Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL.

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the ß barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

Cholesterol Binds the Amphipathic Helix of IFITM3 and Regulates Antiviral Activity.

The interferon-induced transmembrane (IFITM) proteins broadly inhibit the entry of diverse pathogenic viruses, including Influenza A virus (IAV), Zika virus, HIV-1, and SARS coronaviruses by inhibiting virus-cell membrane fusion. IFITM3 was previously shown to disrupt cholesterol trafficking, but the functional relationship between IFITM3 and cholesterol remains unclear. We previously showed that inhibition of IAV entry by IFITM3 is associated with its ability to promote cellular membrane rigidity, and these activities are functionally linked by a shared requirement for the amphipathic helix (AH) found in the intramembrane domain (IMD) of IFITM3. Furthermore, it has been shown that the AH of IFITM3 alters lipid membranes in vitro in a cholesterol-dependent manner. Therefore, we aimed to elucidate the relationship between IFITM3 and cholesterol in more detail. Using a fluorescence-based in vitro binding assay, we found that a peptide derived from the AH of IFITM3 directly interacted with the cholesterol analog, NBD-cholesterol, while other regions of the IFITM3 IMD did not, and native cholesterol competed with this interaction. In addition, recombinant full-length IFITM3 protein also exhibited NBD-cholesterol binding activity. Importantly, previously characterized mutations within the AH of IFITM3 that strongly inhibit antiviral function (F63Q and F67Q) disrupted AH structure in solution, inhibited cholesterol binding in vitro, and restricted bilayer insertion in silico. Our data suggest that direct interactions with cholesterol may contribute to the inhibition of membrane fusion pore formation by IFITM3. These findings may facilitate the design of therapeutic peptides for use in broad-spectrum antiviral therapy.

Recent advances in small-angle scattering and its expanding impact in structural biology.

Applications of small-angle scattering (SAS) in structural biology have benefited from continuing developments in instrumentation, tools for data analysis, modeling capabilities, standards for data and model presentation, and data archiving. The interplay of these capabilities has enabled SAS to contribute to advances in structural biology as the field pushes the boundaries in studies of biomolecular complexes and assemblies as large as whole cells, membrane proteins in lipid environments, and dynamic systems on time scales ranging from femtoseconds to hours. This review covers some of the important advances in biomolecular SAS capabilities for structural biology focused on over the last 5 years and presents highlights of recent applications that demonstrate how the technique is exploring new territories.

A NanoBiT assay to monitor membrane proteins trafficking for drug discovery and drug development.

Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.

Modified uvsY by N-terminal hexahistidine tag addition enhances efficiency of recombinase polymerase amplification to detect SARS-CoV-2 DNA.

BACKGROUND: Recombinase (uvsY and uvsX) from bacteriophage T4 is a key enzyme for recombinase polymerase amplification (RPA) that amplifies a target DNA sequence at a constant temperature with a single-stranded DNA-binding protein and a strand-displacing polymerase. The present study was conducted to examine the effects of the N- and C-terminal tags of uvsY on its function in RPA to detect SARS-CoV-2 DNA. METHODS: Untagged uvsY (uvsY-Δhis), N-terminal tagged uvsY (uvsY-Nhis), C-terminal tagged uvsY (uvsY-Chis), and N- and C-terminal tagged uvsY (uvsY-NChis) were expressed in Escherichia coli and purified. RPA reaction was carried out with the in vitro synthesized standard DNA at 41 °C. The amplified products were separated on agarose gels. RESULTS: The minimal initial copy numbers of standard DNA from which the amplified products were observed were 6 × 105, 60, 600, and 600 copies for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The minimal reaction time at which the amplified products were observed were 20, 20, 30, and 20 min for the RPA with uvsY-Δhis, uvsY-Nhis, uvsY-Chis, and uvsY-NChis, respectively. The RPA with uvsY-Nhis exhibited clearer bands than that with either of other three uvsYs. CONCLUSIONS: The reaction efficiency of RPA with uvsY-Nhis was the highest, suggesting that uvsY-Nhis is suitable for use in RPA.

Ni2+ Catalyzed Cleavage of TrpLE-Fused Small Transmembrane Peptides.

In addition to a membrane anchor, the transmembrane domain (TMD) of single-pass transmembrane proteins (SPTMPs) recently has shown essential roles in the cross-membrane activity or receptor assembly/clustering. However, these small TMD peptides are generally hydrophobic and dynamic, difficult to be expressed and purified. Here, we have integrated the power of TrpLE fusion protein and a sequence-specific nickel-assisted cleavage (SNAC)-tag to produce small TMD peptides in a highly efficient way under mild conditions, which uses Ni2+ as the cleavage reagent, avoiding the usage of toxic cyanogen bromide (CNBr). Furthermore, this method simplifies the downstream protein purification and reconstitution. Two representative TMDs, including the Spike-TMD from severe acute respiratory syndrome coronavirus 2 (SARS2), were successfully produced with high-quality nuclear magnetic resonance (NMR) spectra. Therefore, our study provides a more efficient and practical approach for general structural characterization of the small TM proteins.

MemDis: Predicting Disordered Regions in Transmembrane Proteins.

Transmembrane proteins (TMPs) play important roles in cells, ranging from transport processes and cell adhesion to communication. Many of these functions are mediated by intrinsically disordered regions (IDRs), flexible protein segments without a well-defined structure. Although a variety of prediction methods are available for predicting IDRs, their accuracy is very limited on TMPs due to their special physico-chemical properties. We prepared a dataset containing membrane proteins exclusively, using X-ray crystallography data. MemDis is a novel prediction method, utilizing convolutional neural network and long short-term memory networks for predicting disordered regions in TMPs. In addition to attributes commonly used in IDR predictors, we defined several TMP specific features to enhance the accuracy of our method further. MemDis achieved the highest prediction accuracy on TMP-specific dataset among other popular IDR prediction methods.

A method for intuitively extracting macromolecular dynamics from structural disorder.

Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an analytical approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.

Intracellular autoactivation of TMPRSS11A, an airway epithelial transmembrane serine protease.

Type II transmembrane serine proteases (TTSPs) are a group of enzymes participating in diverse biological processes. Some members of the TTSP family are implicated in viral infection. TMPRSS11A is a TTSP expressed on the surface of airway epithelial cells, which has been shown to cleave and activate spike proteins of the severe acute respiratory syndrome (SARS) and the Middle East respiratory syndrome coronaviruses (CoVs). In this study, we examined the mechanism underlying the activation cleavage of TMPRSS11A that converts the one-chain zymogen to a two-chain enzyme. By expression in human embryonic kidney 293, esophageal EC9706, and lung epithelial A549 and 16HBE cells, Western blotting, and site-directed mutagenesis, we found that the activation cleavage of human TMPRSS11A was mediated by autocatalysis. Moreover, we found that TMPRSS11A activation cleavage occurred before the protein reached the cell surface, as indicated by studies with trypsin digestion to remove cell surface proteins, treatment with cell organelle-disturbing agents to block intracellular protein trafficking, and analysis of a soluble form of TMPRSS11A without the transmembrane domain. We also showed that TMPRSS11A was able to cleave the SARS-CoV-2 spike protein. These results reveal an intracellular autocleavage mechanism in TMPRSS11A zymogen activation, which differs from the extracellular zymogen activation reported in other TTSPs. These findings provide new insights into the diverse mechanisms in regulating TTSP activation.

TMEM106B in humans and Vac7 and Tag1 in yeast are predicted to be lipid transfer proteins.

TMEM106B is an integral membrane protein of late endosomes and lysosomes involved in neuronal function, its overexpression being associated with familial frontotemporal lobar degeneration, and point mutation linked to hypomyelination. It has also been identified in multiple screens for host proteins required for productive SARS-CoV-2 infection. Because standard approaches to understand TMEM106B at the sequence level find no homology to other proteins, it has remained a protein of unknown function. Here, the standard tool PSI-BLAST was used in a nonstandard way to show that the lumenal portion of TMEM106B is a member of the late embryogenesis abundant-2 (LEA-2) domain superfamily. More sensitive tools (HMMER, HHpred, and trRosetta) extended this to predict LEA-2 domains in two yeast proteins. One is Vac7, a regulator of PI(3,5)P2 production in the degradative vacuole, equivalent to the lysosome, which has a LEA-2 domain in its lumenal domain. The other is Tag1, another vacuolar protein, which signals to terminate autophagy and has three LEA-2 domains in its lumenal domain. Further analysis of LEA-2 structures indicated that LEA-2 domains have a long, conserved lipid-binding groove. This implies that TMEM106B, Vac7, and Tag1 may all be lipid transfer proteins in the lumen of late endocytic organelles.

A Fluorescence Polarization-Based High-Throughput Screen to Identify the First Small-Molecule Modulators of the Human Adenylyltransferase HYPE/FICD.

The covalent transfer of the AMP portion of ATP onto a target protein-termed adenylylation or AMPylation-by the human Fic protein HYPE/FICD has recently garnered attention as a key regulatory mechanism in endoplasmic reticulum homeostasis, neurodegeneration, and neurogenesis. As a central player in such critical cellular events, high-throughput screening (HTS) efforts targeting HYPE-mediated AMPylation warrant investigation. Herein, we present a dual HTS assay for the simultaneous identification of small-molecule activators and inhibitors of HYPE AMPylation. Employing the fluorescence polarization of an ATP analog fluorophore-Fl-ATP-we developed and optimized an efficient, robust assay that monitors HYPE autoAMPylation and is amenable to automated, high-throughput processing of diverse chemical libraries. Challenging our pilot screen with compounds from the LOPAC, Spectrum, MEGx, and NATx libraries yielded 0.3% and 1% hit rates for HYPE activators and inhibitors, respectively. Further, these hits were assessed for dose-dependency and validated via orthogonal biochemical AMPylation assays. We thus present a high-quality HTS assay suitable for tracking HYPE's enzymatic activity, and the resultant first small-molecule manipulators of HYPE-promoted autoAMPylation.

Characteristics of Angiotensin I-converting enzyme 2, type II transmembrane serine protease 2 and 4 in tree shrew indicate it as a potential animal model for SARS-CoV-2 infection.

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.

Structural basis of coronavirus E protein interactions with human PALS1 PDZ domain.

SARS-CoV-2 infection leads to coronavirus disease 2019 (COVID-19), which is associated with severe and life-threatening pneumonia and respiratory failure. However, the molecular basis of these symptoms remains unclear. SARS-CoV-1 E protein interferes with control of cell polarity and cell-cell junction integrity in human epithelial cells by binding to the PALS1 PDZ domain, a key component of the Crumbs polarity complex. We show that C-terminal PDZ binding motifs of SARS-CoV-1 and SARS-CoV-2 E proteins bind the PALS1 PDZ domain with 29.6 and 22.8 µM affinity, whereas the related sequence from MERS-CoV did not bind. We then determined crystal structures of PALS1 PDZ domain bound to both SARS-CoV-1 and SARS-CoV-2 E protein PDZ binding motifs. Our findings establish the structural basis for SARS-CoV-1/2 mediated subversion of Crumbs polarity signalling and serve as a platform for the development of small molecule inhibitors to suppress SARS-CoV-1/2 mediated disruption of polarity signalling in epithelial cells.

Can We AlphaFold Our Way Out of the Next Pandemic?

The announcement of the outstanding performance of AlphaFold 2 in the CASP 14 protein structure prediction competition came at the end of a long year defined by the COVID-19 pandemic. With an infectious organism dominating the world stage, the developers of Alphafold 2 were keen to play their part, accurately predicting novel structures of two proteins from SARS-CoV-2. In their blog post of December 2020, they highlighted this contribution, writing "we've also seen signs that protein structure prediction could be useful in future pandemic response efforts". So, what role does structural biology play in guiding vaccine immunogen design and what might be the contribution of AlphaFold 2?

SARS-CoV-2 exacerbates proinflammatory responses in myeloid cells through C-type lectin receptors and Tweety family member 2.

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.

Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza.

Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R↓ sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients.

The SARS-CoV-2 beta coronavirus is the etiological driver of COVID-19 disease, which is primarily characterized by shortness of breath, persistent dry cough, and fever. Because they transport oxygen, red blood cells (RBCs) may play a role in the severity of hypoxemia in COVID-19 patients. The present study combines state-of-the-art metabolomics, proteomics, and lipidomics approaches to investigate the impact of COVID-19 on RBCs from 23 healthy subjects and 29 molecularly diagnosed COVID-19 patients. RBCs from COVID-19 patients had increased levels of glycolytic intermediates, accompanied by oxidation and fragmentation of ankyrin, spectrin beta, and the N-terminal cytosolic domain of band 3 (AE1). Significantly altered lipid metabolism was also observed, in particular, short- and medium-chain saturated fatty acids, acyl-carnitines, and sphingolipids. Nonetheless, there were no alterations of clinical hematological parameters, such as RBC count, hematocrit, or mean corpuscular hemoglobin concentration, with only minor increases in mean corpuscular volume. Taken together, these results suggest a significant impact of SARS-CoV-2 infection on RBC structural membrane homeostasis at the protein and lipid levels. Increases in RBC glycolytic metabolites are consistent with a theoretically improved capacity of hemoglobin to off-load oxygen as a function of allosteric modulation by high-energy phosphate compounds, perhaps to counteract COVID-19-induced hypoxia. Conversely, because the N-terminus of AE1 stabilizes deoxyhemoglobin and finely tunes oxygen off-loading and metabolic rewiring toward the hexose monophosphate shunt, RBCs from COVID-19 patients may be less capable of responding to environmental variations in hemoglobin oxygen saturation/oxidant stress when traveling from the lungs to peripheral capillaries and vice versa.

Colorimetric Test for Fast Detection of SARS-CoV-2 in Nasal and Throat Swabs.

Mass testing is fundamental to face the pandemic caused by the coronavirus SARS-CoV-2 discovered at the end of 2019. To this aim, it is necessary to establish reliable, fast, and cheap tools to detect viral particles in biological material so to identify the people capable of spreading the infection. We demonstrate that a colorimetric biosensor based on gold nanoparticle (AuNP) interaction induced by SARS-CoV-2 lends itself as an outstanding tool for detecting viral particles in nasal and throat swabs. The extinction spectrum of a colloidal solution of multiple viral-target gold nanoparticles-AuNPs functionalized with antibodies targeting three surface proteins of SARS-CoV-2 (spike, envelope, and membrane)-is red-shifted in few minutes when mixed with a solution containing the viral particle. The optical density of the mixed solution measured at 560 nm was compared to the threshold cycle (Ct) of a real-time PCR (gold standard for detecting the presence of viruses) finding that the colorimetric method is able to detect very low viral load with a detection limit approaching that of the real-time PCR. Since the method is sensitive to the infecting viral particle rather than to its RNA, the achievements reported here open a new perspective not only in the context of the current and possible future pandemics, but also in microbiology, as the biosensor proves itself to be a powerful though simple tool for measuring the viral particle concentration.

Prediction of the Receptorome for the Human-Infecting Virome.

The virus receptors are key for the viral infection of host cells. Identification of the virus receptors is still challenging at present. Our previous study has shown that human virus receptor proteins have some unique features including high N-glycosylation level, high number of interaction partners and high expression level. Here, a random-forest model was built to identify human virus receptorome from human cell membrane proteins with an accepted accuracy based on the combination of the unique features of human virus receptors and protein sequences. A total of 1424 human cell membrane proteins were predicted to constitute the receptorome of the human-infecting virome. In addition, the combination of the random-forest model with protein-protein interactions between human and viruses predicted in previous studies enabled further prediction of the receptors for 693 human-infecting viruses, such as the enterovirus, norovirus and West Nile virus. Finally, the candidate alternative receptors of the SARS-CoV-2 were also predicted in this study. As far as we know, this study is the first attempt to predict the receptorome for the human-infecting virome and would greatly facilitate the identification of the receptors for viruses.