A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.

Abstract

SummaryPeptide binding by class II major histocompatibility complex proteins is generally enhanced at low pH in the range of hydrogen ion concentrations found in the endosomal compartments of antigen-presenting cells. We and others have proposed that class II molecules undergo a reversible conformational change at low pH that is associated with enhanced peptide loading. However, no one has previously provided direct evidence for a structural change in class II proteins in the mildly acidic pH conditions in which enhanced peptide binding is observed. In this study, susceptibility to denaturation induced by sodium dodecyl sulfate (SDS) detergent or heat was used to probe the conformation of class II at different hydrogen ion concentrations. Class II molecules became sensitive to denaturation at pH 5.5-6.5 depending on the allele and experimental conditions. The observed structural transition was fully reversible if acidic pH was neutralized before exposure to SDS or heat. Experiments with the environment-sensitive fluorescent probe ANS (8-anilino-l-naphthalene-sulfonic acid) provided further evidence for a reversible structural transition at mildly acidic pH associated with an increase in exposed hydrophobicity in class II molecules. IA d conformation was found to change at a higher pH than IE d, IE k, or IA k, which correlates with the different pH optimal for peptide binding by these molecules. We conclude that pH regulates peptide binding by influencing the structure of class II molecules.

SKN-1 domain folding and basic region monomer stabilization upon DNA binding

“…The Skn domain is a native molten globule that folds to perform a specific function (BR stabilization). This is consistent with proposals that the molten globule state is involved in some molecular recognition and membrane insertion events (van der Goot et al 1991;Gonzá lez-Mañ as et al 1992;Mach and Middaugh 1995;Tortorella et al 1995;Boniface et al 1996;De Filippis et al 1996;Evans et al 1996;Runnels et al 1996). The Skn domain binds DNA with an affinity comparable to that of full-length SKN-1 (Blackwell et al 1994), indicating that the remainder of SKN-1 is dispensable for binding, but other SKN-1 residues (or another protein) could potentially stabilize a Skn-domain fold off DNA.…”

Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide–MHC complexes

“…Register shifting or multi-mode binding has been seen in peptides that bind both class I and class II MHC proteins (Borbulevych et al, 2007; Landais et al, 2009; Günther et al, 2010). The binding of ANS, which like SYPRO orange interacts with exposed hydrophobic surfaces, has been used to examine pH dependent conformational changes in class II MHC proteins (Boniface et al, 1996; Runnels et al, 1996). Insight into these complexities would not be available from other methods to study MHC-peptide binding such as IC 50 measurements or CD spectroscopy.…”

Contribution of conformational stability of hen lysozyme to induction of type 2 T‐helper immune responses

“…The environmentally sensitive fluorescent probe, ANS, is useful for determining the degree of exposure of hydrophobic sites as the structure of a protein is perturbed. ANS is weakly fluorescent in an aqueous environment, but becomes appreciably fluorescent with blue‐shifted emission spectra in non‐polar environments 25,26 . To measure the binding of ANS to HEL derivatives, 5 µ m of each HEL derivative and 200 µ m of ANS in 20 m m N ‐2‐hydroxyethylpiperazine‐ N ′‐2‐ethanesulphonic acid (HEPES)‐buffered saline, pH 7·5, were incubated for 10 min at 37°.…”