David M. Hoover

Many chemokines including CCL20/MIP-3α display antimicrobial activity

Previous studies have demonstrated that β‐defensins exhibit chemotactic activity by sharing the chemokine receptor CCR6 with the CC chemokine ligand CCL20/macrophage‐inflammatory protein‐3α (MIP‐3α). Structural analysis of CCL20/MIP‐3α revealed that most of the positively charged residues are concentrated at one area of its topological surface, a characteristic considered to be important for the antimicrobial activity of defensins. Here, we report that similar to defensins, CCL20/MIP‐3α has antimicrobial effects on Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Streptococcus pyogenes, Enterococcus faecium, Staphylococcus aureus, and Candida albicans. Additionally, by screening a total of 30 human chemokines, we have identified an additional 17 human chemokines, which exhibit antimicrobial activity in vitro. Collectively, about two‐thirds of the chemokines investigated so far has the capacity to kill microorganisms in vitro, suggesting that antimicrobial activity may be another host‐defense function for certain chemokines. Comparison of the structural characteristics between antimicrobial and nonantimicrobial chemokines suggests that topological formation of a large, positively charged electrostatic patch on the surface of the molecule is likely to be a common structural feature of antimicrobial chemokines.

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human β-defensin 3

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into α and β families. Structures of several β-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys1–Cys5, Cys2–Cys4, and Cys3–Cys6. We found that human β-defensin 3 (hBD3), a recently described member of the growing β family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys1–Cys5 and of Cys1–Cys6, we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native β pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by α-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human β-defensins and the design of novel peptide antibiotics.

The Structure of Human Beta-Defensin-2 Shows Evidence of Higher Order Oligomerization

Defensins are small cationic peptides that are crucial components of innate immunity, serving as both antimicrobial agents and chemoattractant molecules. The specific mechanism of antimicrobial activity involves permeabilization of bacterial membranes. It has been postulated that individual monomers oligomerize to form a pore through anionic membranes, although the evidence is only indirect. Here, we report two high resolution x-ray structures of human β-defensin-2 (hBD2). The phases were experimentally determined by the multiwavelength anomalous diffraction method, utilizing a novel, rapid method of derivatization with halide ions. Although the shape and charge distribution of the monomer are similar to those of other defensins, an additional α-helical region makes this protein topologically distinct from the mammalian α- and β-defensin structures reported previously. hBD2 forms dimers topologically distinct from that of human neutrophil peptide-3. The quaternary octameric arrangement of hBD2 is conserved in two crystal forms. These structures provide the first detailed description of dimerization of β-defensins, and we postulate that the mode of dimerization of hBD2 is representative of other β-defensins. The structural and electrostatic properties of the hBD2 octamer support an electrostatic charge-based mechanism of membrane permeabilization by β-defensins, rather than a mechanism based on formation of bilayer-spanning pores.

Studies of the Biological Properties of Human beta-Defensin 1.

Defensins are small (30–45 amino acid residues) cationic proteins with broad antimicrobial activity against many bacteria and fungi, some enveloped viruses, and other activities such as chemoattraction of a range of different cell types to the sites of inflammation. These proteins represent attractive targets for developing novel antimicrobial agents and modulators of immune responses with therapeutic applicability. In this report, we present the results of functional and structural studies of 26 single-site mutants of human β-defensin 1 (hBD1). All mutants were assayed for antimicrobial activity against Escherichia coli (ATCC strain 25922) and for chemotactic activity with CCR6-transfected HEK293 cells. To analyze the structural implications of mutagenesis and to verify the correctness of the disulfide connectivity, we used x-ray crystallography to conduct complete structural studies for 10 mutants in which the topology of disulfides was the same as in the native hBD1. Mutations did not induce significant changes of the tertiary structure, suggesting that the observed alterations of biological properties of the mutants were solely associated with changes in the respective side chains. We found that cationic residues located near the C terminus (Arg29, Lys31, Lys33, and Lys36) of hBD1 define most of the anti-E. coli in vitro activity of this protein. In turn, nearly all mutations altering the CCR6-mediated chemotaxis are located at one area of the protein, defined by the N-terminal α-helical region (Asp1... Ser8) and a few topologically adjacent residues (Lys22, Arg29, and Lys33). These experimental results allow for the first time drafting of the CCR6-epitope for a defensin molecule.

Total chemical synthesis and high-resolution crystal structure of the potent anti-HIV protein AOP-RANTES.

BACKGROUND RANTES is a CC-type chemokine protein that acts as a chemoattractant for several kinds of leukocytes, playing an important pro-inflammatory role. Entry of human immunodeficiency virus-1 (HIV-1) into cells depends on the chemokine receptor CCR5. RANTES binds CCR5 and inhibits HIV-1 entry into peripheral blood cells. Interaction with chemokine receptors involves a distinct set of residues at the amino terminus of RANTES. This finding was utilized in the development of a chemically modified aminooxypentane derivative of RANTES, AOP-RANTES, that was originally produced from the recombinant protein using semisynthetic methods. RESULTS AOP-RANTES has been produced by a novel total chemical synthesis that provides efficient, direct access to large amounts of this anti-HIV protein analog. The crystal structure of chemically synthesized AOP-RANTES has been solved and refined at 1.6 A resolution. The protein is a dimer, with the amino-terminal pentane oxime moiety clearly defined. CONCLUSIONS Total chemical synthesis of AOP-RANTES provides a convenient method of producing the multi-milligram quantities of this protein needed to investigate the molecular basis of receptor binding and antiviral activity. This work provides the first truly high-resolution structure of a RANTES protein, although the structure of RANTES was known from previous nuclear magnetic resonance (NMR) determinations.

The Crystal Structure of the Chemokine Domain of Fractalkine Shows a Novel Quaternary Arrangement

Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX3C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX3CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 Å resolution. The CDF monomers form a dimer through an intermolecular β-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX3C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivois not well understood.

Structures of thymus and activation-regulated chemokine (TARC).

Thymus and activation-regulated chemokine (TARC) is a CC chemokine that is mainly expressed in the thymus. TARC interacts primarily with the CCR4 receptor and to a lesser extent with the CCR8 receptor. The structures of TARC have been solved by molecular replacement in two space groups, triclinic (P1) and tetragonal (P4(1)), and refined to resolutions of 1.72 and 2.1 A, respectively, with R factors of 19.8% (R(free) = 24.1%) and 19.8% (R(free) = 27.7%), respectively. The search model originated from the crystal structure of another chemokine, RANTES, and proved to be only modestly similar to the refined structure of TARC. Whereas the tetragonal structure was easily solved using the program AMoRe, solution of the triclinic structure proved to be quite challenging and was obtained by combining the results from four different molecular-replacement programs (AMoRe, CNS, BEAST and EPMR), with subsequent extension of the gathered information. The tertiary structure of TARC is similar to that of other CC chemokines, with a three-stranded antiparallel beta-sheet flanked by a C-terminal helix. Both quaternary structures consist of dimers, which in the triclinic crystals pack further into tetramers. The TARC dimers are similar to those observed previously in the crystal structures of both MCP-1 and RANTES.

Crystallization and preliminary X-ray studies of thymus and activation-regulated chemokine (TARC).

Thymus and activation-regulated chemokine (TARC) is a CC chemokine that is most highly expressed in the thymus. TARC interacts primarily with the CCR4 receptor and to a lesser extent with the CCR8 receptor. Three different crystal forms of synthetically prepared TARC were grown in triclinic, hexagonal and tetragonal systems. The X-ray data for the triclinic crystals (unit-cell parameters a = 56.46, b = 76.48, c = 88.37 A, alpha = 85.8, beta = 72.8, gamma = 70.0 degrees ) extend to 1.85 A on a conventional radiation source. The hexagonal crystals diffracted to 2.2 A at a synchrotron-radiation source and belong to either space group P6(1)22 or P6(5)22, with unit-cell parameters a = 61.8, c = 315. A. The tetragonal crystals diffracted to about 5 A at a synchrotron-radiation source and had approximate unit-cell parameters a = b = 47.7, c = 58.2 A.