An Infrared 2D-COS Study of Fibril Formation

Aprovat per Decret de l'Alcaldia de 06-07-200

Exploring Polar Headgroup Interactions Between Sphingomyelin and Ceramide with Infrared Spectroscopy

Ceramide is a major actor in the sphingolipid signaling pathway elicited by various kinds of cell stress. Under those conditions ceramide (Cer) is produced in the plasma membrane as a product of sphingomyelin (SM) hydrolysis, and this may lead to apoptosis. Thus, SM and Cer coexist in the membrane for some time, and they are known to separate laterally from the (more abundant) glycerolipids, giving rise to highly rigid domains or platforms. The properties of these domains/platforms are rather well understood, but the underlying SM:Cer molecular interactions have not been explored in detail. Infrared (IR) spectroscopy is a powerful analytical technique that provides information on all the chemical groupings in a molecule, and that can be applied to membranes and lipid bilayers in aqueous media. IR spectra can be conveniently retrieved as a function of temperature, thus revealing the thermotropic transitions of SM and its mixtures with Cer. Four regions of the IR spectrum of these sphingolipids have been examined, two of them dominated by the hydrophobic regions in the molecules, namely the C-H stretching vibrations (2800-3000 cm(-1)), and the CH2 scissoring vibrations (1455-1485 cm(-1)), and two others arising from chemical groups at the lipid-water interface, the sphingolipid amide I band (1600-1680 cm(-1)), and the phosphate vibrations in the 1000-1110 cm(-1) region. The latter two regions have been rarely studied in the past. The IR data from the hydrophobic components show a gel (or ripple)-fluid transition of SM at 40 degrees C, that is shifted up to about 70 degrees C when Cer is added to the bilayers, in agreement with previous studies using a variety of techniques. IR information concerning the polar parts is more interesting. The amide I (carbonyl) band of pure SM exhibits a maximum at 1638 cm(-1) at room temperature, and its position is shifted by about 10 cm(-1) in the presence of Cer. Cer causes also a change in the overall band shape, but no signs of band splitting are seen, suggesting that SM and Cer carbonyl groups are interacting tightly, presumably through H-bonds. The 1086 cm(-1) band, corresponding to PO2- vibrations, appears more stable in SM than in DPPC, and it is further stabilized by Cer, again suggesting an important role of H-bonds in the formation of SM:Cer clusters. Thus, SM and Cer can interact through their polar headgroups, in a way that is not accessible to other lipid classes.This work was supported in part by the Spanish Ministerio de Ciencia e Innovacion (MCI), Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PGC2018-099857-B-I00), by the Basque Government (Grants No. IT1264-19, IT1196-19 and IT1270-19), by the Fundacion Biofisica Bizkaia and by the Basque Excellence Research Centre (BERC) program of the Basque Government

The Binding of Aβ42 Peptide Monomers to Sphingomyelin/Cholesterol/Ganglioside Bilayers Assayed by Density Gradient Ultracentrifugation

The binding of Aβ42 peptide monomers to sphingomyelin/cholesterol (1:1 mol ratio) bilayers containing 5 mol% gangliosides (either GM1, or GT1b, or a mixture of brain gangliosides) has been assayed by density gradient ultracentrifugation. This procedure provides a direct method for measuring vesicle-bound peptides after non-bound fraction separation. This centrifugation technique has rarely been used in this context previously. The results show that gangliosides increase by about two-fold the amount of Aβ42 bound to sphingomyelin/cholesterol vesicles. Complementary studies of the same systems using thioflavin T fluorescence, Langmuir monolayers or infrared spectroscopy confirm the ganglioside-dependent increased binding. Furthermore these studies reveal that gangliosides facilitate the aggregation of Aβ42 giving rise to more extended β-sheets. Thus, gangliosides have both a quantitative and a qualitative effect on the binding of Aβ42 to sphingomyelin/cholesterol bilayers.This work was supported in part by grants from the Spanish Ministry of Economy (grant FEDER MINECO PGC2018-099857-B-I00) and the Basque Government (grants No. IT1264-19 and IT1270-19)

Conformational Plasticity Underlies Membrane Fusion Induced by an HIV Sequence Juxtaposed to the Lipid Envelope

Envelope glycoproteins from genetically-divergent virus families comprise fusion peptides (FPs) that have been posited to insert and perturb the membranes of target cells upon activation of the virus-cell fusion reaction. Conserved sequences rich in aromatic residues juxtaposed to the external leaflet of the virion-wrapping membranes are also frequently found in viral fusion glycoproteins. These membrane-proximal external regions (MPERs) have been implicated in the promotion of the viral membrane restructuring event required for fusion to proceed, hence, proposed to comprise supplementary FPs. However, it remains unknown whether the structure-function relationships governing canonical FPs also operate in the mirroring MPER sequences. Here, we combine infrared spectroscopy-based approaches with cryo-electron microscopy to analyze the alternating conformations adopted, and perturbations generated in membranes by CpreTM, a peptide derived from the MPER of the HIV-1 Env glycoprotein. Altogether, our structural and morphological data support a cholesterol-dependent conformational plasticity for this HIV-1 sequence, which could assist cell-virus fusion by destabilizing the viral membrane at the initial stages of the processThis study was supported by the Spanish MCIU (Grants RTI2018-095624-B-C21; MCIU/AEI/FEDER, UE to JLN and BA; and PID2019-111096GA-I00; MCIU/AEI/FEDER, UE to AC) and Basque Government (Grant: IT1196-19). Technical assistance from MI Collado and M Carril with 31P-NMR measurements and data processing is greatly acknowledge

Conjugative Coupling Proteins and the Role of Their Domains in Conjugation, Secondary Structure and in vivo Subcellular Location

Type IV Coupling Proteins (T4CPs) are essential elements in many type IV secretion systems (T4SSs). The members of this family display sequence, length, and domain architecture heterogeneity, being the conserved Nucleotide-Binding Domain the motif that defines them. In addition, most T4CPs contain a Transmembrane Domain (TMD) in the amino end and an All-Alpha Domain facing the cytoplasm. Additionally, a few T4CPs present a variable domain at the carboxyl end. The structural paradigm of this family is TrwB(R388), the T4CP of conjugative plasmid R388. This protein has been widely studied, in particular the role of the TMD on the different characteristics of TrwB(R388). To gain knowledge about T4CPs and their TMD, in this work a chimeric protein containing the TMD of TraJ(pKM101)and the cytosolic domain of TrwB(R388)has been constructed. Additionally, one of the few T4CPs of mobilizable plasmids, MobB(CloDF13)of mobilizable plasmid CloDF13, together with its TMD-less mutant MobB Delta TMD have been studied. Mating studies showed that the chimeric protein is functionalin vivoand that it exerted negative dominance against the native proteins TrwB(R388)and TraJ(pKM101). Also, it was observed that the TMD of MobB(CloDF13)is essential for the mobilization of CloDF13 plasmid. Analysis of the secondary structure components showed that the presence of a heterologous TMD alters the structure of the cytosolic domain in the chimeric protein. On the contrary, the absence of the TMD in MobB(CloDF13)does not affect the secondary structure of its cytosolic domain. Subcellular localization studies showed that T4CPs have a unipolar or bipolar location, which is enhanced by the presence of the remaining proteins of the conjugative system. Unlike what has been described for TrwB(R388), the TMD is not an essential element for the polar location of MobB(CloDF13). The main conclusion is that the characteristics described for the paradigmatic TrwB(R388)T4CP should not be ascribed to the whole T4CP family. Specifically, it has been proven that the mobilizable plasmid-related MobB(CloDF13)presents different characteristics regarding the role of its TMD. This work will contribute to better understand the T4CP family, a key element in bacterial conjugation, the main mechanism responsible for antibiotic resistance spread.This work was in part supported by grants from the University of the Basque Country (GIU18/229 and COLAB19/08) and the Industry Department of the Basque Government (ELKARTEK 2020 KK-2020/00007). IA-R was a pre-doctoral student supported by the Basque Government

Truncation-Driven Lateral Association of α-Synuclein Hinders Amyloid Clearance by the Hsp70-Based Disaggregase

The aggregation of α-synuclein is the hallmark of a collective of neurodegenerative disorders known as synucleinopathies. The tendency to aggregate of this protein, the toxicity of its aggregation intermediates and the ability of the cellular protein quality control system to clear these intermediates seems to be regulated, among other factors, by post-translational modifications (PTMs). Among these modifications, we consider herein proteolysis at both the N- and C-terminal regions of α-synuclein as a factor that could modulate disassembly of toxic amyloids by the human disaggregase, a combination of the chaperones Hsc70, DnaJB1 and Apg2. We find that, in contrast to aggregates of the protein lacking the N-terminus, which can be solubilized as efficiently as those of the WT protein, the deletion of the C-terminal domain, either in a recombinant context or as a consequence of calpain treatment, impaired Hsc70-mediated amyloid disassembly. Progressive removal of the negative charges at the C-terminal region induces lateral association of fibrils and type B* oligomers, precluding chaperone action. We propose that truncation-driven aggregate clumping impairs the mechanical action of chaperones, which includes fast protofilament unzipping coupled to depolymerization. Inhibition of the chaperone-mediated clearance of C-truncated species could explain their exacerbated toxicity and higher propensity to deposit found in vivo.This work was supported by grants PID2019-111068GB-I00 (to A.M.) (AEI/FEDER, UE) and PID2019-105872GB-I00 (to J.M.V.) (AEI/FEDER, UE) from the Ministry of Science and Innovation and by the Basque Government (grant IT1201-19 to AM). The Centro Nacional de Biotecnología (CNB) is a Severo Ochoa Center of Excellence (MINECO award SEV 2017-0712). N.O. holds a contract funded by Fundacion Biofisika Bizkaia. Acknowledgment

Effects of a N-Maleimide-derivatized Phosphatidylethanolamine on the Architecture and Properties of Lipid Bilayers

N-maleimide-derivatized phospholipids are often used to facilitate protein anchoring to membranes. In autophagy studies, this is applied to the covalent binding of Atg8, an autophagy protein, to a phosphatidylethanolamine (PE) in the nascent autophagosome. However, the question remains on how closely the N-maleimide PE derivative (PE-mal) mimicks the native PE in the bilayer. In the present paper, spectroscopic and calorimetric techniques have been applied to vesicles containing either PE or PE-mal (together with other phospholipids) to compare the properties of the native and derivatized forms of PE. According to differential scanning calorimetry, and to infrared spectroscopy, the presence of PE-mal did not perturb the fatty acyl chains in the bilayer. Fluorescence spectroscopy and microscopy showed that PE-mal did not alter the bilayer permeability either. However, fluorescence emission polarization of the Laurdan and DPH probes indicated an increased order, or decreased fluidity, in the bilayers containing PE-mal. In addition, the infrared spectral data from the phospholipid phosphate region revealed a PE-mal-induced conformational change in the polar heads, accompanied by increased hydration. Globally considered, the results suggest that PE-mal would be a reasonable substitute for PE in model membranes containing reconstituted proteins.This work was funded in part by the Spanish Ministry of Science, Innovation, and Universities (MCIU), Agencia Estatal de Investigación (AEI), Fondo Europeo de Desarrollo Regional (FEDER) (grant No. PID2021-124461NB-I00), the Basque Government (grant No. IT1625-22), Fundación Ramón Areces (CIVP20A6619), Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. E.J.G.-R. was supported by Fundación Ramón Areces. Y.R.V. was a recipient of a pre-doctoral FPU fellowship from the Spanish Ministry of Science, Innovation, and Universities (FPU18/00799). U.B. thanks the University of the Basque Country for a pre-doctoral contract

Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes

Molecular Characterization of the Viroporin Function of Foot-and-Mouth Disease Virus Nonstructural Protein 2B

Nonstructural protein 2B of foot-and-mouth disease (FMD) virus (FMDV) is comprised of a small, hydrophobic, 154-amino-acid protein. Structure-function analyses demonstrated that FMDV 2B is an ion channel-forming protein. Infrared spectroscopy measurements using partially overlapping peptides that spanned regions between amino acids 28 and 147 demonstrated the adoption of helical conformations in two putative transmembrane regions between residues 60 and 78 and between residues 119 and 147 and a third transmembrane region between residues 79 and 106, adopting a mainly extended structure. Using synthetic peptides, ion channel activity measurements in planar lipid bilayers and imaging of single giant unilamellar vesicles (GUVs) revealed the existence of two sequences endowed with membrane-porating activity: one spanning FMDV 2B residues 55 to 82 and the other spanning the C-terminal region of 2B from residues 99 to 147. Mapping the latter sequence identified residues 119 to 147 as being responsible for the activity. Experiments to assess the degree of insertion of the synthetic peptides in bilayers and the inclination angle adopted by each peptide regarding the membrane plane normal confirm that residues 55 to 82 and 119 to 147 of 2B actively insert as transmembrane helices. Using reverse genetics, a panel of 13 FMD recombinant mutant viruses was designed, which harbored nonconservative as well as alanine substitutions in critical amino acid residues in the area between amino acid residues 28 and 147. Alterations to any of these structures interfered with pore channel activity and the capacity of the protein to permeabilize the endoplasmic reticulum (ER) to calcium and were lethal for virus replication. Thus, FMDV 2B emerges as the first member of the viroporin family containing two distinct pore domains