Glycines: role in α-helical membrane protein structures and a potential indicator of native conformation

Abstract

Among the growing number of membrane protein structures in the Protein Data Bank, there are many transmembrane domains that appear to be native-like; at the same time, there are others that appear to have less than complete native-like character. Hence, there is an increasing need for validation tools that distinguish native-like from non-native-like structures. Membrane mimetics used in protein structural characterizations differ in numerous physicochemical properties from native membranes and provide many opportunities for introducing non-native-like features into membrane protein structures. One possible approach for validating membrane protein structures is based on the use of glycine residues in transmembrane domains. Here, we have reviewed the membrane protein structure database and identified a set of benchmark proteins that appear to be native-like. In these structures, conserved glycine residues rarely face the lipid interstices, and many of them participate in close helix-helix packing. Glycine-based validation allowed the identification of non-native-like features in several membrane proteins and also shows the potential for verifying the native-like character for numerous other membrane protein structures.

Figure 1

Putative nonnative-like structural perturbations of three membrane proteins. The membrane central plane was located as described in Supplemental Material. This membrane central plane is shown as a blue dashed line; interfacial regions are represented by 8 Å wide pale blue colored bands; and the conservative hydrophobic thickness in between is 25 Å. (a) The histidine kinase receptor KdpD TM domain (solution NMR structure, PDB entry 2KSF). This four-helix bundle has two very short helices and multiple hydrophilic residues exposed to the hydrophobic region of the would-be membrane. The short helices dictate that hydrophilic backbone amides of the inter-helical loops are also exposed to the membrane interstices. (b) 5-lipoxygenase-activating protein (4.0-Å resolution structure, PDB entry 2Q7M). The three chains are displayed in different colors. Helix 4 appears shifted along the helical axis, exposing two charged residues (Lys116 and Arg117 in space filling mode with carbon atoms green, nitrogen atoms blue, oxygen atoms red, and hydrogen atoms white) to the very center of the membrane and the inter-helical loop between helices 3 and 4 is drawn into the lipid interstices exposing more hydrophilic sites to the hydrophobic region of the membrane. (c-d) Two structures of trimeric acid-sensing ion channel (1.9-Å resolution structure, PDB entry 2QTS in (c); and 3-Å resolution structure, PDB entry 3HGC in (d)). They have similar symmetric extramembranous domains but different TM domains. The TM domain in (c) has a sufficient hydrophobic dimension but is asymmetric, probably the result of substantial crystal contacts, while the TM domain in (d) is more symmetric, but does not span the hydrophobic dimension of native membranes.

Figure 2

Transmembrane domains of benchmark membrane protein structures. C_α atoms of glycines are shown as spheres and color-coded according to their conservation scores (red: highly conserved; blue; not conserved; pale colors: intermediate; see Supplemental Material for details). Default parameters for residue conservation were used for all the proteins except for the ligand-gated ion channel (PDB entry 3EAM), where the minimal sequence identity for sequence alignment was lowered from the default 35% to 25%. (a-z) Structures corresponding to entries a-z in Table 1. Note that the outward facing surface of the helices in these proteins is rarely interrupted by a glycine sphere.

Figure 3

Scatter plots displaying relative accessibility and distance (expressed as |z|, i.e. the absolute value of z) from the membrane central plane for the conserved residues in the benchmark proteins. Relative accessibility (i.e., percentage of the nominal maximum area (irrespective of secondary structure); see Supplemental Material) was calculated for either a whole residue or for the backbone polar atoms (C, O, and N) only. Oligomeric protein structures were used to calculate the solvent accessibility of each residue. However, for each oligomeric protein, only a single chain was used to count the number of glycines and other residues as well as for the backbone statistics. (a-c) Whole-residue relative accessibility for glycine, aspartate, and alanine residues. The hydrophobic region was conservatively defined as ±10 Å from the bilayer center. Surface exposure above 20% was considered significant. (d) Backbone relative accessibility for all 20 types of residues. Lipid-facing surface exposure of the backbone above 15% was considered significant. The two backbone sites in the entire benchmark set that have significant exposure are both proline residues (Pro315 of the B12 ABC transporter, PDB entry 1LV7; and Pro300 of a ligand gated channel, PDB entry 3EAM). The glycine residue with the greatest exposure (Gly87 of the NaK channel, PDB entry 2AHY) is highlighted as a blue ‘+’.

Figure 4

Involvement of glycines in helix packing. A total of 220 helix-helix pairs in the 26 benchmark proteins were identified, (a) Number of helix-helix pairs binned according to distance of the closest contact and grouped according to whether glycine is involved. (b-f) Examples of helix pairs showing helix packing facilitated by glycine residues, highlighted here in space-filling mode. (b) Helix 1 (yellow) residue 27 and helix 6 (green) residues 204, 211, 214, and 218 from PDB entry 2NS1. Gly27 and Gly211 both appear to induce helix kinks that facilitate helix-helix interactions along the entire length of the TM helices despite substantial crossing angles at both crossing points. (c) Helix 7 (gray), helix 8 (yellow) residues 264 and 268, and helix 9 (green) residue 288 from PDB entry 2NS1. (d) Helix 3 (yellow) residues 97 and 104 and helix 4 (green) residues 123 and 130 from PDB entry 3O7Q. The i to i + 7 glycine residues on both helices, along with a small crossing angle, result in a large van der Waals interaction surface. (e) Helix 11 (yellow) residues 402, 406, and 410 and helix 12 (green) residues 421, 424, and 428 from PDB entry 3MKT. Here a GxxxGxxxG motif interacts with a GxxGxxxG motif. (f) Helix 4 (yellow) residues 151, 155, and 159 and helix 5 (green) residues 176 and 180 from PDB entry 3ND0. Here a GxxxGxxxG motif interacts with a GxxxG motif.

Figure 5

Application of the glycine-based validation tool. Conserved glycine residues that are exposed to the lipid interstices are highlighted in a space-filling mode. (a) Gly444 from PDB entry 2KSF. (b) Glycine residues 435, 439, and 443 from each monomer of PDB entry 2QTS. (c) Glycine residues 333, 336, 340, and 341 of the multi-drug transporter, EmrD (3.5-Å resolution structure, PDB entry 2GFP).