3 research outputs found

    10E8 antigorputzaren potentziaren optimizazioa mintzarekiko elkarrekintza hidrofobikoak eta elektrostatikoak sustatzen dituzten mutazioen konbinazioaren bidez

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    [EU] Lan honetan 10E8 Fab mutanteak eraiki ziren mintzarekin elkarrekintza hidrofobikoak eta elektrostatikoak sustatzeko eta horrela, Fab basatiaren afinitatea emendatzeko. Bibliografian deskribatutako 10E8 S30R-N52R antigorputza, 10E8 S100cF antigorputza eta bibliografian egin ez den aurreko bien mutazioak konbinatzen dituen 10E8 S30R-N52R S100cF antigorputza eraiki ziren. S100cF eta S30R-N52R jada deskribatuta egonik, lan hau haien konbinazioaren (S30R-N52R S100cF) eraginaren karakterizaziora bideratuta dago. Helburua konbinazioen bidez 10E8 antigorputzaren potentzia emendatzea posiblea den aztertzea da, prozesuan mutazioen konbinazioak dituen eraginak zehaztuz, eragin horien baturaren elkarrekite fisikoa ulertuz eta atzetik dagoen elkarrekintza mekanismoa proposatuz. Horretarako, antigorputzak adierazi, disoluzioan eta mintz mikroingurunean lotura aztertu eta haien neutralaziazio gaitasuna zehaztu zen. Mutazioen konbinaketaren bidez 10E8 antigorputzaren potentzia nabarmenki emendatzea lortu da. Mutazioak konbinatzerakoan afinitatearen hobekuntzan eragin sinergikoa behatu zen, eta honen oinarria izan daitekeen mekanismo molekularra proposatu da

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

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    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.This study was supported by the Spanish MCIN (Grants PID2021-126014OB-I00 MCIN/AEI/FEDER, UE to JLN and BA; and PID2021-122212OA-I00 MCIN/AEI/FEDER, UE to ER), Basque Government (Grant: IT1449-22) and JSPS KAKENHI 20H03228 (to J.M.M.C.)
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