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

Structural and thermodynamic basis for the recognition of the substrate-binding cleft on hen egg lysozyme by a single-domain antibody

Single-domain antibodies (VHHs or nanobodies), developed from heavy chain-only antibodies of camelids, are gaining attention as next-generation therapeutic agents. Despite their small size, the high affinity and specificity displayed by VHHs for antigen molecules rival those of IgGs. How such small antibodies achieve that level of performance? Structural studies have revealed that VHHs tend to recognize concave surfaces of their antigens with high shape-complementarity. However, the energetic contribution of individual residues located at the binding interface has not been addressed in detail, obscuring the actual mechanism by which VHHs target the concave surfaces of proteins. Herein, we show that a VHH specific for hen egg lysozyme, D3-L11, not only displayed the characteristic binding of VHHs to a concave region of the surface of the antigen, but also exhibited a distribution of energetic hot-spots like those of IgGs and conventional protein-protein complexes. The highly preorganized and energetically compact interface of D3-L11 recognizes the concave epitope with high shape complementarity by the classical lock-and-key mechanism. Our results shed light on the fundamental basis by which a particular VHH accommodate to the concave surface of an antigens with high affinity in a specific manner, enriching the mechanistic landscape of VHHs

Unexpectedly enhanced stereoselectivity of peroxidase-catalyzed sulfoxidation in branched alcohols

Lyophilized horseradish peroxidase (HRP) exhibits poor stereoselectivity in the sulfoxidation of thioanisole when the enzyme is either redissolved in water or suspended in organic solvents. However, when HRP is co-lyophilized in the presence of lyoprotectants or ligands, its stereoselectivity, although still low in most organic solvents, increases up to 4-fold if assayed in secondary or tertiary alcohols (but not in their linear isomers). A mechanistic hypothesis is presented explaining this puzzling phenomenon on the basis of a model of the active site of the enzyme-substrate complex derived from its X-ray crystal structure by means of molecular dynamics and energy minimization

Asymmetric sulfoxidations mediated by α-chymotrypsin

The oxidation of aryl alkyl sulfides with H2O2 in aqueous solution is a reasonably facile reaction producing racemic sulfoxides. We show that in the presence of the hydrolytic enzyme α-chymotrypsin such a sulfoxidation is accelerated and, more importantly, becomes stereoselective. With phenyl isobutyl sulfide as a model, the chymotrypsin-mediated, highly asymmetric oxidation is shown to occur in the hydrophobic binding pocket of the enzyme active site. The stereoselectivity of the chymotrypsin-mediated sulfoxidations is correctly explained by means of structure-based molecular modeling of the enzyme-sulfide complexes

Binding of hydrophobic hydroxamic acids enhances peroxidase's stereoselectivity in nonaqueous sulfoxidations

Horseradish peroxidase exhibits a meager stereoselectivity (E) in the sulfoxidation of thioanisole (1a) in 99.8% (v/v) methanol. The E value, however, is greatly enhanced when the enzyme forms a complex with benzohydroxamic acid (2a). These findings are rationalized by means of molecular dynamics simulations and energy minimization which correctly explain (i) why the free enzyme is not stereoselective, (ii) why 2a inhibits peroxidase-catalyzed sulfoxidation of 1a but the enzymatic formation of one enantiomer of the sulfoxide product is inhibited much more than that of the other, thereby raising peroxidase's E, and (iii) why in the presence of 2a the enzyme favors production of the S sulfoxide of 1a. The generality of the observed ligand-induced stereoselectivity enhancement is demonstrated with other hydrophobic hydroxamic acids, as well as with additional thioether substrates

Bidirectional Transformation of a Metamorphic Protein between the Water-Soluble and Transmembrane Native States

The bidirectional transformation of a protein between its native water-soluble and integral transmembrane conformations is demonstrated for FraC, a hemolytic protein of the family of pore-forming toxins. In the presence of biological membranes, the water-soluble conformation of FraC undergoes a remarkable structural reorganization generating cytolytic transmembrane nanopores conducive to cell death. So far, the reverse transformation from the native transmembrane conformation to the native water-soluble conformation has not been reported. We describe the use of detergents with different physicochemical properties to achieve the spontaneous conversion of transmembrane pores of FraC back into the initial water-soluble state. Thermodynamic and kinetic stability data suggest that specific detergents cause an asymmetric change in the energy landscape of the protein, allowing the bidirectional transformation of a membrane protein

Haemolytic actinoporins interact with carbohydrates using their lipid-binding module

Pore-forming toxins (PFTs) are proteins endowed with metamorphic properties that enable them to stably fold in water solutions as well as in cellular membranes. PFTs produce lytic pores on the plasma membranes of target cells conducive to lesions, playing key roles in the defensive and offensive molecular systems of living organisms. Actinoporins are a family of potent haemolytic toxins produced by sea anemones vigorously studied as a paradigm of α-helical PFTs, in the context of lipid–protein interactions, and in connection with nanopore technologies. We have recently reported that fragaceatoxin C (FraC), an actinoporin, engages biological membranes with a large adhesive motif allowing the simultaneous attachment of up to four lipid molecules prior to pore formation. Since actinoporins also interact with carbohydrates, we sought to understand the molecular and energetic basis of glycan recognition by FraC. By employing structural and biophysical methodologies, we show that FraC engages glycans with low affinity using its lipid-binding module. Contrary to other PFTs requiring separate domains for glycan and lipid recognition, the small single-domain actinoporins economize resources by achieving dual recognition with a single binding module. This mechanism could enhance the recruitment of actinoporins to the surface of target tissues in their marine environment. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’

Supplementary data from Haemolytic actinoporins interact with carbohydrates using their lipid-binding module

Table S1. Structural homology of FraC; Figure S1. Kinetics of hemolysis by FraC in the presence of saccharides; Figure S2. Depiction of the lipid/carbohydrate binding region of FraC; Figure S3. Comparison of FraC with a fungal lectin