Jacek Lubkowski

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.

Human α-defensins block papillomavirus infection

Sexually transmitted human papillomaviruses (HPVs) are the primary cause of cervical cancer. Recent advances in techniques for production of papillomaviral vectors [known as pseudoviruses (PsVs)] have made it possible to perform high-throughput screens for compounds that might block the initial stages of papillomavirus infection. We have used PsVs to screen a variety of compounds that might function as inhibitors of HPV infection, with emphasis on human peptides previously implicated in innate antimicrobial immunity. Little is known about the possible activity of these peptides against nonenveloped viruses, such as HPVs. Our screen revealed that human α-defensins 1-3 [known as human neutrophil peptides (HNPs) 1-3] and human α-defensin 5 (HD-5) are potent antagonists of infection by both cutaneous and mucosal papillomavirus types. In contrast, human β-defensins 1 and 2 displayed little or no anti-HPV activity. HD-5 was particularly active against sexually transmitted HPV types, with 50% inhibitory doses in the high ng/ml range. Microscopic studies of PsV inhibition by the α-defensins revealed that they block virion escape from endocytic vesicles but not virion binding or internalization. Consistent with this finding, PsVs remained susceptible to inhibition by α-defensins for many hours after initial binding to cells. HNPs 1-3 and HD-5 have been reported to be present in the female genital tract at levels that overlap those that inhibit HPVs in vitro, suggesting that they could present a natural barrier to the sexual transmission of HPV and could serve as the basis of a broad-spectrum topical microbicide.

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

Six α‐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 α‐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 α‐ and β‐defensins. Like HNP3 but in contrast to murine or rabbit α‐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 α‐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 α‐defensins.

The structure of human beta-defensin-1. New insights into structural properties of beta-defensins

Defensins are a class of small cationic peptides found in higher organisms that serve as both antimicrobial and cell signaling molecules. The exact mechanism of the antimicrobial activity of defensins is not known, but two models have been postulated, one involving pore formation and the other involving nonspecific electrostatic interaction with the bacterial membrane. Here we report the high resolution structures of human β-defensin-1 (hBD1) in two crystallographic space groups. The structure of a single molecule is very similar to that of human β-defensin-2 (hBD2), confirming the presence of an N-terminal α-helix. However, while the packing of hBD1 is conserved across both space groups, there is no evidence for any larger quaternary structure similar to octameric hBD2. Furthermore, the topology of hBD1 dimers that are formed between monomers in the asymmetric unit is distinct from both hBD2 and other mammalian α-defensins. The structures of hBD1 and hBD2 provide a first step toward understanding the structural basis of antimicrobial and chemotactic properties of human β-defensins.

Filamentous phage infection: crystal structure of g3p in complex with its coreceptor, the C-terminal domain of TolA.

BACKGROUND Infection of male Escherichia coli cells by filamentous Ff bacteriophages (M13, fd, and f1) involves interaction of the phage minor coat gene 3 protein (g3p) with the bacterial F pilus (primary receptor), and subsequently with the integral membrane protein TolA (coreceptor). G3p consists of three domains (N1, N2, and CT). The N2 domain interacts with the F pilus, whereas the N1 domain--connected to N2 by a flexible glycine-rich linker and tightly interacting with it on the phage--forms a complex with the C-terminal domain of TolA at later stages of the infection process. RESULTS The crystal structure of the complex between g3p N1 and TolA D3 was obtained by fusing these domains with a long flexible linker, which was not visible in the structure, indicating its very high disorder and presumably a lack of interference with the formation of the complex. The interface between both domains, corresponding to approximately 1768 A2 of buried molecular surface, is clearly defined. Despite the lack of topological similarity between TolA D3 and g3p N2, both domains interact with the same region of the g3p N1 domain. The fold of TolA D3 is not similar to any previously known protein motifs. CONCLUSIONS The structure of the fusion protein presented here clearly shows that, during the infection process, the g3p N2 domain is displaced by the TolA D3 domain. The folds of g3p N2 and TolA D3 are entirely different, leading to distinctive interdomain contacts observed in their complexes with g3p N1. We can now also explain how the interactions between the g3p N2 domain and the F pilus enable the g3p N1 domain to form a complex with TolA.

Toward Understanding the Cationicity of Defensins ARG AND LYS VERSUS THEIR NONCODED ANALOGS

Human defensins are a family of small antimicrobial proteins found predominantly in leukocytes and epithelial cells that play important roles in the innate and adaptive immune defense against microbial infection. The most distinct molecular feature of defensins is cationicity, manifested by abundant Arg and/or Lys residues in their sequences. Sequence analysis indicates that Arg is strongly selected over Lys in α-defensins but not in β-defensins. To understand this Arg/Lys disparity in defensins, we chemically synthesized human α-defensin 1 (HNP1) and several HNP1 analogs where three Arg residues were replaced by each of the following six α-amino acids: Lys, ornithine (Orn), diaminobutyric acid (Dab), diaminopropionic acid (Dap), N,N-dimethyl-Lys (diMeLys), and homo-Arg (homoArg). In addition, we prepared human β-defensin 1 (hBD1) and Lys→ArghBD1 in which all four Lys residues were substituted for Arg. Bactericidal activity assays revealed the following. 1) Arg-containing HNP1 and Lys→ArghBD1 are functionally better than Lys-HNP1 and hBD1, respectively; the difference between Arg and Lys is more evident in the α-defensin than in the β-defensin and is more evident at low salt concentrations than at high salt concentrations. 2) For HNP1, the Arg/Lys disparity is much more pronounced with Staphylococcus aureus than with Escherichia coli, and the Arg-rich HNP1 kills bacteria faster than its Lys-rich analog. 3) Arg and Lys appear to have optimal chain lengths for bacterial killing as shortening Lys or lengthening Arg in HNP1 invariably becomes functionally deleterious. Our findings provide insights into the Arg/Lys disparity in defensins, and shed light on the cationicity of defensins with respect to their antimicrobial activity and specificity.

TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans

Triggering receptor expressed on myeloid cells-like (TREM-like) transcript-1 (TLT-1), a type 1 single Ig domain orphan receptor specific to platelet and megakaryocyte alpha-granules, relocates to the platelet surface upon platelet stimulation. We found here that patients diagnosed with sepsis, in contrast to healthy individuals, had substantial levels of soluble TLT-1 (sTLT-1) in their plasma that correlated with the presence of disseminated intravascular coagulation. sTLT-1 bound to fibrinogen and augmented platelet aggregation in vitro. Furthermore, the cytoplasmic domain of TLT-1 could also bind ezrin/radixin/moesin family proteins, suggesting its ability to link fibrinogen to the platelet cytoskeleton. Accordingly, platelets of Treml1-/- mice failed to aggregate efficiently, extending tail-bleeding times. Lipopolysaccharide-treated Treml1-/- mice developed higher plasma levels of TNF and D-dimers than wild-type mice and were more likely to succumb during challenge. Finally, Treml1-/- mice were predisposed to hemorrhage associated with localized inflammatory lesions. Taken together, our findings suggest that TLT-1 plays a protective role during inflammation by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. Therefore, therapeutic modulation of TLT-1-mediated effects may provide clinical benefit to patients with hypercoagulatory conditions, including those associated with inflammation.

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Escherichia coli asparaginase II catalyzes the hydrolysis of l‐asparagine to l‐aspartate via a threonine‐bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threomines, Thr‐89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 Å resolution, shows high overall similarity to the wild‐type enzyme, but an aspartyl moiety is covalently bound to Thr‐12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.