Abstract
ABSTRACTConformational stability of human serum transferrin (Tf) at varying pH, salt, and excipient concentrations were investigated using molecular dynamics (MD) simulations and the results are compared with previously published small angle X-ray scattering (SAXS) experiments. SAXS study showed that at pH 5, Tf is predominantly present in partially open (PO) form, and the factions of PO differ based on the physicochemical condition and drifts towards closed form (HO) as the pH increases. Tf is a bilobal glycoprotein that is composed of homologous halves termed as N-lobe and C-lobe. The current study shows that the protonation of Y188 and K206 at pH 5 is the primary conformational drive into PO, which shifts towards the closed (HO) conformer as the pH increases. Furthermore, at pH 6.5, PO is unfavorable due to negative charge-charge repulsion at the N/C-lobe interface linker region causing increased hinge distance when compared to HO, which has favorable attractive electrostatics. Subsequently, the effect of salt concentration at 70 and 140 mM NaCl was studied. At 70 mM NaCl and pH 5, chloride ions bind strongly in the N-lobe iron-binding site, whereas these interactions are weak at pH 6.5. With increasing salt concentration at pH 5, regions surrounding the N-lobe iron-binding site are saturated and as a consequence sodium and chloride ions accumulate into the bulk. Additionally, protein-excipient interactions were investigated. At pH 5, excipients interact in similar loop regions, E89-T93, D416-D420, located in the C-lobe and N-lobe of the HO conformer, respectively. It is anticipated that interactions of additives in these two loop regions cause conformational changes that lead to iron coordinating residues in the N-lobe to drift away from iron and thus drive HO to PO conversion. Furthermore, at pH 6.5 and 140 mM histidine or phosphate, these interactions are negligible leading to the stabilization of HO.
Publisher
Cold Spring Harbor Laboratory