Florence Pojer

Cryo-EM structures and binding of mouse and human ACE2 to SARS-CoV-2 variants of concern indicate that mutations enabling immune escape could expand host range

Kelvin Ka Ching Lau, Alexander Myasnikov, Sergey Nazarov, Dongchun Ni, Florence Pojer, Henning Paul-Julius Stahlberg, Didier Trono, Priscilla Turelli, Emiko Uchikawa

nvestigation of potential hosts of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is crucial to understanding future risks of spillover and spillback. SARS-CoV-2 has been reported to be transmitted from humans to various animals after requiring relatively few mutations. There is significant interest in describing how the virus interacts with mice as they are well adapted to human environments, are used widely as infection models and can be infected. Structural and binding data of the mouse ACE2 receptor with the Spike protein of newly identified SARS-CoV-2 variants are needed to better understand the impact of immune system evading mutations present in variants of concern (VOC). Previous studies have developed mouse-adapted variants and identified residues critical for binding to heterologous ACE2 receptors. Here we report the cryo-EM structures of mouse ACE2 bound to trimeric Spike ectodomains of four different VOC: Beta, Omicron BA.1, Omicron BA.2.12.1 and Omicron BA.4/5. These variants represent the oldest to the newest variants known to bind the mouse ACE2 receptor. Our high-resolution structural data complemented with bio-layer interferometry (BLI) binding assays reveal a requirement for a combination of mutations in the Spike protein that enable binding to the mouse ACE2 receptor.

2023

Uncovering and engineering the mechanical properties of the adhesion GPCR ADGRG1 GAIN domain

Patrick Daniel Barth, Louis Georges Baptiste Dumas, Mahdi Hijazi, Matthieu François Marfoglia, Florence Pojer

Key cellular functions depend on the transduction of extracellular mechanical signals by specialized membrane receptors including adhesion G-protein coupled receptors (aGPCRs). While recently solved structures support aGPCR activation through shedding of the extracellular GAIN domain, the molecular mechanisms underpinning receptor mechanosensing remain poorly understood. When probed using single-molecule atomic force spectroscopy and molecular simulations, ADGRG1 GAIN dissociated from its tethered agonist at forces significantly higher than other reported signaling mechanoreceptors. Strong mechanical resistance was achieved through specific structural deformations and force propagation pathways under mechanical load. ADGRG1 GAIN variants computationally designed to lock the alpha and beta subdomains and rewire mechanically-induced structural deformations were found to modulate the GPS-Stachel rupture forces. Our study provides unprecedented insights into the molecular underpinnings of GAIN mechanical stability and paves the way for engineering mechanosensors, better understanding aGPCR function, and informing drug-discovery efforts targeting this important receptor class.

2023

A high-throughput cell- and virus-free assay shows reduced neutralization of SARS-CoV-2 variants by COVID-19 convalescent plasma

Valeria Cagno, Jérémy Campos, Céline Pellaton, Florence Pojer, Charlène Mireille Raymonde Raclot, Didier Trono, Priscilla Turelli

The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies in the serum of an individual indicates previous infection or vaccination. However, it provides limited insight into the protective nature of this immune response. Neutralizing antibodies recognizing the viral spike protein are more revealing, yet their measurement traditionally requires virus- and cell-based systems that are costly, time-consuming, inflexible, and potentially biohazardous. Here, we present a cell-free quantitative neutralization assay based on the competitive inhibition of trimeric SARS-CoV-2 spike protein binding to the angiotensin-converting enzyme 2 (ACE2) receptor. This high-throughput method matches the performance of the gold standard live virus infection assay, as verified with a panel of 206 seropositive donors with varying degrees of infection severity and virus-specific immunoglobulin G titers, achieving 96.7% sensitivity and 100% specificity. Furthermore, it allows for the parallel assessment of neutralizing activities against multiple SARS-CoV-2 spike protein variants of concern. We used our assay to profile serum samples from 59 patients hospitalized with coronavirus disease 2019 (COVID-19). We found that although most sera had high activity against the 2019-nCoV parental spike protein and, to a lesser extent, the alpha (B.1.1.7) variant, only 58% of serum samples could efficiently neutralize a spike protein derivative containing mutations present in the beta (B.1.351) variant. Thus, we have developed an assay that can evaluate effective neutralizing antibody responses to SARS-CoV-2 spike protein variants of concern after natural infection and that can be applied to characterize vaccine-induced antibody responses or to assess the potency of monoclonal antibodies.

AMER ASSOC ADVANCEMENT SCIENCE2021

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response

Bruno Emanuel Ferreira De Sousa Correia, David Hacker, Kelvin Ka Ching Lau, Florence Pojer

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and β-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small β-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The β-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the β-annulus peptide for vaccine design.

2021

Azole-Based Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors

George Coukos, Somi Reddy Majjigapu, Olivier Michielin, Florence Pojer, Pierre Vogel

The heme enzyme indoleamine 2,3-dioxygenase 1 (IDO1) plays an essential role in immunity, neuronal function, and aging through catalysis of the rate-limiting step in the kynurenine pathway of tryptophan metabolism. Many IDO1 inhibitors with different chemotypes have been developed, mainly targeted for use in anti-cancer immunotherapy. Lead optimization of direct heme iron-binding inhibitors has proven difficult due to the remarkable selectivity and sensitivity of the heme-ligand interactions. Here, we present experimental data for a set of closely related small azole compounds with more than 4 orders of magnitude differences in their inhibitory activities, ranging from millimolar to nanomolar levels. We investigate and rationalize their activities based on structural data, molecular dynamics simulations, and density functional theory calculations. Our results not only expand the presently known four confirmed chemotypes of sub-micromolar heme binding IDO1 inhibitors by two additional scaffolds but also provide a model to predict the activities of novel scaffolds.

AMER CHEMICAL SOC2021

Palmitoylated acyl protein thioesterase APT2 deforms membranes to extract substrate acyl chains

Muhammad Umair Anwar, Martina Audagnotto, Matteo Dal Peraro, Francisco De Magalhães Sarmento R De Mesquita, Giulia Fonti, Laurence Gouzi Abrami, Sylvia Ho, Florence Pojer, Patrick Alain Sandoz, Françoise Gisou van der Goot Grunberg

Acyl protein thioesterase APT2 interacts with membranes via its charged beta-tongue, becomes palmitoylated by ZDHHC3/7 and deforms the bilayer to extract substrate acyl chains. APT2 deacylation leads to its membrane release and degradation. Many biochemical reactions require controlled recruitment of proteins to membranes. This is largely regulated by posttranslational modifications. A frequent one is S-acylation, which consists of the addition of acyl chains and can be reversed by poorly understood acyl protein thioesterases (APTs). Using a panel of computational and experimental approaches, we dissect the mode of action of the major cellular thioesterase APT2 (LYPLA2). We show that soluble APT2 is vulnerable to proteasomal degradation, from which membrane binding protects it. Interaction with membranes requires three consecutive steps: electrostatic attraction, insertion of a hydrophobic loop and S-acylation by the palmitoyltransferases ZDHHC3 or ZDHHC7. Once bound, APT2 is predicted to deform the lipid bilayer to extract the acyl chain bound to its substrate and capture it in a hydrophobic pocket to allow hydrolysis. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated deacylation of substrates throughout the endomembrane system.

NATURE RESEARCH2021

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

Matteo Dal Peraro, Sandrine Madeleine Suzanne Georgeon, Oliver Hantschel, Tim Kükenshöner, Grégory La Sala, Allan Joaquim Lamontanara, Maria Josefina Marcaida Lopez, Daniel Pereira Duarte, Florence Pojer

Bruton's tyrosine kinase (Btk) is critical for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, Btk signaling sustains growth of several B-cell neoplasms which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the structural mechanism by which certain XLA mutations in the SH2 domain strongly perturb Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain required for Btk activation and to which multiple XLA mutations map. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevents activation of wild-type and TKI-resistant Btk, inhibiting Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition. Constitutive Btk signaling drives several B-cell cancers. Here the authors demonstrate key allosteric intramolecular interactions between the SH2 domain and the kinase domain of Btk, and propose an alternative approach for inhibition of both wild-type and tyrosine kinase inhibitor-resistant Btk.

NATURE PUBLISHING GROUP2020

High resolution CryoEM structure of the ring-shaped virulence factor EspB from Mycobacterium tuberculosis

Stewart Cole, Jérémie Piton, Florence Pojer

The EspB protein of Mycobacterium tuberculosis is a 60 kDa virulence factor, implicated in conjugation and exported by the ESX-1 system of which it may also be a component. Previous attempts to obtain high-resolution maps of EspB by cryo-electron microscopic examination of single particles have been thwarted by severe orientation bias of the particles. This was overcome by using detergent as a surfactant thereby allowing reconstruction of the EspB structure at 3.37 Å resolution. The final structure revealed the N-terminal domain of EspB to be organized as a cylindrical heptamer with dimensions of 90 Å x 90 Å and a central channel of 45 Å diameter whereas the C-terminal domain was unstructured. New atomic insight was obtained into the helical packing required for protomer interactions and the overall electrostatic potential. The external surface is electronegatively charged while the channel is lined with electropositive patches. EspB thus has many features of a pore-like transport protein that might allow the passage of an ESX-1 substrate such as the 35 Å diameter EsxA-EsxB heterodimer or B-form DNA consistent with its proposed role in DNA uptake.

2020

Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains

Oliver Hantschel, Grégory La Sala, Kelvin Ka Ching Lau, Florence Pojer

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms. STAT3 is an attractive therapeutic target but its homology with other STAT proteins complicates the development of selective inhibitors. Here, the authors develop monobodies with high affinity and selectivity for STAT3 and show that they can interfere with cellular STAT3 activity.

2020