Specific recognition of two MAX effectors by integrated HMA domains in plant immune receptors involves distinct binding surfaces.

The structurally conserved but sequence-unrelated MAX (Magnaporthe oryzae avirulence and ToxB-like) effectors AVR1-CO39 and AVR-PikD from the blast fungus M. oryzae are recognized by the rice nucleotide-binding domain and leucine-rich repeat proteins (NLRs) RGA5 and Pikp-1, respectively. This involves, in both cases, direct interaction of the effector with a heavy metal-associated (HMA) integrated domain (ID) in the NLR. Here, we solved the crystal structures of a C-terminal fragment of RGA5 carrying the HMA ID (RGA5_S), alone, and in complex with AVR1-CO39 and compared it to the structure of the Pikp1HMA/AVR-PikD complex. In both complexes, HMA ID/MAX effector interactions involve antiparallel alignment of ß-sheets from each partner. However, effector-binding occurs at different surfaces in Pikp1HMA and RGA5HMA, indicating that these interactions evolved independently by convergence of these two MAX effectors to the same type of plant target proteins. Interestingly, the effector-binding surface in RGA5HMA overlaps with the surface that mediates RGA5HMA self-interaction. Mutations in the HMA-binding interface of AVR1-CO39 perturb RGA5HMA-binding, in vitro and in vivo, and affect the recognition of M. oryzae in a rice cultivar containing Pi-CO39 Our study provides detailed insight into the mechanisms of effector recognition by NLRs, which has substantial implications for future engineering of NLRs to expand their recognition specificities. In addition, we propose, as a hypothesis for the understanding of effector diversity, that in the structurally conserved MAX effectors the molecular mechanism of host target protein-binding is conserved rather than the host target proteins themselves.

Pressure and Chemical Unfolding of an α-Helical Bundle Protein: The GH2 Domain of the Protein Adaptor GIPC1.

When combined with NMR spectroscopy, high hydrostatic pressure is an alternative perturbation method used to destabilize globular proteins that has proven to be particularly well suited for exploring the unfolding energy landscape of small single-domain proteins. To date, investigations of the unfolding landscape of all-ß or mixed-α/ß protein scaffolds are well documented, whereas such data are lacking for all-α protein domains. Here we report the NMR study of the unfolding pathways of GIPC1-GH2, a small α-helical bundle domain made of four antiparallel α-helices. High-pressure perturbation was combined with NMR spectroscopy to unravel the unfolding landscape at three different temperatures. The results were compared to those obtained from classical chemical denaturation. Whatever the perturbation used, the loss of secondary and tertiary contacts within the protein scaffold is almost simultaneous. The unfolding transition appeared very cooperative when using high pressure at high temperature, as was the case for chemical denaturation, whereas it was found more progressive at low temperature, suggesting the existence of a complex folding pathway.

Monitoring Unfolding of Titin I27 Single and Bi Domain with High-Pressure NMR Spectroscopy.

A complete description of the pathways and mechanisms of protein folding requires a detailed structural and energetic characterization of the folding energy landscape. Simulations, when corroborated by experimental data yielding global information on the folding process, can provide this level of insight. Molecular dynamics (MD) has often been combined with force spectroscopy experiments to decipher the unfolding mechanism of titin immunoglobulin-like single or multidomain, the giant multimodular protein from sarcomeres, yielding information on the sequential events during titin unfolding under stretching. Here, we used high-pressure NMR to monitor the unfolding of titin I27 Ig-like single domain and tandem. Because this method brings residue-specific information on the folding process, it can provide quasiatomic details on this process without the help of MD simulations. Globally, the results of our high-pressure analysis are in agreement with previous results obtained by the combination of experimental measurements and MD simulation and/or protein engineering, although the intermediate folding state caused by the early detachment of the AB ß-sheet, often reported in previous works based on MD or force spectroscopy, cannot be detected. On the other hand, the A'G parallel ß-sheet of the ß-sandwich has been confirmed as the Achilles heel of the three-dimensional scaffold: its disruption yields complete unfolding with very similar characteristics (free energy, unfolding volume, kinetics rate constants) for the two constructs.

New recognition specificity in a plant immune receptor by molecular engineering of its integrated domain.

Plant nucleotide-binding and leucine-rich repeat domain proteins (NLRs) are immune sensors that recognize pathogen effectors. Here, we show that molecular engineering of the integrated decoy domain (ID) of an NLR can extend its recognition spectrum to a new effector. We relied for this on detailed knowledge on the recognition of the Magnaporthe oryzae effectors AVR-PikD, AVR-Pia, and AVR1-CO39 by, respectively, the rice NLRs Pikp-1 and RGA5. Both receptors detect their effectors through physical binding to their HMA (Heavy Metal-Associated) IDs. By introducing into RGA5_HMA the AVR-PikD binding residues of Pikp-1_HMA, we create a high-affinity binding surface for this effector. RGA5 variants carrying this engineered binding surface perceive the new ligand, AVR-PikD, and still recognize AVR-Pia and AVR1-CO39 in the model plant N. benthamiana. However, they do not confer extended disease resistance specificity against M. oryzae in transgenic rice plants. Altogether, our study provides a proof of concept for the design of new effector recognition specificities in NLRs through molecular engineering of IDs.

Rational design of the zonulin inhibitor AT1001 derivatives as potential anti SARS-CoV-2.

Although vaccines are greatly mitigating the worldwide pandemic diffusion of SARS-Cov-2, therapeutics should provide many distinct advantages as complementary approach to control the viral spreading. Here, we report the development of new tripeptide derivatives of AT1001 against SARS-CoV-2 Mpro. By molecular modeling, a small compound library was rationally designed and filtered for enzymatic inhibition through FRET assay, leading to the identification of compound 4. X-ray crystallography studies provide insights into its binding mode and confirm the formation of a covalent bond with Mpro C145. In vitro antiviral tests indicate the improvement of biological activity of 4 respect to AT1001. In silico and X-ray crystallography analysis led to 58, showing a promising activity against three SARS-CoV-2 variants and a valuable safety in Vero cells and human embryonic lung fibroblasts. The drug tolerance was also confirmed by in vivo studies, along with pharmacokinetics evaluation. In summary, 58 could pave the way to develop a clinical candidate for intranasal administration.

Structural Insights into of the Allosteric Activation of the LicT Antiterminator by PTS-Mediated Phosphorylation.

LicT belongs to an essential family of bacterial transcriptional antitermination proteins controlling the expression of sugar-metabolizing operons. When activated, they bind to nascent mRNAs, preventing premature arrest of transcription. The RNA binding capacity of the N-terminal domain CAT is controlled by phosphorylations of two homologous regulation modules by the phosphotransferase system (PTS). Previous studies on truncated and mutant proteins provided partial insight into the mechanism of signal transduction between the effector and regulatory modules. We report here the conformational and functional investigation on the allosteric activation of full-length LicT. Combining fluorescence anisotropy and NMR, we find a tight correlation between LicT RNA binding capacity and CAT closure upon PTS-mediated phosphorylation and phosphomimetic mutations. Our study highlights fine structural differences between activation processes. Furthermore, the NMR study of full-length proteins points to the back and forth propagation of structural restraints from the RNA binding to the distal regulatory module.