New and Notable: Key New Insights into Membrane Targeting by Proteins

The sensing and targeting of membranes by certain protein motifs is a critically important issue in molecular biophysics and structural biology. Two back-to-back articles (1,2) in this issue of Biophysical Journal contribute greatly to the advancing state of knowledge on membrane targeting by proteins, as will be described below. It is known that proteins can electrostatically target specific rare lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol-(3–5)-trisphosphate (PI(3,4,5)P3), and/or more common lipids such as phosphatidylserine. This behavior is found, for instance, in the targeting of membranes by the pleckstrin homology domain membrane-targeting motif; proteins having this domain regulate a number of key cellular processes such as growth, metabolism, and chemotaxis (see, e.g., Knight and Falke (3)). In other cases, certain proteins may sense membrane composition and its intrinsic heterogeneity, i.e., the membrane rafts. An important example of this behavior from the recent literature is seen in the finding that the HIV-1 group-specific antigen (Gag) protein can sense both the acyl chains of phosphatidylserine and the local cholesterol concentration, as well as other aspects of the membrane such as PIP2 concentration (4). The sensing by proteins of other more physical features of membranes such as their curvature appears to be a critical feature of protein-mediated membrane remodeling, such as that found in the process of endocytosis (5). In this case, the Bin/amphiphysin/Rvs (BAR) domains are seen to be important, including the case of the N-BAR domains, which contain N-terminal amphipathic helices (6). The amphipathic helix is in fact implicated in a number of membrane sensing proteins (7), including the amphipathic lipid packing sensor (ALPS) motif found, for example, in the enzyme ArfGAP1, which is involved in coat protein disassembly important in membrane trafficking. Important experimental work has shown that membrane curvature sensing by amphipathic helices can be largely defined by a higher density of binding sites on curved membranes (8). This behavior leads to a model in which membrane curvature-induced defects in the lipid packing density are key to membrane curvature sensing by amphipathic helices, e.g., in N-BAR domain proteins like endophilin. This model was subsequently confirmed computationally by the molecular-dynamics (MD) simulations of Cui et al. (9), who showed that curved membranes do indeed have a larger number of packing defects and that the N-terminal amphipathic helix (H0) of endophilin has a favorable free energy to bind and then fold into such defects (see Fig. 1). Moreover, the probability of the packing defect size was found to follow a simple exponential law with a decay constant that depends on the curvature. Figure 1 MD results by Cui et al. (9) of N-BAR H0 helix binding and folding into curved membranes. (a) Typical configuration of the H0 for a flat or slightly bent region of the membrane. (Red dots) ASA packing defects. Hydrophobic insertions into ... Now, by virtue of the results presented in back-to-back articles in this issue of Biophysical Journal (1,2), our understanding of membrane targeting by proteins has been significantly expanded. In the first article, by Vamparys et al. (1), the important result is presented that membrane packing defects can be created in the membrane surface by conical lipids such as dioleoylglycerol (DOG) in a lipid bilayer consisting of predominantly cylindrical lipids such as phosphatidylcholine (PC). Remarkably, the behavior of these defects satisfies the same exponential law as found by Cui et al. (9) for curved membranes. Vamparys et al. also quantify defects by two additional criteria in addition to the one used by Cui et al. involving the accessible surface area (ASA). The first additional criterion is a chemical defect, i.e., a lipid of a different kind, in this case DOG, among the PC lipids. The second additional criterion is a geometrical defect for voids deeper than the glycerol backbone. In general, chemical defects are the largest in size, while geometrical defects are the smallest. ASA defects fall in the middle range and are rather similar to the geometrical defects. All defects satisfy an exponential probability law. Another finding of Vamparys et al. (1) is that the defects do not appear to colocalize with the DOG conical lipids in their simulations. Indeed, the defects are evenly distributed on the mixed DOPC/DOG bilayer, even though the presence of DOG appears to favor a greater number of defects overall. This behavior seems at odds with concepts of lipid rafts, which have properties different from other regions of the membrane, but it is also possible that the finite size of the simulations and the periodic boundary conditions led to this observed behavior. In simulations carried at larger lengthscales, one might presumably see some clear degree of localization of the defects along with the lipids that cause them and that are locally enriched in a lipid raft region. Along these lines, another interesting point to consider is what might happen if both conical lipids and membrane curvature are present in a given membrane region. Would the defect formation be super-enhanced? Such behavior could have significant implications for membrane targeting by amphipathic-helix containing proteins. In the second of the two back-to-back articles in this issue, Vanni et al. (2) explore the way in which the biologically important ALPS motif senses and adsorbs on the lipid bilayer via insertions of its hydrophobic residues into packing defects. In these simulations, it was also found that such insertions cause only minor changes to the statistical distribution of underlying packing defects. It should be noted that Vanni et al. are limited in the timescale of their simulations and thus did not observe the folding of the amphipathic helix into the membrane as is observed experimentally by circular-dichroism spectroscopy. They are, however, able to observe repeated spontaneous hydrophobic residue insertions into the bilayer that correlate the initial peptide binding with the behavior of the packing defects in the membranes. The results of Vanni et al. (2) can be placed in perspective with the earlier MD results by Cui et al. (9) on N-BAR H0 helix binding and folding into curved membranes. Fig. 1 a shows the typical configuration of the H0 for a flat or slightly bent region of the membrane. The ASA packing defects are shown by red dots and the hydrophobic residue insertions into these defects are seen for the H0 helix. The free energy surface for the H0 on the membrane surface as a function of two variables that describe its conformations is shown at the bottom of the panel, including the helical state as one limit (upper-right region) and the fully unstructured state (lower-left region). This rugged free energy surface is shown in colors and was calculated using an implementation of the metadynamics (10) enhanced sampling MD method. The lowest free energy state of the helix on the flat membrane is in the middle region, which corresponds to a mostly unstructured helix with some number of insertions into the smaller membrane defects by its hydrophobic residues. This situation largely corresponds to the conformations for the ALPS peptide observed by Vanni et al. (2). Fig. 1 b shows the H0 helix folded into a large defect on the highly curved surface of the membrane. Correspondingly, the free energy surface shows a distinct minimum around the folded state of the H0, with a number of higher free energy local minima for more unstructured states of H0 (but still having multiple insertions by some of its hydrophobic residues). A comparison of the result in Fig. 1 a with Fig. 1 b, as well as with the MD results of Vanni et al. (2) for the ALPS motif on a DOPC/DOG surface with packing defects, points to a question that must be addressed in the future. As shown by Cui et al. (9) for curved membrane surfaces, and now by Vamparys et al. (2) for mixed membranes with conical lipids, the size of the packing defects follows an exponential law. In turn, this means that the probability is extremely small for a defect of sufficient size to spontaneously exist that can accommodate a fully folded amphipathic helix. As noted by Cui et al. (9), the complete folding process of the helix must therefore involve a restructuring of the smaller defects into larger ones on the membrane surface through an interaction with the peptide as well as an additional energy driving force coming from the creation of the helical turns of the peptide as it folds. This process would appear to be a critical albeit largely unexplored step in the process of amphipathic helix insertion into defect-ridden membranes. A more extensive elaboration of this complex behavior will be a challenge for future research, inspired in no small way by the work of Vamparys et al. (1) and Vanni et al. (2) in this issue of Biophysical Journal.