Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting

Abstract

Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold.

Stabilization of soluble high‐affinity T‐cell receptor with de novo disulfide bonds

“…Surprisingly, of the 18 mutants proposed by the algorithm, only eight could be expressed, and only one mutant with novel disulfide bonds in both α‐ and β‐chains was stabilized. The success of prediction was lower than achieved with DSDBASE algorithm with other proteins, such as the Fc fragment or the large extracellular loop of CD81, an α‐helical protein of the tetraspanin family . One reason might be that the chosen algorithm mainly takes into account the parameters of secondary structure provided, and the regions of TCR where the proposed stabilization mutations cluster can be less prominently assigned to such particular elements.…”

An engineered CD81‐based combinatorial library for selecting recombinant binders to cell surface proteins: Laminin binding CD81 enhances cellular uptake of extracellular vesicles

“…Availability of several high‐resolution crystal structures has corroborated CD81 LEL as an independent folding unit (Cunha et al., 2018 ; Kitadokoro, 2001 ; Kitadokoro et al., 2001 ; Nelson et al., 2018 ). Previously, we have attempted to improve the biophysical properties of CD81 LEL and constructed mutants that exhibit in one case an extremely high level of thermostability and in one case reversible thermal denaturation (Vogt et al., 2018 ). As these should be more permissive for mutagenesis required to introduce a novel antigen binding site, we have identified amino acid residues that could form a novel antigen‐binding surface based on the above mutants.…”

“…The crystallographic structure of a soluble form of the tetraspanin CD81 LEL domain is a five‐helix bundle stabilized by two disulfide bridges (Kitadokoro, 2001 ; Kitadokoro et al., 2001 ). The protein fold as well as its key structural features are conserved among tetraspanins, while a high degree of variability in its length and composition can be found in its central part that in CD81 comprises three helices (Seigneuret, 2006 ; Seigneuret et al., 2001 ) Previously, we have designed highly stabilized mutants of CD81 LEL via introduction of de novo disulfide bonds (Vogt et al., 2018 ). Two such CD81 LEL variants, the most stable one and the variant that exhibited reversible refolding after denaturation, were chosen to serve as scaffolds for libraries of antigen‐binding units, obtained by randomization of selected solvent‐exposed residues.…”

The large extracellular loop of CD63 interacts with gp41 of HIV-1 and is essential for establishing the virological synapse

“…Misfolded regions can be used to determine important interaction regions within a protein. For CD81, a member of the tetraspanin superfamily, it has been shown that cysteines within the LEL are important for stabilizing the protein folding 49 . The same observation was made with FRET experiments where the point mutations C145A and C146A led to a decrease in PPI strength (Fig.…”