Transient Co-expression of Membrane Protein Complexes in Mammalian Cells.

Membrane proteins are essential components of biological membranes with key roles in cellular processes such as nutrient transport, cell communication, signaling, or energy conversion. Due to their crucial functions, membrane proteins and their complexes are often targets for therapeutic interventions. Expression and purification of membrane proteins are often a bottleneck to yield sufficient material for structural studies and further downstream characterization. Taking advantage of the Expi293 expression system for the production of eukaryotic proteins, we present a very efficient and fast protocol for the co-expression of a membrane complex. Here, we use transient transfection to co-express the membrane transporter PHT1 with its adaptor protein TASL. To allow the simultaneous screening of different proteins, constructs, or interaction partners, we make use of the Twin-Strep magnetic system. The protocol can be applied for small-scale screening of any membrane protein alone or co-expressed with interacting partners followed by large-scale production and purification of a potential membrane protein complex.

Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS).

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses.

Molecular basis of TASL recruitment by the peptide/histidine transporter 1, PHT1.

PHT1 is a histidine /oligopeptide transporter with an essential role in Toll-like receptor innate immune responses. It can act as a receptor by recruiting the adaptor protein TASL which leads to type I interferon production via IRF5. Persistent stimulation of this signalling pathway is known to be involved in the pathogenesis of systemic lupus erythematosus (SLE). Understanding how PHT1 recruits TASL at the molecular level, is therefore clinically important for the development of therapeutics against SLE and other autoimmune diseases. Here we present the Cryo-EM structure of PHT1 stabilized in the outward-open conformation. By combining biochemical and structural modeling techniques we propose a model of the PHT1-TASL complex, in which the first 16 N-terminal TASL residues fold into a helical structure that bind in the central cavity of the inward-open conformation of PHT1. This work provides critical insights into the molecular basis of PHT1/TASL mediated type I interferon production.

Sybodies as Novel Bioreceptors toward Field-Effect Transistor-Based Detection of SARS-CoV-2 Antigens.

The SARS-CoV-2 pandemic has increased the demand for low-cost, portable, and rapid biosensors, driving huge research efforts toward new nanomaterial-based approaches with high sensitivity. Many of them employ antibodies as bioreceptors, which have a costly development process that requires animal facilities. Recently, sybodies emerged as a new alternative class of synthetic binders and receptors with high antigen binding efficiency, improved chemical stability, and lower production costs via animal-free methods. Their smaller size is an important asset to consider in combination with ultrasensitive field-effect transistors (FETs) as transducers, which respond more intensely when biorecognition occurs near their surface. This work demonstrates the immobilization of sybodies against the spike protein of the virus on silicon surfaces, which are often integral parts of the semiconducting channel of FETs. Immobilized sybodies maintain the capability to capture antigens, even at low concentrations in the femtomolar range, as observed by fluorescence microscopy. Finally, the first proof of concept of sybody-modified FET sensing is provided using a nanoscopic silicon net as the sensitive area where the sybodies are immobilized. The future development of further sybodies against other biomarkers and their generalization in biosensors could be critical to decrease the cost of biodetection platforms in future pandemics.

In vitro and ex vivo proteomics of Mycobacterium marinum biofilms and the development of biofilm-binding synthetic nanobodies.

The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.

MLL1 is regulated by KSHV LANA and is important for virus latency.

Mixed lineage leukemia 1 (MLL1) is a histone methyltransferase. Kaposi's sarcoma-associated herpesvirus (KSHV) is a leading cause of malignancy in AIDS. KSHV latently infects tumor cells and its genome is decorated with epigenetic marks. Here, we show that KSHV latency-associated nuclear antigen (LANA) recruits MLL1 to viral DNA where it establishes H3K4me3 modifications at the extensive KSHV terminal repeat elements during primary infection. LANA interacts with MLL1 complex members, including WDR5, integrates into the MLL1 complex, and regulates MLL1 activity. We describe the 1.5-Å crystal structure of N-terminal LANA peptide complexed with MLL1 complex member WDR5, which reveals a potential regulatory mechanism. Disruption of MLL1 expression rendered KSHV latency establishment highly deficient. This deficiency was rescued by MLL1 but not by catalytically inactive MLL1. Therefore, MLL1 is LANA regulable and exerts a central role in virus infection. These results suggest broad potential for MLL1 regulation, including by non-host factors.

Deamidation drives molecular aging of the SARS-CoV-2 spike protein receptor-binding motif.

The spike protein is the main protein component of the SARS-CoV-2 virion surface. The spike receptor-binding motif mediates recognition of the human angiotensin-converting enzyme 2 receptor, a critical step in infection, and is the preferential target for spike-neutralizing antibodies. Posttranslational modifications of the spike receptor-binding motif have been shown to modulate viral infectivity and host immune response, but these modifications are still being explored. Here we studied asparagine deamidation of the spike protein, a spontaneous event that leads to the appearance of aspartic and isoaspartic residues, which affect both the protein backbone and its charge. We used computational prediction and biochemical experiments to identify five deamidation hotspots in the SARS-CoV-2 spike protein. Asparagine residues 481 and 501 in the receptor-binding motif deamidate with a half-life of 16.5 and 123 days at 37 °C, respectively. Deamidation is significantly slowed at 4 °C, indicating a strong dependence of spike protein molecular aging on environmental conditions. Deamidation of the spike receptor-binding motif decreases the equilibrium constant for binding to the human angiotensin-converting enzyme 2 receptor more than 3.5-fold, yet its high conservation pattern suggests some positive effect on viral fitness. We propose a model for deamidation of the full SARS-CoV-2 virion illustrating how deamidation of the spike receptor-binding motif could lead to the accumulation on the virion surface of a nonnegligible chemically diverse spike population in a timescale of days. Our findings provide a potential mechanism for molecular aging of the spike protein with significant consequences for understanding virus infectivity and vaccine development.

Structural comparison of GLUT1 to GLUT3 reveal transport regulation mechanism in sugar porter family.

The human glucose transporters GLUT1 and GLUT3 have a central role in glucose uptake as canonical members of the Sugar Porter (SP) family. GLUT1 and GLUT3 share a fully conserved substrate-binding site with identical substrate coordination, but differ significantly in transport affinity in line with their physiological function. Here, we present a 2.4 Å crystal structure of GLUT1 in an inward open conformation and compare it with GLUT3 using both structural and functional data. Our work shows that interactions between a cytosolic "SP motif" and a conserved "A motif" stabilize the outward conformational state and increases substrate apparent affinity. Furthermore, we identify a previously undescribed Cl- ion site in GLUT1 and an endofacial lipid/glucose binding site which modulate GLUT kinetics. The results provide a possible explanation for the difference between GLUT1 and GLUT3 glucose affinity, imply a general model for the kinetic regulation in GLUTs and suggest a physiological function for the defining SP sequence motif in the SP family.

Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2.

The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Therapeutic neutralizing antibodies constitute a key short-to-medium term approach to tackle COVID-19. However, traditional antibody production is hampered by long development times and costly production. Here, we report the rapid isolation and characterization of nanobodies from a synthetic library, known as sybodies (Sb), that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Several binders with low nanomolar affinities and efficient neutralization activity were identified of which Sb23 displayed high affinity and neutralized pseudovirus with an IC50 of 0.6 µg/ml. A cryo-EM structure of the spike bound to Sb23 showed that Sb23 binds competitively in the ACE2 binding site. Furthermore, the cryo-EM reconstruction revealed an unusual conformation of the spike where two RBDs are in the 'up' ACE2-binding conformation. The combined approach represents an alternative, fast workflow to select binders with neutralizing activity against newly emerging viruses.

Crystal structure of the plant symporter STP10 illuminates sugar uptake mechanism in monosaccharide transporter superfamily.

Plants are dependent on controlled sugar uptake for correct organ development and sugar storage, and apoplastic sugar depletion is a defense strategy against microbial infections like rust and mildew. Uptake of glucose and other monosaccharides is mediated by Sugar Transport Proteins, proton-coupled symporters from the Monosaccharide Transporter (MST) superfamily. We present the 2.4 Å structure of Arabidopsis thaliana high affinity sugar transport protein, STP10, with glucose bound. The structure explains high affinity sugar recognition and suggests a proton donor/acceptor pair that links sugar transport to proton translocation. It contains a Lid domain, conserved in all STPs, that locks the mobile transmembrane domains through a disulfide bridge, and creates a protected environment which allows efficient coupling of the proton gradient to drive sugar uptake. The STP10 structure illuminates fundamental principles of sugar transport in the MST superfamily with implications for both plant antimicrobial defense, organ development and sugar storage.

KSHV but not MHV-68 LANA induces a strong bend upon binding to terminal repeat viral DNA.

Latency-associated nuclear antigen (LANA) is central to episomal tethering, replication and transcriptional regulation of γ2-herpesviruses. LANA binds cooperatively to the terminal repeat (TR) region of the viral episome via adjacent LANA binding sites (LBS), but the molecular mechanism by which LANA assembles on the TR remains elusive. We show that KSHV LANA and MHV-68 LANA proteins bind LBS DNA using strikingly different modes. Solution structure of LANA complexes revealed that while kLANA tetramer is intrinsically bent both in the free and bound state to LBS1-2 DNA, mLANA oligomers instead adopt a rigid linear conformation. In addition, we report a novel non-ring kLANA structure that displays more flexibility at its assembly interface than previously demonstrated. We identified a hydrophobic pivot point located at the dimer-dimer assembly interface, which gives rotational freedom for kLANA to adopt variable conformations to accommodate both LBS1-2 and LBS2-1-3 DNA. Alterations in the arrangement of LBS within TR or at the tetramer assembly interface have a drastic effect on the ability of kLANA binding. We also show kLANA and mLANA DNA binding functions can be reciprocated. Although KSHV and MHV-68 are closely related, the findings provide new insights into how the structure, oligomerization, and DNA binding of LANA have evolved differently to assemble on the TR DNA.