Crystal structures of human alpha-defensins HNP4, HD5, and HD6

Abstract

Six alpha-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human alpha-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known alpha- and beta-defensins. Like HNP3 but in contrast to murine or rabbit alpha-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human alpha-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human alpha-defensins.

Figure 1.

Structure-related properties of human α-defensins. (A) Alignment of the amino acid sequences. Conserved Cys residues are shown in yellow, positively-charged residues are printed in blue, and anionic residues in red. The distribution of β-strands and turns (T's) common to four α-defensins is shown above the sequence alignment (assignment was prepared with the program DSSP; Kabsch and Sander 1983). (B) Histograms of buried solvent-accessible surfaces for individual residues in folded monomers of defensins. Values of buried surfaces were calculated with program Naccess (www.biochem.ucl.ac.uk/~roman/naccess/naccess.html) and averaged over all crystallographically-independent monomers. The histogram is followed by the legend, describing the color assignment used. (C) Histograms of the solvent-accessible surfaces for individual residues buried upon dimerization. (D) Distributions of the average B-factor values for the main chain atoms of individual residues, divided by the B-factor values averaged over all main chain atoms of each defensin (see also Table 2). (E) As in D, except for the side chain atoms. Values used for the preparation of panels D and E represent the averages over all structurally independent monomers of each defensin. (F) Distributions of the discrepancies (in Å) calculated for the equivalent C_α-atoms of superimposed, structurally-independent monomers of defensins. For each defensin, values represent the averages obtained from all possible superimpositions. (G) Histograms of the discrepancies (in Å) calculated for the equivalent C_α-atoms of superimposed monomers from different defensins. Values used in this figure represent the averages (i.e., each color bar for HNP4 represents an average of 36 equivalent discrepancies). (H) Ribbon diagrams of the monomers of four human α-defensins shown in equivalent orientation. The side chains of all charged and Cys residues are shown in a ball-and-stick representation with labels. For HNP4, the structure of the last residue, Asp34, was not determined due to disorder. For completeness, however, this residue is also drafted in dotted lines and labeled in italics. The panel was prepared with programs Ribbons (Carson 1991) and POV-Ray (http://www.povray.org).

Figure 2.

A superposition of the C_α-traces of the intimate monomers of HD5 (monomers A and C) and HD6 (monomers A and B). Based on the comparison of the equivalent backbone torsion angles for each of the two monomers compared, only the subset of C_α-atoms (1–18 and 29–32 for HD5, 1–18 and 27–32 for HD6) corresponding to the most conformationally-similar residues has been used during alignment. For better readability, all C_α-atoms are labeled, and the disulfide bridges are shown as balls-and-sticks. Two structural differences between the two monomers, common for both defensins, include different conformations of the second disulfide bridge (Cys₅–Cys₂₀ in HD5, and Cys₆–Cys₂₀ in HD6) and orientations of the β2-hairpins. The latter discrepancy can be measured by the distance between the equivalent atoms at the tip of the hairpin in both monomers and varies from 5 Å (HD5) to nearly 7 Å (HD6). Several residues within this hairpin have different backbone conformations in both monomers, as seen clearly for the fragment Thr₂₁–Asn₂₆ in HD6. In both proteins, the differences are the result of a conformational change of Gly₁₈ (a “hinge” residue). The lesser difference is seen within the loop Ala₁₁–Glu₁₄ in both monomers of HD5. Conformational changes shown in this figure lead to the asymmetry of the dimers (see text for more details).

Figure 3.

Dimers of human α-defensins. Dimers of four α-defensins, HNP3 (shown in two shades of red), HNP4 (yellow and brown), HD5 (blue), and HD6 (green), are aligned based on the C_α-atoms from the left (“fixed”) monomer only. The discrepancies between the right (“riding”) monomers illustrate the extent of topological changes within dimers. The largest departure, seen for HD6, is the result of a relative shift of two monomers along their β2 strands.

Figure 4.

Two types of dimerization found in the crystals of human HD6. In addition to four intermonomer H-bonds contributed by the strands β2 and observed for all α-defensins in crystals (here marked with green dashed lines for the monomers b [yellow] and d [blue]), each monomer of HD6 forms the second cluster of four H-bonds (marked with black dashed lines) through the backbone atoms of Phe₂ and Cys₄. The second network is shown for monomers b (yellow) and c (red). Each mode of dimerization is associated with the formation of four H-bonds and a comparable reduction of the solvent-accessible surface per monomer (584 Ų and 834 Ų, for β2-strand- and N termini-mediated dimerizations, respectively).

Figure 5.

Distribution of the electrostatic potential on solvent-accessible surfaces of human α-defensins. The stereo representations of the solvent-accessible surfaces have been determined with program MSMS (Sanner et al. 1996), using the solvent sphere with radius of 1.4 Å. Surfaces are colored according to the values of Coulomb potential calculated on the basis of the charged residues and the termini. The regions colored in blue are associated with positively-charged groups, red with negative charges, and white-gray areas are electrically neutral. Monomers of all defensins are shown in equivalent orientations and are seen from two opposite views. For convenience, the C_α-representations of the molecules are also shown inside the semitransparent surfaces, and the charged residues are annotated. Drawings for HNP2 and HNP3 are based PDB entries 1ZMI and 1DFN, respectively. The cross-section of the defensin molecule is shown in the lower right panel as a composite of the cross-sections for individual proteins, and it is a flat schematic representation of both faces of the solvent-accessible surfaces. For ease of analysis (see text), solvent-accessible surfaces were arbitrarily divided into five regions (Reg1 through Reg5). Boundaries of the regions were chosen based on careful comparison of electrostatic potentials and shapes of the surfaces, and bear no relevance to the detailed topological features of the proteins. For convenience, areas of the composite cross-sections, charged in at least one defensin, are colored.