Structural perspectives on antimicrobial chemokines

Abstract

Chemokines are best known as signaling proteins in the immune system. Recently however, a large number of human chemokines have been shown to exert direct antimicrobial activity. This moonlighting activity appears to be related to the net high positive charge of these immune signaling proteins. Chemokines can be divided into distinct structural elements and some of these have been studied as isolated peptide fragments that can have their own antimicrobial activity. Such peptides often encompass the α-helical region found at the C-terminal end of the parent chemokines, which, similar to other antimicrobial peptides, adopt a well-defined membrane-bound amphipathic structure. Because of their relatively small size, intact chemokines can be studied effectively by NMR spectroscopy to examine their structures in solution. In addition, NMR relaxation experiments of intact chemokines can provide detailed information about the intrinsic dynamic behavior; such analyses have helped for example to understand the activity of TC-1, an antimicrobial variant of CXCL7/NAP-2. With chemokine dimerization and oligomerization influencing their functional properties, the use of NMR diffusion experiments can provide information about monomer-dimer equilibria in solution. Furthermore, NMR chemical shift perturbation experiments can be used to map out the interface between self-associating subunits. Moreover, the unusual case of XCL1/lymphotactin presents a chemokine that can interconvert between two distinct folds in solution, both of which have been elucidated. Finally, recent advances have allowed for the determination of the structures of chemokines in complex with glycosaminoglycans, a process that could interfere with their antimicrobial activity. Taken together, these studies highlight several different structural facets that contribute to the way in which chemokines exert their direct microbicidal actions.

CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

“…To determine whether cysteines are critical for activity in small peptides, we synthesized mutated variants with alanine in place of cysteine at positions 3 and 5. Cysteines were not absolutely required because mutated CXCL14 [1][2][3][4][5][6][7][8][9][10][11][12][13] (AA), where the two cysteines were replaced by alanine, retained part of the activity at 1 and 0.5 mM. However, the activity of the alanine mutated CXCL14 1-8 (AA) was totally abolished, suggesting that cysteines are partly important for the bactericidal activity especially with smaller peptides (Fig.…”

“…Truncated forms of CXCL14 were custom synthesized by Thermo Fisher Scientific (Ulm, Germany) or Shanghai Biochemical (Shanghai, China) and designated as follows: CXCL14 1-8 (SKCKCSRK); CXCL14 1-9 (SKCKCSRKG); CXCL14 1-10 (SKCKCSRKGP); CXCL14 [1][2][3][4][5][6][7][8][9][10][11] (SKCKCSRKGPK); CXCL14 1-13 (SKCKCSRKGPKIR); CXCL14 (YSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHCLHPK); CXCL14 55-73 (LQSTKRFIKWYNAWNEKRR); and CXCL14 55-77 (LQS-TKRFIKWYNAWNEKRRVYEE). The accurate concentrations of all the peptides were determined by amino acid analysis (Analytical Research and Services, University of Bern, Bern, Switzerland).…”

“…Next, we wanted to further refine the structure associated with the antimicrobial activity by shortening of the N-terminal CXCL14 peptides, and therefore synthesized CXCL14 1-11 , CXCL14 1-10 , CXCL14 [1][2][3][4][5][6][7][8][9] , and CXCL14 1-8 peptides. As shown in Fig.…”

“…However, removing glycine from CXCL14 1-9 exposed lysine as the C-terminal amino acid in CXCL14 [1][2][3][4][5][6][7][8] , and this form regained antimicrobial activity against E. coli with similar potency as CXCL14 [1][2][3][4][5][6][7][8][9][10][11] , but it was significantly lower than CXCL14 [1][2][3][4][5][6][7][8][9][10][11][12][13] . The active peptides CXCL14 1-8 , CXCL14 [1][2][3][4][5][6][7][8][9][10][11] , and CXCL14 1-13 have two cysteines as common features, as well as a cationic amino acid (lysine and arginine, respectively) at the C-terminal end. To determine whether cysteines are critical for activity in small peptides, we synthesized mutated variants with alanine in place of cysteine at positions 3 and 5.…”

“…Later it was recognized that chemokines have additional functions including roles in embryogenesis, cancer, and wound healing, and more recent studies also established that many chemokines exhibit broad-spectrum antimicrobial activity (2)(3)(4). Antimicrobial activity appears particularly relevant for mucosaassociated chemokines that are expressed in barrier tissues and, in fact, the four primarily mucosa-associated chemokines, CCL25, CCL28, CXCL14, and CXCL17, have been shown to function as antimicrobial peptides (AMPs) (5)(6)(7)(8)(9)(10).…”