A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

Superantigen Recognition and Interactions: Functions, Mechanisms and Applications

Superantigens are unconventional antigens which recognise immune receptors outside their usual recognition sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulins on B-cells, affecting opsonisation, IgG-mediated phagocytosis, and driving apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

Dual domain recognition determines SARS-CoV-2 PLpro selectivity for human ISG15 and K48-linked di-ubiquitin

The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) genome is evolving as the viral pandemic continues its active phase around the world. The Papain-like protease (PLpro) is a domain of Nsp3 – a large multi-domain protein that is an essential component of the replication-transcription complex, making it a good therapeutic target. PLpro is a multi-functional protein encoded in coronaviruses that can cleave viral polyproteins, poly-ubiquitin and protective Interferon Stimulated Gene 15 product, ISG15, which mimics a head-to-tail linked ubiquitin (Ub) dimer. PLpro across coronavirus families showed divergent selectivity for recognition and cleavage of these protein substrates despite sequence conservation. However, it is not clear how sequence changes in SARS-CoV-2 PLpro alter its selectivity for substrates and what outcome this has on the pathogenesis of the virus. We show that SARS-CoV-2 PLpro preferentially binds ISG15 over Ub and K48-linked Ub2. We determined crystal structures of PLpro in complex with human K48-Ub2 and ISG15 revealing that dual domain recognition of ISG15 drives substrate selectivity over Ub and Ub2. We also characterized the PLpro substrate interactions using solution NMR, cross-linking mass spectrometry to support that ISG15 is recognized via two domains while Ub2 binds primarily through one Ub domain. Finally, energetic analysis of the binding interfaces between PLpro from SARS-CoV-1 and SARS-CoV-2 with ISG15 and Ub2 define the sequence determinants for how PLpros from different coronaviruses recognize two topologically distinct substrates and how evolution of the protease altered its substrate selectivity. Our work reveals how PLpro substrate selectivity may evolve in PLpro coronaviruses variants enabling design of more effective therapeutics.

Regulation of human mTOR complexes by DEPTOR

The vertebrate-specific DEP domain-containing mTOR interacting protein (DEPTOR), an oncoprotein or tumor suppressor, has important roles in metabolism, immunity, and cancer. It is the only protein that binds and regulates both complexes of mammalian target of rapamycin (mTOR), a central regulator of cell growth. Biochemical analysis and cryo-EM reconstructions of DEPTOR bound to human mTOR complex 1 (mTORC1) and mTORC2 reveal that both structured regions of DEPTOR, the PDZ domain and the DEP domain tandem (DEPt), are involved in mTOR interaction. The PDZ domain binds tightly with mildly activating effect, but then acts as an anchor for DEPt association that allosterically suppresses mTOR activation. The binding interfaces of the PDZ domain and DEPt also support further regulation by other signaling pathways. A separate, substrate-like mode of interaction for DEPTOR phosphorylation by mTOR complexes rationalizes inhibition of non-stimulated mTOR activity at higher DEPTOR concentrations. The multifaceted interplay between DEPTOR and mTOR provides a basis for understanding the divergent roles of DEPTOR in physiology and opens new routes for targeting the mTOR-DEPTOR interaction in disease.

Structure-activity relationships of B.1.617 and other SARS-CoV-2 spike variants

The surge of COVID-19 infection cases is spurred by emerging SARS-CoV-2 variants such as B.1.617. Here we report 38 cryo-EM structures, corresponding to the spike protein of the Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Kappa (B.1.617.1) variants in different functional states with and without its receptor, ACE2. Mutations on the N-terminal domain not only alter the conformation of the highly antigenic supersite of the Delta variant, but also remodel the glycan shield by deleting or adding N-glycans of the Delta and Gamma variants, respectively. Substantially enhanced ACE2 binding was observed for all variants, whose mutations on the receptor binding domain modulate the electrostatics of the binding interfaces. Despite their abilities to escape host immunity, all variants can be potently neutralized by three unique antibodies.

Scaffolding mechanism of arrestin-2 in the cRaf/MEK1/ERK signaling cascade

Arrestins were initially identified for their role in homologous desensitization and internalization of G protein–coupled receptors. Receptor-bound arrestins also initiate signaling by interacting with other signaling proteins. Arrestins scaffold MAPK signaling cascades, MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK. In particular, arrestins facilitate ERK1/2 activation by scaffolding ERK1/2 (MAPK), MEK1 (MAP2K), and Raf (MAPK3). However, the structural mechanism underlying this scaffolding remains unknown. Here, we investigated the mechanism of arrestin-2 scaffolding of cRaf, MEK1, and ERK2 using hydrogen/deuterium exchange–mass spectrometry, tryptophan-induced bimane fluorescence quenching, and NMR. We found that basal and active arrestin-2 interacted with cRaf, while only active arrestin-2 interacted with MEK1 and ERK2. The ATP binding status of MEK1 or ERK2 affected arrestin-2 binding; ATP-bound MEK1 interacted with arrestin-2, whereas only empty ERK2 bound arrestin-2. Analysis of the binding interfaces suggested that the relative positions of cRaf, MEK1, and ERK2 on arrestin-2 likely facilitate sequential phosphorylation in the signal transduction cascade.

Superantigen Recognition and Interactions: Functions, Mechanisms and Applications

Superantigens are unconventional antigens which recognise immune receptors outside the usual binding sites e.g. complementary determining regions (CDRs), to elicit a response within the target cell. T-cell superantigens crosslink T-cell receptors and MHC Class II molecules on antigen-presenting cells, leading to lymphocyte recruitment, induction of cytokine storms and T-cell anergy or apoptosis among many other effects. B-cell superantigens, on the other hand, bind immunoglobulin receptors on B-cells affecting opsonisation, IgG-mediated phagocytosis, and drive B-cells into apoptosis. Here, through a review of the structural basis for recognition of immune receptors by superantigens, we show that their binding interfaces share specific physicochemical characteristics when compared with other protein-protein interaction complexes. Given that antibody-binding superantigens have been exploited extensively in industrial antibody purification, these observations could facilitate further protein engineering to optimize the use of superantigens in this and other areas of biotechnology.

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grassspecific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.

Spherical Neutralizing Aptamer Inhibits SARS-CoV-2 Infection

<p>New neutralizing agents against SARS-CoV-2 and the associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a <u>s</u>pherical cocktail <u>n</u>eutralizing <u>a</u>ptamer-gold nano<u>p</u>article (SNAP) to synergistically block the interaction of SARS-CoV-2 receptor-binding domain (RBD) and angiotensin-converting enzyme-2 (ACE2). Taking advantage of the simultaneous recognition of multi-homologous and multi-heterogenous neutralizing aptamers and dimensionally matched nano-scaffolds, the SNAP exhibits increased affinity to the RBD with a dissociation constant value of 5.46 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 aM. Additional benefits include the multi-epitope blocking capability of the aptamer cocktail and the steric hindrance of the nano-scaffold, which further covers the ACE2 binding interfaces and affects the conformational transition of the spike protein. As a result, the SNAP strategy exhibits broad neutralizing activity, almost completely blocking the infection of<a> N501Y</a> and D614G mutant strains. Overall, the SNAP strategy provides a new direction for development of anti-virus infection mechanisms, both to fight the COVID-19 pandemic and serve as a powerful technical reserve for future unknown pandemics.</p>

Spherical Neutralizing Aptamer Inhibits SARS-CoV-2 Infection

<p>New neutralizing agents against SARS-CoV-2 and the associated mutant strains are urgently needed for the treatment and prophylaxis of COVID-19. Herein, we develop a <u>s</u>pherical cocktail <u>n</u>eutralizing <u>a</u>ptamer-gold nano<u>p</u>article (SNAP) to synergistically block the interaction of SARS-CoV-2 receptor-binding domain (RBD) and angiotensin-converting enzyme-2 (ACE2). Taking advantage of the simultaneous recognition of multi-homologous and multi-heterogenous neutralizing aptamers and dimensionally matched nano-scaffolds, the SNAP exhibits increased affinity to the RBD with a dissociation constant value of 5.46 pM and potent neutralization against authentic SARS-CoV-2 with a half-maximal inhibitory concentration of 142.80 aM. Additional benefits include the multi-epitope blocking capability of the aptamer cocktail and the steric hindrance of the nano-scaffold, which further covers the ACE2 binding interfaces and affects the conformational transition of the spike protein. As a result, the SNAP strategy exhibits broad neutralizing activity, almost completely blocking the infection of<a> N501Y</a> and D614G mutant strains. Overall, the SNAP strategy provides a new direction for development of anti-virus infection mechanisms, both to fight the COVID-19 pandemic and serve as a powerful technical reserve for future unknown pandemics.</p>