Explore the vast range of titles available.

CheV is one of the least understood chemosensory signaling proteins. Our demonstration that CheV interacts only with certain chemoreceptors offers fundamental new insights. These findings, combined with the observation that CheV is present in bacteria with numerous chemoreceptors, suggest that CheV plays a role in coordinating chemotactic outputs in complex chemosensory systems. Understanding the mechanisms by which chemotactic responses are defined in bacteria with a high number of chemoreceptors is a major research priority in the field of chemotaxis. While previous studies, including this one, show that the ability to be phosphorylated is crucial for CheV function, the molecular consequences of CheV phosphorylation have remained unclear. Our discovery that phosphorylation is essential for CheV binding to certain chemoreceptors fills in this critical gap in understanding the molecular mechanism of CheV. This study is likely to inspire further research into CheV function in other bacteria using similar approaches.

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR–CHR binding.

Abstract Bacteria sense and respond to changing environmental conditions using a diverse range of receptors. Currently, the signals recognized by most receptors remain unknown, thereby limiting our understanding of their function. Since its introduction a decade ago, ligand screening by the thermal-shift assay has identified the signal molecules recognized by numerous receptors, solute-binding proteins, and transcriptional regulators. This progress is summarized in this review. Signal identification is facilitated by the fact that ligand-binding domains can be generated as individual soluble proteins that retain the signal-binding capabilities of the full-length proteins. Various issues relevant to the reliability of the thermal shift assay are discussed, including false-positive and false-negative results, the value of a protein pH screen prior to ligand screening, and the need to verify results with methods for the direct study of ligand binding, such as isothermal titration calorimetry. This review was inspired by the XVIII conference on Bacterial Locomotion and Signal Transduction (Cancun, January 2025), where several notable advances were reported based on the application of the thermal shift assay. The thermal shift assay has enabled major advances in the study of bacterial signal transduction and receptor function.

Since the beginning of the COVID-19 pandemic, considerable efforts have been made to develop protective vaccines against SARS-CoV-2 infection. However, immunity tends to decline within a few months, and new virus variants are emerging with increased transmissibility and capacity to evade natural or vaccine-acquired immunity. Therefore, new robust strategies are needed to combat SARS-CoV-2 infection. The viral spike composed of S1 and S2 subunits mediates viral attachment and membrane fusion to infect the host cell. In this process, interaction between the highly conserved heptad repeat 1 and 2 regions (HR1 and HR2) of S2 is crucial and for this reason; these regions are promising targets to fight SARS-CoV-2. Here, we describe the design and characterization of chimeric proteins that structurally imitate the S2 HR1 region in a trimeric coiled-coil conformation. We biophysically characterized the proteins and determined their capacity to bind the HR2 region, as well as their inhibitory activity of SARS-CoV-2 infection in vitro. HR1 mimetic proteins showed conformational heterogeneity and a propensity to form oligomers. Moreover, their structure is composed of subdomains with varied stability. Interestingly, the full HR1 proteins showed high affinity for HR2-derived peptides and SARS-CoV-2 inhibitory activity, whereas smaller proteins mimicking HR1 subdomains had a decreased affinity for their complementary HR2 region and did not inhibit the virus. The results provide insight into effective strategies to create mimetic proteins with broad inhibitory activity and therapeutic potential against SARS-CoV-2.

Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.

The functional and pathological implications of the enormous genetic diversity of the human genome are mostly unknown, primarily due to our unability to predict pathogenicity in a high-throughput manner. In this work, we characterized the phenotypic consequences of eight naturally-occurring missense variants on the multifunctional and disease-associated NQO1 protein using biophysical and structural analyses on several protein traits. Mutations found in both exome-sequencing initiatives and in cancer cell lines cause mild to catastrophic effects on NQO1 stability and function. Importantly, some mutations perturb functional features located structurally far from the mutated site. These effects are well rationalized by considering the nature of the mutation, its location in protein structure and the local stability of its environment. Using a set of 22 experimentally characterized mutations in NQO1, we generated experimental scores for pathogenicity that correlate reasonably well with bioinformatic scores derived from a set of commonly used algorithms, although the latter fail to semiquantitatively predict the phenotypic alterations caused by a significant fraction of mutations individually. These results provide insight into the propagation of mutational effects on multifunctional proteins, the implementation of in silico approaches for establishing genotype-phenotype correlations and the molecular determinants underlying loss-of-function in genetic diseases.

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype–phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

Phosphorylation is fundamental to modulate protein function and stabilty. There have detected about 300000 site-specic Phosphorylation sites in over 20000 human proteins. However, only 5% of the sites have been experimenrally characterized. In this work, we investigated a phosphorylation event in AGT, an important enzyme due to its detoxifying role of glyoxylate and hundreds of mutations cause a rare disease (primary hyperoxaluria type I or PH1). We analyzed the effect of phosphomimetic mutations Ser81 on the WT proten, the common polymorpshim LM, and the most common disease-associted variants (LM-G170R and LM-I244T). Using biochemical, biophysical and cell biology approaches, we show that phosphorylation at S81 dramatically affects PLP/PMP binding pose and disrupts enzyme activity, without pertubing its subcellular location to peroxisomes. This reversible phenotype is similar to the irreversible effects of some PH1-causing mutaions. Thus, we provide evidence for a novel regulatory mechanism for PH1, in health and disease.

Chemotaxis pathways are among the most complex signaling systems in bacteria. A central feature of these pathways is the ternary complex formed by chemoreceptors, the autokinase CheA, and the coupling proteins CheW and CheV. Whereas CheW is present in all chemotaxis pathways, CheV is primarily found in bacteria that contain many chemoreceptors. CheV is a fusion of a CheW-like domain to a phosphorylatable receiver domain. The roles of CheV and its phosphorylation are currently uncertain. Pseudomonas aeruginosa contains many chemoreceptors for which the cognate signals have been identified. Quantitative capillary chemotaxis assays of a cheV mutant revealed that responses to certain chemoeffectors, such as nitrate and α-ketoglutarate, were drastically reduced, while responses to others, such as amino acids and inorganic phosphate, were comparable to the wild type, indicating that CheV selectively acts on specific chemoreceptors. To study the mechanism of CheV action, we conducted protein-protein interaction experiments using isothermal titration calorimetry. These studies showed that unphosphorylated CheV fails to bind to cytosolic fragments of the McpN and PctA chemoreceptors, which mediate responses to nitrate and amino acids, respectively. In contrast, the phosphorylation-mimic CheV D238E bound with very high affinity (KD = 8 nM) to McpN but failed to interact with PctA. Thus, CheV in P. aeruginosa binds to some chemoreceptors but not to others in a phosphorylation-dependent manner. These results suggest that CheV is a regulatory protein that modulates signaling through specific chemoreceptors. CheV may thus facilitate the coordination of chemotaxis responses in complex, multi-chemoreceptor systems.

MRGPRX2, a G-protein-coupled-seven transmembrane domain receptor, is mainly expressed in mast cells and neurons and is involved in skin immunity and pain. It is implicated in the pathophysiology of non-IgE-mediated immediate hypersensitivity and has been related to adverse drug reactions. Moreover, a role has been proposed in asthma, atopic dermatitis, contact dermatitis, and chronic spontaneous urticaria. Although it has a prominent role in disease, its signaling transduction is poorly understood. This study shows that MRGPRX2 activation with substance P increased Lysyl t-RNA synthetase (LysRS) translocation to the nucleus. LysRS is a moonlighting protein with a dual role in protein translation and IgE signaling in mast cells. Upon allergen- IgE-FcεRI crosslinking, LysRS is translocated to the nucleus and activates microphthalmia-associated transcription factor (MITF) activity. In this study, we found that MRGPRX2 triggering led to MITF phosphorylation and increased MITF activity. Therefore, overexpression of LysRS increased MITF activity after MRGPRX2 activation. MITF silencing reduced MRGPRX2-dependent calcium influx and mast cell degranulation. Furthermore, a MITF pathway inhibitor, ML329, impaired MITF expression, calcium influx, and mast cell degranulation. Moreover, drugs such as atracurium, vancomycin, and morphine, reported to induce MRGPRX2-dependent degranulation, increased MITF activity. Altogether, our data show that MRGPRX2 signaling enhances MITF activity, and its abrogation by silencing or inhibition resulted in defective MRGPRX2 degranulation. We conclude that MRGPRX2 signaling involves the LysRS and MITF pathway. Thus, MITF and MITF-dependent targets may be considered therapeutic approaches to treat pathologies where MRGPRX2 is implicated.