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Antimicrobial Agents and Chemotherapy, March 2008, p. 883-894, Vol. 52, No. 3
0066-4804/08/$08.00+0     doi:10.1128/AAC.00805-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Macrophage Inflammatory Protein 3: Protein and Peptide Nuclear Magnetic Resonance Solution Structures, Dimerization, Dynamics, and Anti-Infective Properties ^,

David I. Chan,¹ Howard N. Hunter,^1, Brian F. Tack,² and Hans J. Vogel^1*

Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada,¹ Departments of Pediatrics and Microbiology, University of Iowa College of Medicine, Iowa City, Iowa 52242²

Received 21 June 2007/ Returned for modification 30 July 2007/ Accepted 7 December 2007

Human macrophage inflammatory protein 3 (MIP-3), also known as CCL20, is a 70-amino-acid chemokine which exclusively binds to chemokine receptor 6. In addition, the protein also has direct antimicrobial, antifungal, and antiviral activities. The solution structure of MIP-3 was solved by the use of two-dimensional homonuclear proton nuclear magnetic resonance (NMR). The structure reveals the characteristic chemokine fold, with three antiparallel β strands followed by a C-terminal helix. In contrast to the crystal structures of MIP-3, the solution structure was found to be monomeric. Another difference between the NMR and crystal structures lies in the angle of the helix with respect to the β strands, which measure 69 and 56.5° in the two structures, respectively. NMR diffusion and pH titration studies revealed a distinct tendency for MIP-3 to form dimers at neutral pH and monomers at lower pH, dependent on the protonation state of His40. Molecular dynamics simulations of both the monomeric and the dimeric forms of MIP-3 supported the notion that the chemokine undergoes a change in helix angle upon dimerization and also highlighted the important hydrophobic and hydrogen bonding contacts made by His40 in the dimer interface. Moreover, a constrained N terminus and a smaller binding groove were observed in dimeric MIP-3 simulations, which could explain why monomeric MIP-3 may be more adept at receptor binding and activation. The solution structure of a synthetic peptide consisting of the last 20 residues of MIP-3 displayed a highly amphipathic helix, reminiscent of various antimicrobial peptides. Antimicrobial assays with this peptide revealed strong and moderate bactericidal activities against Escherichia coli and Staphylococcus aureus, respectively. This confirms that the C-terminal -helical region of MIP-3 plays a significant part in its broad anti-infective activity.

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* Corresponding author. Mailing address: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada. Phone: (403) 220-6006. Fax: (403) 289-9311. E-mail: vogel{at}ucalgary.ca

Published ahead of print on 17 December 2007.

Supplemental material for this article may be found at http://aac.asm.org/.

Present address: Department of Chemistry, York University, Toronto, Ontario, Canada.

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Antimicrobial Agents and Chemotherapy, March 2008, p. 883-894, Vol. 52, No. 3
0066-4804/08/$08.00+0     doi:10.1128/AAC.00805-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.