Structure and dynamics of a human myelin protein P2 portal region mutant indicate opening of the β barrel in fatty acid binding proteins

Abstract

BackgroundMyelin is a multilayered proteolipid sheath wrapped around selected axons in the nervous system. Its constituent proteins play major roles in forming of the highly regular membrane structure. P2 is a myelin-specific protein of the fatty acid binding protein (FABP) superfamily, which is able to stack lipid bilayers together, and it is a target for mutations in the human inherited neuropathy Charcot-Marie-Tooth disease. A conserved residue that has been proposed to participate in membrane and fatty acid binding and conformational changes in FABPs is Phe57. This residue is thought to be a gatekeeper for the opening of the portal region upon ligand entry and egress.ResultsWe performed a structural characterization of the F57A mutant of human P2. The mutant protein was crystallized in three crystal forms, all of which showed changes in the portal region and helix α2. In addition, the behaviour of the mutant protein upon lipid bilayer binding suggested more unfolding than previously observed for wild-type P2. On the other hand, membrane binding rendered F57A heat-stable, similarly to wild-type P2. Atomistic molecular dynamics simulations showed opening of the side of the discontinuous β barrel, giving important indications on the mechanism of portal region opening and ligand entry into FABPs. The results suggest a central role for Phe57 in regulating the opening of the portal region in human P2 and other FABPs, and the F57A mutation disturbs dynamic cross-correlation networks in the portal region of P2.ConclusionsOverall, the F57A variant presents similar properties to the P2 patient mutations recently linked to Charcot-Marie-Tooth disease. Our results identify Phe57 as a residue regulating conformational changes that may accompany membrane surface binding and ligand exchange in P2 and other FABPs.Electronic supplementary materialThe online version of this article (10.1186/s12900-018-0087-2) contains supplementary material, which is available to authorized users.

Sub-atomic resolution crystal structures reveal conserved geometric outliers at functional sites

“…Anisotropy analysis of the structure (Figure 1e) shows an overall directionality of atomistic anisotropy, which could be related to functional open-close movements in the protein. Opening of the β barrel is thought to be central to ligand entry and egress, and could play a role in lipid membrane binding [18]. C-H...O bonds between side chains and main-chain carbonyl moieties are also frequently observed at the ends of secondary structure elements ( Figure 3).…”

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

“…Knowledge of how ligands enter the cavity is important for manipulating FABP's function by blocking the ligand entrance. Previous molecular dynamics (MD) studies suggested three possible ligand entry sites for ligand and water to enter into or exit from the protein cavity: E I , located in the cap region involving the second α-helix (α2) and βC-βD and βE-βF turns; E II , the gap between βD and βE; and E III , in the area around the N-terminus (5)(6)(7)(8). Very recently, our studies on human intestinal FABP (hIFABP) have showed that opening the cap by swinging the two helices away from the barrel is dispensable for ligands to enter the cavity, and further demonstrated the existence of a minor conformational state that undergoes transient local unfolding in α2 on sub-millisecond timescale and thus provides a temporary opening in the E I region for ligand entry (9).…”

“…Recent MD simulations on human heart FABP show that the gap between βD and βE can open to become significantly wider (5,8). In addition, MD simulations on a human myelin protein P2, which is a member of the FABP superfamily, indicates that a large-scale opening of the barrel between βD and βE can occur through flapping βE and βF out (7). Upon this opening, the internalized cavity becomes accessible by ligands.…”

Identification of Key Residues in Proteins Through Centrality Analysis and Flexibility Prediction with RINspector

“…The combination of the three functionalities allows the experimenter to predict the influence of mutation of central residues on local protein flexibility. RINspector has been used in different research projects to help in the identification of important residues in proteins that may be located at their core or surface, alone or in a complex (Brysbaert et al., ; de Ruyck et al., ; Laulumaa et al., ).…”

“…These structures can be used for modeling at different levels, like homology modeling, protein-ligand docking, protein-protein docking, dynamic predictions, and simulations, or used to identify regions or residues of interest that may lead to mutagenesis experiments or be employed for targeted drug design. We have recently developed a tool called RINspector (Brysbaert, Lorgouilloux, Vranken, & Lensink, 2017, 2018, the goal of which is to identify key residues in a structure that are amenable to mutagenesis experiments. It is included as an app in the Cytoscape network visualization and analysis software (Shannon et al, 2003;Su, Morris, Demchak, & Bader, 2014), accessible through its apps manager.…”