Our theoretical analyses of the membrane binding modes of Sec14-like proteins using the PPM method 11
underline the essential role of hydrophobic residues of two “gating” helices located at the mouth of the ligand binding pocket in recruitment of these proteins to membranes. The subsequent mutagenesis studies support the role of hydrophobic residues in membrane binding of α-TTP and subsequent transfer of NBD-Toc from α-TTP into the lipid bilayer. Indeed, mutations of residues from both lid helices (F165, F169, I202, V206 and M209) to either Ala or Asp significantly affected the rate of NDB-Toc transfer to SUVs (, ) and protein absorption to phospholipid bilayers as measured by DPI (, ) or vesicle filtration experiments (, ). The largest effect on the protein absorption was obtained when replacements involved residues that are predicted to be fully immersed in the lipid bilayer. This included Phe165
from helix A8 that we predict anchors α-TTP to membranes in both “open” and “closed” states. On the other hand, mutations of Ile202
, and Met209
residues in the flexible A10 helix, predicted to be fully lipid-exposed only in the “open” state, exhibited either normal membrane binding behavior (V206A, M209A) or moderate functional impairment (I202D, V206D, M209D). In all experiments with the artificial lipid bilayers it appeared that mutations of Phe165
affect ligand transfer and membrane binding the most. Likewise, α-TTP(F169D) was more defective in assisting tocopherol secretion from cultured hepatocytes than the F169A mutant (). The larger effect of substitution to negatively charged Asp as compared to Ala substitution can be explained by a deionization penalty of the charged Asp residues when penetrating into the nonpolar membrane interior.
Our data supported and extend our previous observation that α-TTP binds tighter and exchanges the α-tocopherol at a higher rate with highly-curved SUVs, as compared to more flat LUVs. Accordingly, all observed effects of residue substitutions were more pronounced with SUVs (, , ). This effect may be related to the decreased lateral pressure in outer leaflets of curved SUV membranes, as compared to the flatter membranes of LUVs, which is known to facilitate insertion of proteins and peptides into membranes.30
However, we also observed a five-to-ten–fold loss of membrane binding affinity for F165A, F169D, and F165D mutants in the DPI experiments, which presumably employ “flat” membranes immobilized on the sensor chip. The fact that the extent of protein-membrane binding assessed by DPI paralleled results obtained by vesicle binding assays is likely due to the extreme sensitivity of the DPI assay. Thus, we found the DPI technique more advantageous, because it is capable of reproducibly detecting small changes in an already small response. Vesicle binding assays that use filtration-based separation of lipid-bound protein appeared to be less sensitive and showed considerably more variability.
In this work we theoretically estimated the transfer free energy of peripheral membrane proteins from water to the lipid bilayer of a defined lipid composition using an improved PPM method that accounts for the hydrophobic, H-bonding and electrostatic interactions of proteins with membranes. The calculated membrane binding affinity (ΔGtransf
) may be overestimated because it does not include the entropy of protein immobilization in membrane12
and the lipid bilayer deformation that is due to the lateral pressure12
. The contribution of lateral pressure is expected to be smaller or negative for curved membrane surfaces32
, facilitating protein binding.30
To eliminate these potential errors, we operated with transfer energy differences of mutants and wild-type protein (ΔΔGtransf
) instead of the absolute values. This allowed us to obtain good correlations (R2
in the range 0.8 – 0.9) between predicted transfer free energy from aqueous to the lipid phase and relative protein adsorption measured by DPI for α-TTP and its mutants (Supplementary Figure 1
, Supplementary Table 1
). This result indicates that our theoretical method is adequate for quantitative analysis of protein-membrane binding energetics of different protein conformations.
The theoretical analysis supports the previously proposed three-step process of inter-membrane transfer of α-tocopherol by α-TTP.22
However, it must be modified to include the transition between protein conformations within the lipid bilayer. The modified scheme of α-tocopherol extraction includes the following steps that are shown in : (1) binding of the ligand-free α-TTP to the membrane (A) followed by opening of the protein (B) in the membrane; (2) ligand binding in membrane with simultaneous closing of the structure (C); and (3) dissociation from the membrane of ligand-loaded α-TTP (D). During step 2 the equilibrium between protein states (B and C) depends on the concentration of α-tocopherol. Additional steps 4 and 5 correspond to the transitions between different protein states in water, such as “closed” ligand-loaded conformations (D) or ligand-free state (A), and a metastable “open” conformation of the ligand-free α-TTP (E). The free energy changes during transitions from water to membrane of 1R5L-like and 1OIZ-like conformations (transitions 3 and 6, respectively) were estimated by the PPM.
Figure 11 Transitions between different states of α-TTP. α-TTP is shown by gray circles with A8 and A10 helices shown by small black circles, α-tocopharol is shown by a triangle, bilayer is shown by a grey rectangle. α-TTP can adopt (more ...)
As follows from our calculations, there is a significant free energy gain associated with the membrane binding of the α-TTP, which is smaller for the ligand-loaded “closed” conformation (ΔGbind2
= −13.6 kcal/mol for 1R5L) than for the ligand-free “open” conformation (ΔGbind3
= −19.8 kcal/mol for 1OIZ). The difference between these values (−6.2 kcal/mol) can be attributed to the energetic cost of the conformational transition (ΔGconf1
) and the energy of ligand binding (ΔGlig
) in water. Thus, the “closed” conformation loaded with the ligand should have a weaker membrane binding affinity than its apo-conformation (ΔGbind2
) and dissociates more easily from the membrane. This result agrees with our previous observation that the apparent affinity of α-TTP to lipid vesicles decreases in the presence of α-tocopherol.22
Analysis of crystal structures demonstrated that for all Sec14-like proteins have a common conformational transition from a “closed” to an “open” state, which involves the dissociation of helices homologous to A8 and A10 of α-TTP. This transition is expected to be energetically unfavorable in water (transition 5, ΔGconf1
), because it leads to the exposure of hydrophobic residues of both helices and the ligand-binding cavity to water. A similar transition in the non-polar lipid environment (transition 2, ΔGconf2
) may be of lower energy, thus enabling the reversibility of the α-tocopherol binding and transport. Energies of the “open” and “closed” states in membranes are likely similar because there is no penalty for exposure of non-polar residues in the lipid environment. Some energy may be lost due to dissociation of tocopherol, but the ligand has been partially replaced by lipids because of the opening and deeper penetration of the protein structure to membrane. The lower transfer energies from water to the lipid bilayer and the deeper immersion (by 2–3 Å) into the lipid core of “open” conformations, as compared to the “closed” ones, seems to be a common feature of all Sec14-like proteins (). As modeled in , the processes observed in DPI or filtration-based essays can be described by transitions 1–2 for the ligand free TTP and by transitions 3–2 for the ligand-loaded TTP. The NBD-Toc transfer to phospholipid vesicles monitored in FRET experiments may be described by transitions 3–2, while the NBD-Toc extraction from the membranes likely corresponds to transitions 2-3-4.
The present work has emphasized the role of surface hydrophobic residues on the ability of the protein to bind and transfer ligand to lipid bilayers. However, the question remains about the possible functional role of multiple solvent-exposed basic residues. We have previously reported22
on the effect of mutations that cause ataxia with vitamin E deficiency (AVED), which include a number of charge replacement mutations. In this work we confirmed that R192H has near normal rates of ligand delivery (83% of wild type TTP) and binds immobilized bilayers with slightly greater efficiency (~150% of wild type TTP) as judge by DPI. The mutations of two basic residues, K211A and K217, located at or near movable A10 helix also have little effect on tocopherol delivery to membranes (83% and 102% of WT), but increased TTP binding in DPI assay (123% and 171% of WT, respectively). This result indicates that all basic residues tested do not contribute to the membrane binding of TTP via non-specific ionic interactions with lipid headgroups, but may be involved in intramolecular interactions that would stabilize the native structure of TTP or some of its conformations, thus resulting in the improved binding or facilitated conformational transition, which are essential for the efficient inter-membrane transfer of the vitamin E.
Our failure to see any change in NBD-transfer rates for wild type TTP when the receiving vesicles contain a variety of anionic lipids (PS, PI, PA, LBPA) also confirmed that non-specific electrostatic effects play only a minor role in binding of TTP to membranes.9
However, our results were obtained with bilayers devoid of other possible protein partners or targeting lipids such as phosphoinositides. It remains possible that PtdIns(3,5)P2
, that is highly enriched on late endosomes33
may be essential for the recruitment of TTP to late endosomes, the major intracellular compartment to where the α-TTP has been originally found in cultured hepatocytes6
. An interaction of TTP with unspecified phosphoinositides has been reported34
and we eagerly await further reports on this phenomenon.
In summary, we report the sensitivity of TTP binding to membranes to the loss of particular surface hydrophobic residues that can insert into the hydrophobic core of membranes, while the protein is insensitive to the presence of anionic lipids9
. That such insertion/binding is more favorable in smaller unilamellar vesicles (i.e.
SUVs versus LUVs) is a phenomenon matched by other membrane binding proteins and peptides.35