Functional characterization of TasA and TapA in the formation of the amyloid fiber in Bacillus subtilis

Introduction Functional amyloids are a very heterogeneous family of amyloid proteins widespread in nature, from humans to bacteria. Unlike their “pathogenic” relatives, implicated in several neurodegenerative diseases, functional amyloids play important roles in several biological processes. In Bacillus subtilis, the protein TasA forms amyloid-like fibers that serve as a scaffold for the rest of the components of the extracellular matrix. Along with TasA, the auxiliary protein TapA promotes and accelerates TasA fiber assembly. Most amyloid proteins contain regions within their sequence in which their aminoacid composition make them prone to aggregation. However, the sequence determinants in TasA or TapA involved in the assembly of the amyloid fiber, its structure and function still remains elusive. Objectives To identify and characterize regions of TasA or TapA important for amyloid fiber assembly and functionality in Bacillus subtilis biofilms. Materials & methods An in silico study was performed in order to define amyloidogenic regions within TasA and TapA sequences. This analysis revealed several regions of interest and was followed by in vitro experiments using synthetic peptides corresponding to the analyzed regions. We used several biophysical techniques in combination with transmission electron microscopy to study their possible amyloid properties. Results Of the predicted amyloidogenic regions of TasA, only two polymerized with enrichment of beta-sheets, characteristic of amyloid proteins. A similar behavior was found in a sequence of the N-terminal half of TapA, which has been previously demonstrated to be determinant in the functionality of TapA. Conclusion Our findings support the utility of the in silico prediction for the search of amyloidogenic domains in proteins. The aggregative properties of all peptides and the additional amyloid-like features of some of them are suggestive of their relevance in the amyloid properties of TasA and suggest in some cases, their putative implication in the TasA-TapA interaction.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Exploring Highly Conserved Regions of SARS-CoV-2 Spike S2 Subunit as Targets for Fusion Inhibition Using Chimeric Proteins

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.Junta de AndaluciaSpain's State Research Agency CV20.26565 ERDF/ESF PID2019.107515RB.C21ANRSFrench National Research Agency (ANR) French National Research Agency (ANR)EHVA ANR-10-LABX-77 68103

The influence of N-terminal acetylation on micelle-induced conformational changes and aggregation of α-Synuclein

The biological function of α-Synuclein has been related to binding to lipids and membranes but these interactions can also mediate α-Synuclein aggregation, which is associated to Parkinson's disease and other neuropathologies. In brain tissue α-Synuclein is constitutively N-acetylated, a modification that plays an important role in its conformational propensity, lipid and membrane binding, and aggregation propensity. We studied the interactions of the lipid-mimetic SDS with N-acetylated and non-acetylated α-Synuclein, as well as their early-onset Parkinson's disease variants A30P, E46K and A53T. At low SDS/protein ratios α-Synuclein forms oligomeric complexes with SDS micelles with relatively low α-helical structure. These micellar oligomers can efficiently nucleate aggregation of monomeric α-Synuclein, with successive formation of oligomers, protofibrils, curly fibrils and mature amyloid fibrils. N-acetylation reduces considerably the rate of aggregation of WT α-Synuclein. However, in presence of any of the early-onset Parkinson's disease mutations the protective effect of N-acetylation against micelle-induced aggregation becomes impaired. At higher SDS/protein ratios, N-acetylation favors another conformational transition, in which a second type of α-helix-rich, non-aggregating oligomers become stabilized. Once again, the Parkinson's disease mutations disconnect the influence of N-acetylation in promoting this transition. These results suggest a cooperative link between the N-terminus and the region of the mutations that may be important for α-Synuclein function

Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up NewWays of Inhibiting HIV-1 Fusion

Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms23052794/s1.Acknowledgments: M.C.-M. acknowledges a grant from Youth Employment Operative Program of the Andalusia Government and the European Social Fund (ESF). S.C. acknowledges an exchange studentship from the ERASMUS+ program of the European Union. The results shown are included as part of M.C.-M. doctoral thesis.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.Grants BIO2016-76640-R and PID2019.107515RB.C21 from the Spain’s State Research AgencySRA/10.13039/501100011033, co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future

The high-resolution NMR structure of the R21A Spc-SH3:P41 complex: Understanding the determinants of binding affinity by comparison with Abl-SH3

BACKGROUND: SH3 domains are small protein modules of 60–85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the α-spectrin SH3 domain (Spc-SH3). RESULTS: Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex. CONCLUSION: Based on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding

Novel chimeric proteins mimicking SARS-CoV-2 spike epitopes with broad inhibitory activity.

SARS-CoV-2 spike (S) protein mediates virus attachment to the cells and fusion between viral and cell membranes. Membrane fusion is driven by mutual interaction between the highly conserved heptad-repeat regions 1 and 2 (HR1 and HR2) of the S2 subunit of the spike. For this reason, these S2 regions are interesting therapeutic targets for COVID-19. Although HR1 and HR2 have been described as transiently exposed during the fusion process, no significant antibody responses against these S2 regions have been reported. Here we designed chimeric proteins that imitate highly stable HR1 helical trimers and strongly bind to HR2. The proteins have broad inhibitory activity against WT B.1 and BA.1 viruses. Sera from COVID-19 convalescent donors showed significant levels of reactive antibodies (IgG and IgA) against the HR1 mimetic proteins, whereas these antibody responses were absent in sera from uninfected donors. Moreover, both inhibitory activity and antigenicity of the proteins correlate positively with their structural stability but not with the number of amino acid changes in their HR1 sequences, indicating a conformational and conserved nature of the involved epitopes. Our results reveal previously undetected spike epitopes that may guide the design of new robust COVID-19 vaccines and therapies.This work was supported by grants CV20.26565 from the Consejería de Economía y Conocimiento, Junta de Andalucía (Spain), PID2019.107515RB.C21 from the Spanish State Research Agency (SRA/10.13039/501100011033), and co-funded by ERDF/ESF, “A way to make Europe”/“Investing in your future. The work performed in C.M.’s laboratory was supported by grants from ANRS (Agence Nationale de Recherches sur le SIDA et les h´epatites virales), the Investissements d’Avenir program managed by the ANR under reference ANR-10-LABX-77 and EHVA (No. 681032, Horizon 2020). Work in S.B.’s laboratory was supported by grants from the Agence Nationale de la Recherche (ANR) (ANR-11-LABX-0070_TRANSPLANTEX), the INSERM (UMR_S1109), the Institut Universitaire de France (IUF), all the University of Strasbourg (IDEX UNISTRA), the European Regional Development Fund (European Union) INTERREG V program (project no. 3.2 TRIDIAG) and MSD-Avenir grant AUTOGEN

Probing vulnerability of the gp41 C-terminal heptad repeat as target for miniprotein HIV inhibitors

One of the therapeutic strategies in HIV neutralization is blocking membrane fusion. In this process, tight interaction between the N-terminal and C-terminal heptad-repeat (NHR and CHR) regions of gp41 is essential to promote membranes apposition and merging. We have previously developed single-chain proteins (named covNHR) that accurately mimic the complete gp41 NHR region in its trimeric conformation. They tightly bind CHR-derived peptides and show a potent and broad HIV inhibitory activity in vitro. However, the extremely high binding affinity (sub-picomolar) is not in consonance with their inhibitory activity (nanomolar), likely due to partial or temporal accessibility of their target in the virus. Here, we have designed and characterized two single-chain covNHR miniproteins each encompassing one of the two halves of the NHR region and containing two of the four sub-pockets of the NHR crevice. The two miniproteins fold as trimeric helical bundles as expected but while the C-terminal covNHR (covNHR-C) miniprotein is highly stable, the N-terminal counterpart (covNHR-N) shows only marginal stability that could be improved by engineering an internal disulfide bond. Both miniproteins bind their respective complementary CHR peptides with moderate (micromolar) affinity. Moreover, the covNHR-N miniproteins can access their target in the context of trimeric native envelope proteins and show significant inhibitory activity for several HIV pseudoviruses. In contrast, covNHR-C cannot bind its target sequence and neither inhibits HIV, indicating a higher vulnerability of C-terminal part of CHR. These results may guide the development of novel HIV inhibitors targeting the gp41 CHR region.Spanish Ministry of Economy and Competitiveness (grant: BIO2016-76640-R), ANRS and the Vaccine Research Institute for the Investissements d'Avenir program to C.M. and by the European Fund for Research and Development from the European Union.Departamento de Química Física, Facultad de Ciencias, Universidad de Granada. Grupo FQM-171 "Biofísica y Biotecnología Molecular

Strategies for achieving intense single-cycle pulses with in-line post-compression setups

Intense few- and single-cycle pulses are powerful tools in different fields of science Today, third- and higher-order terms in the remnant spectral phase of the pulses remain a major obstacle for obtaining high-quality few- and single-cycle pulses from in-line post-compression setups. In this Letter, we show how input pulse shaping can successfully be applied to standard post-compression setups to minimize the occurrence of high-order phase components during nonlinear propagation and to directly obtain pulses with durations down to 3 fs. Furthermore, by combining this pulse shaping of the input pulse with new-generation broadband chirped mirrors and material addition for remnant third-order phase correction, pulses down to 2.2 fs duration have been measured.Ministerio de Economía y Competitividad (MINECO) (FIS2013-44174-P, FIS2015-71933-REDT, FIS2016-75652-P, FIS2017-87970-R); Consejería de Educación, Junta de Castilla y León (SA046U16, SA116U13); Fundação para a Ciência e a Tecnologia (FCT) (M-ERANET2/0002/2016); European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-022096)