Structure determination of human and murine beta-defensins reveals structural conservation in the absence of significant sequence similarity

Abstract

Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian beta-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human beta-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that beta-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the beta-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because beta-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.

Fig. 1.

Sequence alignment of mammalian β-defensins (BD). The alignment includes the sequences of four human defensins (hBD-1, hBD-2, hBD-3, hBD-4), six murine defensins (mBD-1 to mBD-4, mBD-7, mBD-8), three bovine defensins (bBD-1, bBD-2, bBD-12), and the bovine tracheal anitmicrobial peptide (bTAP). Strictly conserved amino acid residues are highlighted by a black box and residues occurring with a frequency of ≥50% are marked by gray boxes. The alignment was generated using the programs ClustalW (Higgins et al. 1992) and Alscript (Barton 1993). Because of the low sequence similarity, DALI analysis (Holm and Sander 1996) of the three-dimensional structures was used to allow a correct placement of the gaps in the sequences of mBD-7 and mBD-8. Elements of secondary structure found in hBD-1, hBD-2, mBD-7, and mBD-8 are schematically indicated below the alignment. The numbering scheme refers to the full-length hBD-2 including the amino-terminal signal sequence.

Fig. 2.

Summary of the NMR spectroscopic data obtained for hBD-2. (A) Contour plot of the Hα–Hα region of a 200 msec mixing time NOESY spectrum of hBD-2 in D₂O. Hα–Hα cross resonances indicative of β-strands are labeled. An asterisk denotes frequency degeneration of the corresponding cross-peak. (B) Summary of the spatial interactions between the amino acids in hBD-2. (▴) NOEs that were observed between the corresponding residues. The length and position of the helix and β-sheets can be deduced from this presentation.

Fig. 3.

Schematic representation of the solution structure of hBD-2. The elements of secondary structure and the disulfide bonds are indicated and highly conserved residues are shown in ball-and-stick representation. The two views in A and B are rotated by ∼180° around the vertical axis. The figure was generated with Molscript (Kraulis 1991) and Raster 3D (Merritt and Murphy 1994).

Fig. 4.

Overlay of a set of 10 hBD-2 structures obtained by NMR spectroscopy (black) with 5 crystal structures (gray) from the work of Hoover et al. (2000) (pdb entries: 1FD3 and 1FD4). Same orientation as in Fig. 3a ▶. Figure drawn with SYBYL 6.5.

Fig. 5.

Schematic representation of the solution structures of mBD-7 (A), mBD-8 (B) and hBD-1 (C). The elements of secondary structure and the disulfide bonds are indicated. Same orientation as in Fig. 3a ▶. The figure was drawn with Molscript (Kraulis 1991) and Raster 3D (Merritt and Murphy 1994). (D) Overlay of the structures of the two human defensins hBD-1 and hBD-2 (dark gray) with the structures of the two murine defensins mBD-7 and mBD-8 (light gray); figure drawn with SYBYL 6.5.

Fig. 6.

Calculation of the accessible surface area per residue for hBD-1 (•), hBD-2 (▪), mBD-7 (▾), and mBD-8 (▴). Values represent average values for the 20 lowest-energy conformers of each defensin and were calculated using MolMol (Koradi et al. 1996). All sequence positions and those residues that exhibit the lowest solvent accessibility are labeled according to the hBD-2 numbering scheme.