Hydrophobic pulses predict transmembrane helix irregularities and channel transmembrane units

Abstract

Background: Few high-resolution structures of integral membranes proteins are available, as crystallization of such proteins needs yet to overcome too many technical limitations. Nevertheless, prediction of their transmembrane (TM) structure by bioinformatics tools provides interesting insights on the topology of these proteins.

Methods: We describe here how to extract new information from the analysis of hydrophobicity variations or hydrophobic pulses (HPulses) in the sequence of integral membrane proteins using the Hydrophobic Pulse Predictor, a new tool we developed for this purpose. To analyze the primary sequence of 70 integral membrane proteins we defined two levels of analysis: G1-HPulses for sliding windows of n = 2 to 6 and G2-HPulses for sliding windows of n = 12 to 16.

Results: The G2-HPulse analysis of 541 transmembrane helices allowed the definition of the new concept of transmembrane unit (TMU) that groups together transmembrane helices and segments with potential adjacent structures. In addition, the G1-HPulse analysis identified helix irregularities that corresponded to kinks, partial helices or unannotated structural events. These irregularities could represent key dynamic elements that are alternatively activated depending on the channel status as illustrated by the crystal structures of the lactose permease in different conformations.

Conclusions: Our results open a new way in the understanding of transmembrane secondary structures: hydrophobicity through hydrophobic pulses strongly impacts on such embedded structures and is not confined to define the transmembrane status of amino acids.

Figure 1

Definition of transmembrane structures. [A;D] is a transmembrane helix (TMH) and [B;C] represents the transmembrane segment (TMS) which is the embedded part of TMH.

Figure 2

G2-HPulse distribution. This figure displays schematically the relative position of G2-HPulses within one of the three intervals: [C;D] is the extracellular end of the first TMH, [E;F] is the extracellular beginning of the next TMH and [D;E] comprises the amino acids positioned between the two TMHs. The length of [C; D], [D; E] and [E; F] is proportional to the number of involved amino acids.

Figure 3

Structure of the rotor ring of the V-type Na-ATPase[15]. A: Embedded amino acids are in yellow (limits predicted by PDBTM). B: Each G2-HPulse is represented by a different color.

Figure 4

Distribution of G1-HPulses compared to the extremities of α-helices. A helix is considered to be in or near the membrane if its distance to the closest TMS is not higher than 40 amino acids: 782 helices were selected. Eleven values were not contained in the range [12] and thus were not displayed.

Figure 5

Prediction of structural irregularities in the different 3D structures of the lactose permease by G1-HPulses. Each G1-HPulse is indicated by a change of color. A, stereo view of the 2CFQ structure (from amino acid 5 to 71): the red α-helix is a partial helix and the transition from the magenta to the blue α-helix is marked by a kink. B, stereo view of the 1PV6 structure (from amino acid 209 to 250): the yellow α-helix is clearly isolated from the red and green α-helices. A kink separates the green and yellow α-helices. C, stereo view of 2V8N (from amino acid 311 to 343): the red and green α-helices form a curved α-helix whose interruption is detected by a G1-HPulse.

Figure 6

Structure of the chimeric Kv channel. A, schematic model of the structure, where arrows represent G1-HPulses (Figure adapted from Long et al. [20]). B, stereo view of S6: each G1-HPulse is indicated by a change of color.

Figure 7

The concept of transmembrane unit (TMU). The hatched area symbolizes the membrane. TMS correspond to the embedded part of α-helices. A TMH is composed of one (H2 and H3) or more (H1a and H1b) α-helices. The TMU groups together structures that are comprised between two G2-HPulses: it can contain a single α-helix, like TMU2, or associate TMH and small structures localized near the membrane, like TMU3 that contains H3 and H2-H3.

Figure 8

Detection of HPulses by a finite state automaton. The consensus is based on the sign of values that have been computed for 5 different lengths of window. The same automaton was used for both G1 and G2, but with different window sizes.