Polar residues drive association of polyleucine transmembrane helices

Abstract

Transmembrane (TM) α-helices contain few strongly hydrophilic residues compared with the composition of proteins in general (1–4), which can be explained in part by the high energetic cost associated with burying polar side chains in a hydrophobic environment (5). Serine and threonine are found in TM helices more frequently than are other polar residues (N, D, Q, E, R, K, and H), partially because of their potential to form intrahelical hydrogen bonds to main-chain carbonyl oxygens (6). In fact, permitting S, T, Y, and C in TM regions seems to improve TM helix prediction (7). Interestingly, despite their rare presence in TM helices, strongly polar residues are highly conserved, especially in multispanning TM proteins, suggesting molecular interactions that either functionally or structurally favor these residues (2, 3). Functional roles of some polar residues are observed in structures of proteins, such as binding of prosthetic groups (H and E) in the photosynthetic reaction center (8), retinal binding (K) and proton transport (D) in bacteriorhodopsin (9, 10), binding of hemes (H) in cytochrome c oxidase (11) and the cytochrome bc₁ complex, and Ca ^{binding (N, D, E, and T) in the Ca ATPase (}12).

Structural contributions of polar residues in the membrane are less well understood. Interhelical polar interactions have been observed in some integral membrane protein structures available at high resolution. Ion pairs (E65/R185 and R70/E180) are suggested to have a role in the stabilization of the structure of the light-harvesting complex (13). The hydrogen bond formed between conserved E97 and H219 in subunit III of cytochrome c oxidase is thought to play a structural role (11). At least one hydrogen bond (main chain to side chain or side chain to side chain) exists between each pair of adjacent helices within the monomer of bacteriorhodopsin; some of the hydrogen bonds are bridged by water molecules (10).

Genetic and biophysical studies of TM proteins have identified critical polar residues that may participate in electrostatic interactions. The Escherichia coli lactose permease has only six irreplaceable residues for its function, all of which are polar and reside within TM helices (14–16). Each one is in proximity to one of the others, probably forming three pairs of hydrogen bonds or salt bridges (E126/R144, E269/H322, and R302/E325). These residues as well as a number of other polar ones are implicated in substrate binding and substrate-induced conformational changes. Voltage-gated Na^{, K, and Ca channels have highly conserved polar residues in their TM helices S2–S4 (}17). These residues in the Shaker K ^{channel are proposed to interact in two clusters (E283/R368/R371 and E293/D316/K374) and may be involved in voltage sensing and/or association of helices (}18–20). In the inwardly rectifying K ^{channel, functionally conserved S95 of M1 and Q164 of M2 are interacting within the same subunit, presumably forming an interhelical hydrogen bond (}21). In the E. coli F₁F_o ATP synthase, H^{-transporting residue D61 of subunit} c is thought to functionally interact with R210 of subunit a during the protonation–deprotonation cycle that drives the c₁₂ oligomer rotor and the subsequent ATP synthesis (22–25).

An interesting group of TM proteins with conserved polar residues belongs to members of the G-protein-coupled receptor superfamily, in particular the family of rhodopsin-like receptors, which has about 1,500 members known to date (26–29). A number of conserved polar residues in the TM helices are found to be vital to ligand binding and/or signal transduction; interactions among these residues are proposed to conformationally constrain the receptors in their inactive states in the absence of light or ligands (28, 30). Incorporating hydrogen bonding interactions between conserved polar residues as constraints has improved molecular modeling of the TM helix bundle of rhodopsin-like receptors (31). A number of such hydrogen bonds involving the most conserved polar residues (N55, N78, D83, E134, R135, and N302) are confirmed by the recent crystallographic structure of bovine rhodopsin in the ground state (32).

The identification of TM polar interactions may provide helpful structural insights into understanding disease mechanisms. The transformation activity of the bovine papillomavirus E5 protein is a result of TM helix association with platelet-derived growth factor β receptor, an interaction partly mediated by polar residues (33). A single mutation (V664E) in the TM helix of the neu/erb-2 protooncogene leads to constitutive activation of the encoded tyrosine kinase receptor (34). It was found later that the glutamic acid is involved in interhelical hydrogen bonding interactions (35). However, it remains unclear whether such interactions directly cause dimerization and/or activation of the receptor.

It is obvious that polar residues in TM helices are important, both functionally and structurally. In many cases, however, these residues may be involved not only in binding of ligands/substrates, but also in inducing conformational changes or maintaining structural integrity crucial to the protein function. Such structure–function dependence complicates the interpretation of structural contributions of polar interactions—roles of individual polar residues can only be inferred from the activity of the whole protein. The extent to which these polar residues specify the arrangement of TM helices or stabilize helix–helix interactions is energetically unclear. Therefore, it would be desirable to develop more direct approaches to identifying polar interactions that define structural features of membrane proteins.

We reported previously that asparagine drives TM helix association, possibly by forming interhelical hydrogen bonds (36), which was demonstrated by examining synthetic peptide and chimeric proteins both in detergent and in biological membrane environments. An accompanying report drew comparable conclusions by studying similar peptides, with the use of analytical ultracentrifugation and fluorescence resonance energy transfer methods (37).

A question that arises from the previous results is whether the interhelical hydrogen bond formation is a characteristic of other polar residues that can act as both hydrogen bond donors and acceptors. Here we report contributions by other polar residues to TM helix association through chimeras, in which each TM helix includes a single polar substitution (N, D, Q, E, H, S, T, or Y) in a polyleucine sequence. The ability of these chimeras to associate was tested in detergent micelles and in biological membranes, and the results were compared with those of the glycophorin A (GpA) chimeras.

Values represent diameter measurements of circular zones of inhibition (ZOI) around the chloramphenicol disk devoid of bacterial growth. The concentration of chloramphenicol in the solid medium diminishes with increasing distance from the disk. Small ZOIs indicate chloramphenicol-resistant bacteria and higher CAT activity.

Acknowledgments

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