Engineered disulfide bonds in recombinant human interferon-γ: the impact of the N-terminal helix A and the AB-loop on protein stability

Abstract

Insertion sites for cysteines with optimal stereochemistry for the formation of unstrained disulfide bridges were identified in recombinant human interferon-gamma (rhu-IFN-gamma) by computer modelling. We have engineered two different disulfide cross-linked mutants, containing a pair of symmetry-related disulfide bonds, which stabilize the N-termini of both monomers of the homodimeric protein. Mutations E7C and S69C allow the formation of an intramonomer disulfide bond between helices A and D. In contrast, the A17C and H111C mutations lead to a covalent cross-link between both monomers. The AB-loop is linked to helix F. The fluorescence properties of native and disulfide cross-linked proteins were studied as a function of guanidine hydrochloride concentration. Melting temperatures (Tm) were calculated from the decrease in CD ellipticity at 220 nm. The induction of the antiviral effect was measured using A549 fibroblast cells infected with encephalomyocarditis virus. The ability to induce the expression of the HLA-DR antigen in Colo 205 cells was determined by fluorescence-activated cell scanning analysis. The stability of both mutants was strongly enhanced against temperature- and cosolvent-induced unfolding. The delta Tm of mutant IFN-gamma E7C/S69C was 15 degrees C. All measured biological activities of this mutant were equal to wild type. In the case of the other mutant IFN-gamma A17C/H111C, the delta Tm value was 25 degrees C. This mutation abolishes nearly the entire biological activity (< 1%) with no detectable changes of secondary structure in the CD spectrum. Our results illustrate the importance of the N-terminal helix A and the AB-loop for the unfolding pathway and thermodynamic stability of rhu-IFN-gamma.

The IQGAP1 N-Terminus Forms Dimers, and the Dimer Interface Is Required for Binding F-Actin and Calcium-Bound Calmodulin

“…We then sought to stabilize the dimer by introducing a disulfide bond. The Cα-Cα distance of cysteines involved in a disulfide bond is usually less than 7.5 Å 32 . Two pairs of residues at the dimer interface satisfied this requirement: K161-K161 (6.7 Å) and A154-A154 (7.1 Å).…”

Solution structure of the TLR adaptor MAL/TIRAP reveals an intact BB loop and supports MAL Cys91 glutathionylation for signaling

“…This rearrangement is caused by cysteine residues 89 and 134 forming a disulfide bond and shifting the location of strand βB. In the solution structure, the C α atoms of these two cysteine residues are 11 Å apart, making disulfide-bond formation unlikely (36). The second disulfide bond found in the crystal structures involves the pair C142 and C174.…”

Interferon‐γ variants with deletions in the AB surface loop

“…The starting model of recombinant human IFN-γ is based on the C A coordinates of the corresponding crystal structure (PDB code 1hig [2]) and has been described in an earlier report [6]. Deletions were constructed using a database search procedure [10]: a non-redundant fragment database, derived by hierarchical clustering (HCAPD database) is searched for suitable loops according to geometrical criteria derived from defined anchor groups in the template protein.…”

“…For computer modelling and documentation, the software packages BRAGI [9] and SYBYL (Tripos Associates Inc.) were used on a HP series 9000 model 735/99 workstation and on a Silicon Graphics Indigo 2 workstation, respectively. The starting model of recombinant human IFN-γ is based on the C A coordinates of the corresponding crystal structure (PDB code 1hig [2]) and has been described in an earlier report [6]. Deletions were constructed using a database search procedure [10]: a non-redundant fragment database, derived by hierarchical clustering (HCAPD database) is searched for suitable loops according to geometrical criteria derived from defined anchor groups in the template protein.…”