title: Exploring platelet chemokine antimicrobial activity: nuclear magnetic resonance backbone dynamics of NAP-2 and TC-1.
abstract: The platelet chemokines neutrophil-activating peptide-2 (NAP-2) and thrombocidin-1 (TC-1) differ by only two amino acids at their carboxy-terminal ends. Nevertheless, they display a significant difference in their direct antimicrobial activities, with the longer NAP-2 being inactive and TC-1 being active. In an attempt to rationalize this difference in activity, we studied the structure and the dynamics of both proteins by nuclear magnetic resonance (NMR) spectroscopy. Using 15N isotope-labeled protein, we confirmed that the two monomeric proteins essentially have the same overall structure in aqueous solution. However, NMR relaxation measurements provided evidence that the negatively charged carboxy-terminal residues of NAP-2 experience a restricted motion, whereas the carboxy-terminal end of TC-1 moves in an unrestricted manner. The same behavior was also seen in molecular dynamic simulations of both proteins. Detailed analysis of the protein motions through model-free analysis, as well as a determination of their overall correlation times, provided evidence for the existence of a monomer-dimer equilibrium in solution, which seemed to be more prevalent for TC-1. This finding was supported by diffusion NMR experiments. Dimerization generates a larger cationic surface area that would increase the antimicrobial activities of these chemokines. Moreover, these data also show that the negatively charged carboxy-terminal end of NAP-2 (which is absent in TC-1) folds back over part of the positively charged helical region of the protein and, in doing so, interferes with the direct antimicrobial activity.
authors.affiliation: Department of Biological Sciences, University of Calgary, 2500 University Dr., NW, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Leonard T
authors.initials: LT
authors.lastname: Nguyen
authors.forename: Paulus H S
authors.initials: PH
authors.lastname: Kwakman
authors.forename: David I
authors.initials: DI
authors.lastname: Chan
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Liu
authors.forename: Leonie
authors.initials: L
authors.lastname: de Boer
authors.forename: Sebastian A J
authors.initials: SA
authors.lastname: Zaat
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
mesh_terms: D000890:Anti-Infective Agents; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D056004:Molecular Dynamics Simulation; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D001620:beta-Thromboglobulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000890:Anti-Infective Agents; D009363:Neoplasm Proteins; C061615:PPBP protein, human; D010455:Peptides; C418741:TCIM protein, human; D001620:beta-Thromboglobulin