Ligand Entry into Fatty Acid Binding Protein via Local Unfolding instead of Gap Widening

Abstract

Fatty acid binding proteins (FABPs) play an important role in transportation of fatty acids. Despite intensive studies, how fatty acids enter the protein cavity for binding is still controversial. Here, a gap-closed variant of human intestinal FABP was generated by mutagenesis, in which the gap is locked by a disulfide bridge. According to its structure determined here by NMR, this variant has no obvious openings as the ligand entrance and the gap cannot be widened by internal dynamics. Nevertheless, it still uptakes fatty acids and other ligands. NMR relaxation dispersion, chemical exchange saturation transfer and hydrogen-deuterium exchange experiments show that the variant exists in a major native state, two minor native-like state, and two locally unfolded states in aqueous solution. Local unfolding of either βB-βD or helix 2 can generate an opening large enough for ligands to enter the protein cavity, but only the fast local unfolding of helix 2 is relevant to the ligand entry process. Statement of SignificanceFatty acid binding proteins transport fatty acids to specific organelles in the cell. To enable the transport, fatty acids must enter and leave the protein cavity. In spite of many studies, how fatty acids enter the protein cavity remains controversial. Using mutagenesis and biophysical techniques, we have resolved the disagreement and further showed that local unfolding of the second helix can generate a transient opening to allow ligands to enter the protein cavity. Since lipid binding proteins are highly conserved in 3D structures and ligand binding, all of them may use the same local unfolding mechanism for ligand uptake and release.

Structural transitions permitting ligand entry and exit in bacterial fatty acid binding proteins

“…NMR dynamics measurements reveal that an opening between the -sheets may function as an alternate portal (Guo et al, 2019;Xiao et al, 2016); however, simply providing an opening for the FA is not sufficient to deliver the FA to the lipid bilayer. Previous MD simulations with FABP4 and a membrane surface (Mihajlovic and Lazaridis, 2007;Xiao et al, 2020) show that the 2-helix bundle associates with the membrane via electrostatic interactions, as suggested by extensive mutagenesis and crosslinking experiments (Corsico et al, 1998;Falomir-Lockhart et al, 2006;Herr et al, 1996), but the alternate portal between the  sheets is pointing away from the bilayer.…”

Brain fatty acid binding protein exhibits non-preferential and mutation-resistant binding towards fatty acids

“…Taken together, we provide support for the presence of an H3-and βD-unfolded states that may be ubiquitous across the FABP family. For FABP2 (32,(34)(35)(36)(37) and FABP5 (38)(39)(40), the unfolding of H3 occurs on a similar timescale to ligand binding, which may indicate that H3-unfolding is an essential step along the binding pathway (34). Regardless, these minor states may be necessary for the other biological activities of FABP7, namely in driving membrane interactions or nuclear localization.…”

“…Unfolding to H3 and the gap region has also been observed for other apo FABPs. Extensive NMR and simulation studies performed on intestinal-FABP (FABP2) (32,(34)(35)(36)(37) and epidermal-FABP (FABP5) (33,(38)(39)(40) reveal that, like FABP7, these proteins also undergo H3 and gap region unfolding. For FABP2, 15 N relaxation dispersion (RD) and chemical shift saturation transfer (CEST) NMR experiments reveal minor populations with structural changes to H3 and gap region residues (34).…”

“…Extensive NMR and simulation studies performed on intestinal-FABP (FABP2) (32,(34)(35)(36)(37) and epidermal-FABP (FABP5) (33,(38)(39)(40) reveal that, like FABP7, these proteins also undergo H3 and gap region unfolding. For FABP2, 15 N relaxation dispersion (RD) and chemical shift saturation transfer (CEST) NMR experiments reveal minor populations with structural changes to H3 and gap region residues (34). X-ray crystallography structures of FABP5 suggest higher H3 flexibility (38), while data from hydrogen-deuterium exchange experiments (34-36) on both proteins show lower protection factors and increased flexibility for residues in H3.…”