19) pandemic may affect the relationship between current cognitive biases and future depression severity in a population with non-clinical depression. This protocol can also be used in other contexts, including cognitive bias-related studies and depression-related functional magnetic resonance imaging (fMRI) studies. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Identification and architecture of a putative secretion tube across mycobacterial outer envelope.

Tuberculosis-causing mycobacteria have thick cell-wall and capsule layers that are formed from complex structures. Protein secretion across these barriers depends on a specialized protein secretion system, but none has been reported. We show that Mycobacterium tuberculosis Rv3705c and its homologous MSMEG_6251 in Mycobacterium smegmatis are tube-forming proteins in the mycobacterial envelope (TiME). Crystallographic and cryo-EM structures of these two proteins show that both proteins form rotationally symmetric rings. Two layers of TiME rings pack together in a tail-to-tail manner into a ring-shaped complex, which, in turn, stacks together to form tubes. M. smegmatis TiME was detected mainly in the cell wall and capsule. Knocking out the TiME gene markedly decreased the amount of secreted protein in the M. smegmatis culture medium, and expression of this gene in knocked-out strain partially restored the level of secreted protein. Our structure and functional data thus suggest that TiME forms a protein transport tube across the mycobacterial outer envelope.

Structural mechanism of cooperative activation of the human calcium-sensing receptor by Ca2+ ions and L-tryptophan.

The human calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) responsible for maintaining Ca2+ homeostasis in the blood. The general consensus is that extracellular Ca2+ is the principal agonist of CaSR. Aliphatic and aromatic L-amino acids, such as L-Phe and L-Trp, increase the sensitivity of CaSR towards Ca2+ and are considered allosteric activators. Crystal structures of the extracellular domain (ECD) of CaSR dimer have demonstrated Ca2+ and L-Trp binding sites and conformational changes of the ECD upon Ca2+/L-Trp binding. However, it remains to be understood at the structural level how Ca2+/L-Trp binding to the ECD leads to conformational changes in transmembrane domains (TMDs) and consequent CaSR activation. Here, we determined the structures of full-length human CaSR in the inactive state, Ca2+- or L-Trp-bound states, and Ca2+/L-Trp-bound active state using single-particle cryo-electron microscopy. Structural studies demonstrate that L-Trp binding induces the closure of the Venus flytrap (VFT) domain of CaSR, bringing the receptor into an intermediate active state. Ca2+ binding relays the conformational changes from the VFT domains to the TMDs, consequently inducing close contact between the two TMDs of dimeric CaSR, activating the receptor. Importantly, our structural and functional studies reveal that Ca2+ ions and L-Trp activate CaSR cooperatively. Amino acids are not able to activate CaSR alone, but can promote the receptor activation in the presence of Ca2+. Our data provide complementary insights into the activation of class C GPCRs and may aid in the development of novel drugs targeting CaSR.

Outbreak of COVID-19 altered the relationship between memory bias and depressive degree in nonclinical depression.

The outbreak of the novel coronavirus disease 2019 (COVID-19) has increased concern about people's mental health under such serious stressful situation, especially depressive symptoms. Cognitive biases have been related to depression degree in previous studies. Here, we used behavioral and brain imaging analysis, to determine if and how the COVID-19 pandemic affects the relationship between current cognitive biases and future depression degree and the underlying neural basis in a nonclinical depressed population. An out-expectation result showed that a more negative memory bias was associated with a greater decrease in future depressive indices in nonclinical depressed participants during the COVID-19 pandemic, which might be due to decreased social stress. These data enhance our understanding of how the depressive degree of nonclinical depressed populations will change during the COVID-19 pandemic and also provide support for social distancing policies from a psychological perspective.

Structural insights into human acid-sensing ion channel 1a inhibition by snake toxin mambalgin1.

Acid-sensing ion channels (ASICs) are proton-gated cation channels that are involved in diverse neuronal processes including pain sensing. The peptide toxin Mambalgin1 (Mamba1) from black mamba snake venom can reversibly inhibit the conductance of ASICs, causing an analgesic effect. However, the detailed mechanism by which Mamba1 inhibits ASIC1s, especially how Mamba1 binding to the extracellular domain affects the conformational changes of the transmembrane domain of ASICs remains elusive. Here, we present single-particle cryo-EM structures of human ASIC1a (hASIC1a) and the hASIC1a-Mamba1 complex at resolutions of 3.56 and 3.90 Å, respectively. The structures revealed the inhibited conformation of hASIC1a upon Mamba1 binding. The combination of the structural and physiological data indicates that Mamba1 preferentially binds hASIC1a in a closed state and reduces the proton sensitivity of the channel, representing a closed-state trapping mechanism.

A genetically encoded small-size fluorescent pair reveals allosteric conformational changes of G proteins upon its interaction with GPCRs by fluorescence lifetime based FRET.

The dynamics of GPCRs (G protein-coupled receptors) coupling for cognate G proteins play a critical role in signal transduction. Herein, we reported a site-specifically labelled small-sized fluorescent pair 7-HC/FlAsH ((7-hydroxycoumarin-4-yl)-ethylglycine/fluorescein arsenical hairpin) for fluorescence lifetime based FRET (fluorescence resonance energy transfer) to reveal conformational differences of Gαi1 (inhibitory G proteins) and Gαs (stimulatory G proteins) upon ß2AR (ß2-adrenergic receptor) coupling. It offers a new generally applicable method to probe protein dynamic interactions or conformational changes.

Synthesis of Disulfide Surrogate Peptides Incorporating Large-Span Surrogate Bridges Through a Native-Chemical-Ligation-Assisted Diaminodiacid Strategy.

The use of synthetic bridges as surrogates for disulfide bonds has emerged as a practical strategy to obviate the poor stability of some disulfide-containing peptides. However, peptides incorporating large-span synthetic bridges are still beyond the reach of existing methods. Herein, we report a native chemical ligation (NCL)-assisted diaminodiacid (DADA) strategy that enables the robust generation of disulfide surrogate peptides incorporating surrogate bridges up to 50 amino acids in length. This strategy provides access to some highly desirable but otherwise impossible-to-obtain disulfide surrogates of bioactive peptide. The bioactivities and structures of the synthetic disulfide surrogates were verified by voltage clamp assays, NMR, and X-ray crystallography; and stability studies established that the disulfide replacements effectively overcame the problems of disulfide reduction and scrambling that often plague these pharmacologically important peptides.

Ion channel modulation by scorpion hemolymph and its defensin ingredients highlights origin of neurotoxins in telson formed in Paleozoic scorpions.

An increasing number of scorpion fossils indicate that the venomous telson developed from the sharp telson in sea scorpions into the extant scorpion-like telson in aquatic scorpions in the Paleozoic Era and then further evolved into the fetal venom system. This hypothesis led us to evaluate the inhibition of scorpion venom-sensitive potassium channels by hemolymph from the scorpion Mesobuthus martensii. Scorpion hemolymph diluted 1:10 inhibited Kv1.1, Kv1.2, Kv1.3 and SK3 potassium channel currents by 76.4%, 90.2%, 85.8%, and 52.8%, respectively. These discoveries encouraged us to investigate the functional similarity between the more ancient defensin ingredients in hemolymph and the evolved neurotoxins in the venom. In addition to the expression of the representative defensin BmKDfsin3 and BmKDfsin5 in both venomous and non-venomous tissues, NMR analysis revealed structural similarities between scorpion defensin and neurotoxin. Functional experiments further indicated that scorpion defensin used the same mechanism as classical neurotoxin to block the neurotoxin-sensitive Kv1.1, Kv1.2, Kv1.3 and SK3 channels. These findings emphasize the likelihood that scorpion defensins evolved into neurotoxins that were adapted to the emergence of the scorpion telson from the sharp telson of sea scorpions into the extant scorpion-like telson in aquatic scorpions in the Paleozoic Era.

Mutation in the intracellular chloride channel CLCC1 associated with autosomal recessive retinitis pigmentosa.

We identified a homozygous missense alteration (c.75C>A, p.D25E) in CLCC1, encoding a presumptive intracellular chloride channel highly expressed in the retina, associated with autosomal recessive retinitis pigmentosa (arRP) in eight consanguineous families of Pakistani descent. The p.D25E alteration decreased CLCC1 channel function accompanied by accumulation of mutant protein in granules within the ER lumen, while siRNA knockdown of CLCC1 mRNA induced apoptosis in cultured ARPE-19 cells. TALEN KO in zebrafish was lethal 11 days post fertilization. The depressed electroretinogram (ERG) cone response and cone spectral sensitivity of 5 dpf KO zebrafish and reduced eye size, retinal thickness, and expression of rod and cone opsins could be rescued by injection of wild type CLCC1 mRNA. Clcc1+/- KO mice showed decreased ERGs and photoreceptor number. Together these results strongly suggest that intracellular chloride transport by CLCC1 is a critical process in maintaining retinal integrity, and CLCC1 is crucial for survival and function of retinal cells.

Cryo-EM structure of the ASIC1a-mambalgin-1 complex reveals that the peptide toxin mambalgin-1 inhibits acid-sensing ion channels through an unusual allosteric effect.

Acid-sensing ion channels (ASICs) are neuronal voltage-independent Na+ channels that are activated by extracellular acidification. ASICs play essential roles in a wide range of physiological processes, including sodium homeostasis, synaptic plasticity, neurodegeneration, and sensory transduction. Mambalgins, a family of three-finger toxins isolated from black mamba venom, specifically inhibit ASICs to exert strong analgesic effects in vivo, thus are thought to have potential therapeutic values against pain. However, the interaction and inhibition mechanism of mambalgin on ASICs remains elusive. Here, we report a cryo-electron microscopy (cryo-EM) structure of chicken ASIC1a (cASIC1a) in complex with mambalgin-1 toxin at 5.4 Å resolution. Our structure provides the first experimental evidence that mambalgin-1 interacts directly with the extracellular thumb domain of cASIC1a, rather than inserting into the acid-sensing pocket, as previously reported. Binding of mambalgin-1 leads to relocation of the thumb domain that could disrupt the acidic pocket of cASIC1a, illustrating an unusual inhibition mechanism of toxins on ASIC channels through an allosteric effect. These findings establish a structural basis for the toxicity of the mambalgins, and provide crucial insights for the development of new optimized inhibitors of ASICs.

Centipedes subdue giant prey by blocking KCNQ channels.

Centipedes can subdue giant prey by using venom, which is metabolically expensive to synthesize and thus used frugally through efficiently disrupting essential physiological systems. Here, we show that a centipede (Scolopendra subspinipes mutilans, ∼3 g) can subdue a mouse (∼45 g) within 30 seconds. We found that this observation is largely due to a peptide toxin in the venom, SsTx, and further established that SsTx blocks KCNQ potassium channels to exert the lethal toxicity. We also demonstrated that a KCNQ opener, retigabine, neutralizes the toxicity of a centipede's venom. The study indicates that centipedes' venom has evolved to simultaneously disrupt cardiovascular, respiratory, muscular, and nervous systems by targeting the broadly distributed KCNQ channels, thus providing a therapeutic strategy for centipede envenomation.

Chemical synthesis of membrane proteins by the removable backbone modification method.

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Temperature-dependent ESR and computational studies on antiferromagnetic electron transfer in the yeast NADH dehydrogenase Ndi1.

Ndi1 is a special type-II complex I nicotinamide-adenine-dinucleotide (NADH):ubiquinone (UQ) oxidoreductase in the yeast respiratory chain, with two bound UQs (UQI and UQII) mediating electron transfer from flavin cofactors to ubiquinone, in the absence of Fe-S chains. Here, we elucidate the underlying mechanism of electron transfer in Ndi1 through temperature-dependent Electron Spin Resonance (ESR) experiments in conjunction with quantum chemical calculations. It is revealed that electron transfer is mediated by antiferromagnetic (AFM) interactions between flavin-adenosine-dinucleotide (FAD) and UQI and between UQI and UQII. The π-stacking interactions among the aromatic complexes also enhance the through-space electron transfer. The FAD/UQI pair works as a rectifier converting double-electron co-transfer into sequential single-electron transfer events. The results not only expand our understanding on the observed AFM interactions among p-orbital aromatic mixed-stack in proteins, but also provide significant insights into the fabrication of materials with special magnetic properties using biological samples.

Combined approaches of EPR and NMR illustrate only one transmembrane helix in the human IFITM3.

Interferon-inducible transmembrane protein IFITM3 was known to restrict the entry of a wide spectrum of viruses to the cytosol of the host. The mechanism used by the protein to restrict viral entry is unclear given the unavailability of the membrane topology and structures of the IFITM family proteins. Systematic site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) studies of IFITM3 in detergent micelles identified a single, long transmembrane helix in the C-terminus and an intramembrane segment in the N-terminal hydrophobic region. Solution NMR studies of the same sample verified the secondary structure distribution and demonstrated two rigid regions interacting with the micellar surface. The resulting membrane topology of IFITM3 supports the mechanism of an enhanced restricted membrane hemi-fusion.