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The “hydrophobicity template” method

(Efremov et al., (1999) Theor. Chem. Acc. 101, 73).

The problem:
Often, the membrane pores are formed by bundles of α-helices arranged in parallel or antiparallel manner. In addition, several globular proteins, like cholera, shiga, and pertussis toxins, reveal pore-forming five α-helix bundles (5HB) surrounded by b-barrels (Li J. 1992, Curr. Opin. Struct. Biol. 2, 545). While most of such toxins do not serve to pump ions, analysis of this spatial motif is important because similar folds were proposed for some membrane ion transporters. Thus, 5HB-structure was discovered for the cartilage oligomeric matrix protein (COMP) (Malashkevich V.N. et al., 1996, Science 274, 761), the membrane parts of the nicotinic achetylcholine receptor (AChR) (Unwin N. 1993, J. Mol. Biol. 229, 1101) and d-endotoxin (Gazit E. et al., 1994, Biochem. J. 304, 895). Molecular models with five TM helices lining ion pore were developed for some other proteins and channel-forming peptides (Montal M. 1996, Curr. Opin. Struct. Biol. 6, 499). Atomic-scale ab initio prediction of the structure of multi-helix bundles is still a challenge task. For this to be done, major factors driving helix association should be delineated. This calls for detailed analysis of hydrophobic organization of high-resolution 5HB-structures.

Objectives:

• to assess hydrophobic properties of pore-lining α-helices in known 5HBs and to delineate a common (if any) hydrophobic template for them;
• to check various TM helices for presence of the similar motif;
• based on the sequence analysis, to develop a method for recognition of the 5HB-fold in channels with unknown structure.

Method:
Detailed analysis of spatial hydrophobic properties of pore-forming helices in proteins with known structure (AB5 toxins) via calculation of the molecular hydrophobicity potential on their surfaces.

Results:

1. Despite the lack of sequence homology, α-helices in 5HBs reveal similar hydrophobic properties on their surfaces: two polar sides separated by strong hydrophobic stretches (Fig. 1). The helices are tightly packed via nonpolar faces, one hydrophilic side is exposed to the central pore, and another one is turned to the exterior of the bundle. This polarity template is rather different from that observed in 4HB-proteins (Fig.2).
2. Hydrophobic properties of several channel-forming TM α-helices (M2 of AChR and GABA_A receptors, α5 of d-endotoxin) are similar to the 5HB polarity template. This confirms that these TM segments can form channels with 5HB-architecture.
3. Application of the method to recognition of 5HBs based on the sequence information only, is proposed. This information is necessary to build molecular models of ion channels because it imposes stringent constraints on the helix orientation in the assembly. In turn, the models provide a basis for rationalization of structural and functional data on the channel as well as for design of further experiments.

Fig. 1 Hydrophobic properties of the pore-forming α-helix A in B-pentamer of verotoxin-1. Top: 2D isopotential map of the molecular hydrophobicity potential (MHP) on the peptide surface. The value on the X axis is the rotation angle about the helix axis; parameter on Y-axis is the distance along helix axis. Only the hydrophobic areas with MHP > 0.09 are shown. Contour intervals are 0.015. The positions of residues are indicated by letters. The grey shadow represents pore-lining surface in the experimental structure. Bottom: Solid line, angular distribution of MHP created by the peptide atoms on its surface; dotted line: angular distribution of MHP created by the neighbouring protein parts on the surface of helix A. The values of MHP are summarized inside the sectors 90°-width.

Fig. 2 Angular distribution of molecular hydrophobicity potential on the surface of pore-forming helices in proteins with five- (A) and four- (B) helix bundle folds. A: helix A of verotoxin-1 (VT-A), helix D of cholera-toxin (CT-D), helix S3 of pertussis toxin (PT-S3), cartilage oligomeric matrix protein (COMP). The plots are aligned relatively that of VT-A. Known pore exposure is shown with filled bars. helices A ¸ D of ectatomin (ECI-A ¸ ECI-D), A, B of myohemerythrin (MHR-A, MHR-B).

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