Ordered opening of LDL receptor binding domain of human apolipoprotein E3 revealed by hydrogen/deuterium exchange mass spectrometry and fluorescence spectroscopy

Abstract

Apolipoprotein E3 (apoE3) is an exchangeable apolipoprotein that plays a critical role in cholesterol homeostasis. The N-terminal (NT) domain of apoE3 (residues 1-191) is folded into a helix bundle comprised of 4 amphipathic α-helices: H1, H2, H3 and H4, flanked by flexible helices N1 and N2, and Hinge Helix 1 (Hinge H1), at the N-and C-terminal sides of the helix bundle, respectively. The NT domain plays a critical role in binding to the low density lipoprotein receptor (LDLR), which eventually leads to lowering of plasma cholesterol levels. In order to be recognized by the LDLR, the helix bundle has to open and undergo a conformational change. The objective of the study was to understand the mechanism of opening of the helix bundle. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) revealed that apoE3 NT domain adopts several disordered and unfolded regions, with H2 exhibiting relatively little protection against exchange-in compared to H1, H3, and H4. Site-directed fluorescence labeling indicated that H2 not only has the highest degree of solvent exposure but also the most flexibility in the helix bundle. It also indicated that the lipoprotein behavior of H1 was significnatly different from that of H2, H3 and H4. These results suggest that the opening of the helix bundle is likely initiated at the flexible end of H2 and the loop linking H2/H3, and involves movement of H2/H3 away from H1/H4. Together, these observations offer mechanistic insight suggesting a regulated helix bundle opening of apoE3 NT domain can be triggered by lipid binding.

Advances in Hydrogen/Deuterium Exchange Mass Spectrometry and the Pursuit of Challenging Biological Systems

“…HDX-MS has also been used to probe peripheral membrane proteins in the context of their native environments such as drug metabolizing enzymes at the lipid membrane surface − and the interactions and structural transitions mediating lipoprotein assembly. ,,, One interesting finding from the slew of HDX-MS studies is that EX1 kinetics, while rare in most globular proteins, have been encountered quite frequently with membrane proteins. EX1 has been observed and characterized in several lipoproteins, interestingly even in the lipid-free state. ,,, Correlated motions occurring near or in the membrane have been noted for several systems, and by tracking the EX1 profiles the kinetics of the conformational transitions could be characterized. ,− ,, Further advances in HDX-MS may enable the study of membrane proteins in their fully native membrane environment in situ . This has been demonstrated in the study of the ADP/ATP carrier (bAnc1p) on intact mitochondria .…”

The LDL receptor binding domain of apolipoprotein E directs the relative orientation of its C-terminal segment in reconstituted nascent HDL

“…The average FP values for the three sites probed in the NT domain in apoE3 and apoE4 are 0.37 and 0.39, respectively, while those in the CT domain for both isoforms was 0.35, Table 2 . Previously, we reported FP values of 0.12 and 0.19 for isolated apoE3 NT domain bearing residues 1–191 in WT and single Cys constructs, respectively [ 21 ] and 0.35 for isolated CT domain (residues 201–299) [ 23 ]. The relatively lower values for isolated NT domain compared to similar sites probed in the full length proteins is expected since the former encompasses a helix bundle and is a compact, globular, monomeric protein.…”

“…In general, the ease of unfolding is correlated with ease of binding to lipids, since less structured segments tend to seek stability by lipid binding interaction. Further, hydrogen-deuterium exchange coupled to mass spectrometry in conjunction with fluorescence polarization data suggest that lipid binding, as predicted by ease of exchange and ease of unfolding, involve multiple steps [ 21 , 23 ]. It was proposed that upon encountering lipids, the oligomeric assembly of apoE dissociates and likely anchors to the surface via the CT domain, with the short and relatively polar helix C3 (residues 271–276, DMQRQW) likely seeking stability prior to C1 and C2.…”

“…Although high resolution structural information of intact apoAI is lacking, biophysical studies in combination with modeling based on partial X-ray crystallographic analyses suggests that the NT domain bears a 4-helix bundle [ 59 , 60 ] similar to apoE3. However, because the NT domain of apoAI is not as stable as that of apoE3, swapping it for apoE3 NT domain (yielding apoAI-NT/apoE-CT) significantly enhanced the vesicle solubilization rate of the chimera, which otherwise shows a very poor ability to bind DMPC vesicles to yield rHDL [ 23 , 21 , 61 ]. Taken together with the current study, it appears that the stable NT domain helix bundle of apoE3 not only mitigates the lipid binding rate of its CT tail, but that it also influences the relative orientation of one monomer with respect to the other on rHDL.…”

“…Single Cys constructs were generated using the QuikChange II Site Directed Mutagenesis kit (Agilent Technology, Stratagene, La Jolla, CA). For apoE3, which has a single Cys at position 112, the endogenous Cys was replaced by Ser (with no significant change in function or fold as described previously [ 21 ]) and will be referred to as apoE3. The selected sites for the various single Cys substitutions were determined from the X-ray structure of apoE3 NT (1–191) (PDB ID 1NFN) [ 9 ], and NMR structures of apoE3 NT (1–183) (PDB ID 2KC3) [ 8 ] and apoE3 rendered monomeric by multiple mutations at the interfacial site (F257A/W264R/V269A/L279Q/V287E) [ 11 ] (PDB ID 2L7B); these sites were distributed to sample different segments of the NT and CT domains.…”