Influence of solubilizing environments on membrane protein structures

Abstract

Membrane protein structures are stabilized by weak interactions and are influenced by additional interactions with the solubilizing environment. Structures of influenza virus A M2 protein, a proven drug target, have been determined in three different environments, thus providing a unique opportunity to assess environmental influences. Structures determined in detergents and detergent micelles can have notable differences from those determined in lipid bilayers. These differences make it imperative to validate membrane protein structures.

Figure I

Comparison of amino acid compositions between water-soluble protein helices and transmembrane protein helices. In green and red are amino acids which are more common in membrane protein TM helices and in water-soluble protein helices, respectively. Adapted from ref. [79] with permission; copyright (2007) John Wiley & Sons, Inc.

Figure I

Solubilization of two helical peptides with different lengths in dodecylphosphocholine (DPC) micelles. (a) WALP16, with the sequence GWW(LA)₅WWA. (b) WALP35, with the sequence GWW(LA)₁₄LWWA. The two peptides were solvated in a 40-DPC micelle surrounded by 5000 water molecules, and in a 65-DPC micelle surrounded by 8125 water molecules, respectively. Shown are snapshots after ~10 ns of molecular dynamics simulations under constant temperature (300 K) and constant pressure (1 bar). The peptides are shown as ribbons; phosphorus atoms of the DPC molecules are shown as spheres. Note the exposure of some DPC hydrocarbon tails to the aqueous environment.

Figure 1

Solid-state NMR structures of the M2 TM domain in liquid crystalline lipid bilayers. (a) Structure in the absence of amantadine (PDB entry 1NYJ). The observed short distance between His37 N_δ1 and Trp41 C_γ is indicated by a red dashed line. (b) Structure in the presence of amantadine (PDB entry 2H95). Note that the helices are kinked in the vicinity of Gly34 (shown as spheres); dashed lines are drawn through the N-terminal and C-terminal halves of one helix to highlight the kink. (c) The amantadine binding site, obtained from molecular dynamics simulations starting from 2H95. The bound amantadine (in space-filling representation) is located below the Val27 (yellow carbons) secondary gate and flanked by Ser31 (blue carbons) and Ala30 (dark green carbons) residues. Two waters that hydrogen bond to the downward-pointing amine are also shown. (d) The structure in (c) viewed from the viral exterior, with Val27 in space-filling representation showing that the secondary gate is closed.

Figure 2

Crystal structures of the M2 TM domain from octylglucoside solutions. (a) Structure of the amantadine-bound G34A mutant at pH 5.3 (PDB entry 3C9J). The bound amantadine is shown in space filling representation. (b)-(e) Different views of the TM domain determined at neutral pH (PDB entry 3BKD). In (b), the helical structure and even the His37 and Trp41 sidechains have nearly the same conformations as in the lowpH form shown in (a), except here one of the helices is significantly kinked near Gly34. (c) Surface rendering showing that the helices splay apart from the middle of the membrane toward the C-terminal end. (d) Similar orientation as in (b) but showing two octylglucoside and a polyethylene glycol molecule inserted into the tetrameric structure. (e) Surface rendering showing the crystal contacts between two tetramers in a unit cell.

Figure 3

Solution NMR structure of the M2 conductance domain determined in DHPC micelles (PDB entry 2RLF). (a) Overview of the NMR structure as a pair of four-helix bundles, one for the TM domain and one for the amphipathic helices C-terminal to the TM domain. Four rimantadine molecules (in space-filling representation) were bound to exterior of the TM helix bundle. The amphipathic helix bundle was found to be water soluble. (b) and (c) Two views of the HxxxW quartet in the NMR structure. In (b) a side view is shown. In (c) a view from the viral interior illustrates the tight packing of the Trp41 sidechains (in space-filling representation) and as such their inability to serve as a gate.

Figure 4

Comparison of TM helix packing in three neutral-pH M2 structures, illustrating the influence of solubilizing environments. (a) X-ray structure crystallized from octylglucoside solutions (PDB entry 3BKD). (b) Solid-state NMR structure determined in liquid crystalline lipid bilayers (PDB entry 1NYJ). (c) Solution NMR structure determined in DHPC micelles (PDB entry 2RLF). Colored bars above and below the structures indicate the separation of the helices on either side of the membrane; stars indicate helix crossing points. (d)-(f) “HOLE” images [78] for the structures in (a)-(c) displaying the variations of the channel pore due to different helix packing. Pore size color code: red (< 1.2 Å, radius of a water molecule); green (< 3 Å, radius of amantadine); and blue (> 3 Å).