Membrane Protein Insertion Regulated by Bringing Electrostatic and Hydrophobic Interactions into Play

Abstract

The study of the membrane insertion of the translocation domain of diphtheria toxin deepens our insight into the interactions between proteins and membranes. During cell intoxication, this domain undergoes a change from a soluble and folded state at alkaline pH to a functional membrane-inserted state at acid pH. We found that hydrophobic and electrostatic interactions occur in a sequential manner between the domain and the membrane during the insertion. The first step involves hydrophobic interactions by the C-terminal region. This is because of the pH-induced formation of a molten globule specialized for binding to the membrane. Accumulation of this molten globule follows a precise molecular mechanism adapted to the toxin function. The second step, as the pH decreases, leads to the functional inserted state. It arises from the changes in the balance of electrostatic attractions and repulsions between the N-terminal part and the membrane. Our study shows how the structural changes and the interaction with membranes of the translocation domain are finely tuned by pH changes to take advantage of the cellular uptake system.Folding and insertion of membrane proteins (1, 2), binding of hormones to membrane receptors (3), action of antibiotic peptides (4, 5), protein translocation, and internalization of toxins (6) are examples of phenomena that require the interactions of polypeptide chains with membranes. Because of the anisotropic nature of membranes, the initial steps of the association and the final structure and localization of polypeptide chains within membranes depend on a combination of hydrophobic and electrostatic interactions (7). Hydrophobic interactions are dominant for the insertion of transmembrane polypeptides. Electrostatic interactions are important for the binding of antibiotic peptides (5,8), the association of proteins with the surface of the membrane (9 -11), and as determinants of the topology of integral membrane proteins after biosynthesis (12). In most cases, electrostatic interactions are the result of the attraction between anionic phospholipid head groups and basic amino acid side chains (13,14). However, there are examples where electrostatic repulsions are involved in the membrane association of peptides, particularly when their structure and localization within the membrane is regulated by the pH (15-19). The interplay of hydrophobicity and electrostatics and their distribution within the polypeptide sequence have only been studied in detail for small peptides (3-5, 7, 13-15, 17-20). In the case of proteins, the role of these effects on the association with and the insertion into membranes are still poorly understood (2,(21)(22)(23)(24).The study of the membrane insertion process of the translocation (T) 1 domain of diphtheria toxin (25) can provide precious insight into the interactions between proteins and membranes and the refolding mechanisms of membrane proteins. During intoxication of cells (25), the toxin reaches the early endosomes through the clathrin-coated pathway (26). Beca...

Topography of the Hydrophilic Helices of Membrane-Inserted Diphtheria Toxin T Domain: TH1−TH3 as a Hydrophilic Tether

“…This is consistent with the model of Zhan et al, who proposed that upon incubation at low pH the three helical layers of the T domain (TH 1-4, TH 5-7 and TH 8-9) come apart to a small degree (41). (It should be noted that with previous studies showing T domain secondary structure in solution remains highly helical both at neutral and low pH (1,45). )…”

Bacterial protein toxins and lipids: pore formation or toxin entry into cells

“…Translocation could proceed by a series of transient association-dissociation events between the T domain and the A chain. It has been proposed that the T domain may act like a transmembrane chaperone, forming a 'sticky pore' that binds to the hydrophobic A chain surface, thus, maintaining it in an unfolded state during translocation (Ren et al, 1999;Chenal et al, 2002;Hammond et al, 2002;Ladokhin et al, 2004). Recently, the DT enzymatic domain has been found to insert into lipid membranes through one or several transient transmembrane β-sheet structures.…”

Solvent reaction field potential inside an uncharged globular protein: A bridge between implicit and explicit solvent models?

“…The electrostatic properties of solvated biomolecules are a subject of intense computational [1][2][3] as well as experimental interest [4][5][6][7]. The polar nature of water molecules influences the structure of proteins, nucleic acids, and lipid membranes.…”