Folding and stability of the leucine-rich repeat domain of internalin B from Listeria monocytogenes

Internalin B (InlB), a surface protein of the human pathogen Listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium. The N-terminal half of InlB (residues 36-321, InlB₃₂₁), which is sufficient for this process, contains a central leucine-rich repeat (LRR) domain that is flanked by a small α-helical cap and an immunoglobulin (Ig)-like domain. Here we investigated the spectroscopic properties, stability and folding of InlB ₃₂₁ and of a shorter variant lacking the Ig-like domain (InlB ₂₄₈). The circular dichroism spectra of both protein variants in the far ultraviolet region are very similar, with a characteristic minimum found at ~200 nm, possibly resulting from the high 3₁₀-helical content in the LRR domain. Upon addition of chemical denaturants, both variants unfold in single transitions with unusually high cooperativity that are fully reversible and best described by two-state equilibria. The free energies of GdmCl-induced unfolding determined from transitions at 20°C are 9.9(±0.8)kcal/mol for InlB₃₂₁ and 5.4(±0.4)kcal/mol for InlB₂₄₈. InlB₃₂₁ is also more stable against thermal denaturation, as observed by scanning calorimetry. This suggests, that the Ig-like domain, which presumably does not directly interact with the host cell receptor during bacterial invasion, plays a critical role for the in vivo stability of InlB.