NMR structures of the histidine-rich peptide LAH4 in micellar environments: membrane insertion, pH-dependent mode of antimicrobial action, and DNA transfection

Abstract

The LAH4 family of histidine-rich peptides exhibits potent antimicrobial and DNA transfection activities, both of which require interactions with cellular membranes. The bilayer association of the peptides has been shown to be strongly pH-dependent, with in-planar alignments under acidic conditions and transmembrane orientations when the histidines are discharged. Therefore, we investigated the pH- and temperature-dependent conformations of LAH4 in DPC micellar solutions and in a TFE/PBS solvent mixture. In the presence of detergent and at pH 4.1, LAH4 adopts helical conformations between residues 9 and 24 concomitantly with a high hydrophobic moment. At pH 6.1, a helix-loop-helix structure forms with a hinge encompassing residues His¹⁰-Ala¹³. The data suggest that the high density of histidine residues and the resulting electrostatic repulsion lead to both a decrease in the pK values of the histidines and a less stable α-helical conformation of this region. The hinged structure at pH 6.1 facilitates membrane anchoring and insertion. At pH 7.8, the histidines are uncharged and an extended helical conformation including residues 4-21 is again obtained. LAH4 thus exhibits a high degree of conformational plasticity. The structures provide a stroboscopic view of the conformational changes that occur during membrane insertion, and are discussed in the context of antimicrobial activity and DNA transfection.

Figure 1

Graphic summary of NOEs observed for LAH4 in (A) 50% TFE-PBS, (B) DPC micellar solutions at pH 6.1 and 317 K and (300 K. The thickness of the lines represents strong, medium, and weak NOE intensities. The helical domains indicated correspond to the statistical analysis of simulated annealing calculations (panel B) or to regions where at least two of the following are observed: d_αN (i, i+3), d_αN (i, i+4), d_αβ (i, i+3), d_NN (i, i+2), strong d_NN (i, i+1), and high density of helical CSI (47).

Figure 2

The NH-Hα region of TOCSY spectra of LAH4 in DPC micellar solutions at 317 K is shown. (A) pH = 6.1. (B) pH = 4.1.

Figure 3

The NH-NH region of the 2D NOESY spectrum in DPC recorded at pH 6.1 and 300 K, with the assignment of cross-peaks shown.

Figure 4

Graphic representation of NOEs observed for LAH4 in DPC micellar solutions at 317 K and pH values of (A) 7.8 and (B) 4.1. The thickness of the lines represents strong, medium, and weak NOE intensities. The helical domains have been identified by the rules specified in the legend to Fig. 1.

Figure 5

Stereo view of the backbone superposition of the 20 lowest-energy structures at pH 4.1, where region 9–24 (A) is considered for superposition of the coordinates, and at pH 6.1, where the regions 3–9 (B) and 14–24 (C) are considered separately for superposition of the structures.

Figure 6

(A) Backbone traces of the mean structures of LAH4 showing the histidine heavy atoms mostly affected by pH changes. The mean structure of the peptide at pH 6.1 is displayed in blue, and the red trace represents the mean structure at pH 4.1. The superposition of both structures is based on the helical region encompassing the region between H14 and L23. (B) Space-filling model of the structure obtained at pH 6.1 and 317 K, representing the positive surface charges of the polypeptide in blue and the negative charges in red. The C-terminus is shown to the left. (C) Model representations of the LAH4 peptide in solution (random coil and monomeric at low pH (22,23)), of the structures in micellar environments (D–F) and in lipid bilayers (G–I). The surface-oriented topology at acidic pH (D and G), the transition state (E and H), and the transmembrane alignments at pH > 7 are shown (F and I) as measured in oriented phospholipid bilayers (G–I) (16) or by analogy (D–F). Panels D–F summarize the structural features observed in this work, whereas panels G–I combine these findings with the membrane topology obtained from oriented phospholipid bilayers (16,18).