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 15 N 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.

Structural Basis of Native CXCL7 Monomer Binding to CXCR2 Receptor N-Domain and Glycosaminoglycan Heparin

“…Comparison of our data to the previously reported relaxation data of CXCL7 in the presence of chloroethanol shows striking differences for the 30s-loop residues. Heteronuclear NOE measurements in the presence of 2-chloroethanol indicate a highly dynamic 30s-loop, especially residues Q33, V34 and E35, showing very low NOE values observed for the very terminal residues, whereas our values are similar to those of structured residues [16,31,32]. These observations suggest that chloroethanol influences dynamic properties, and so, these data may not fully reflect the dynamics of the native protein.…”

“…Our structure showed a backbone RMSD of 0.82 Å compared to the monomer units of the tetramer over the same regions, and differences in these regions are mainly due to extended β-strands and a slight change in the orientation of the helix. The largest differences between our monomer and the tetramer structure were observed for the N-terminus and 30s-loop residues, which can be attributed to their conformational flexibility [16,31,32] (Figure 3A). In general, we observed that the more dynamic a region, the greater the difference in its backbone RMSD.…”

Native Thrombocidin-1 and Unfolded Thrombocidin-1 Exert Antimicrobial Activity via Distinct Structural Elements

“…This truncation is required for strong antimicrobial activity (2), but it does not substantially affect chemokine activity (39). In NAP-2, the negatively charged C terminus folds back over the positively charged surface and may thus interfere with antimicrobial activity (40). The C-terminal truncation generating TC-1 reduces the negative charge of the C terminus and thus abolishes interference of the C terminus with the positive patch (40).…”

Structural perspectives on antimicrobial chemokines

“…(D) Electrostatic surface diagram showing the extension of the positive patch created through TC-1 dimerization. Panels (B) and (D) reprinted with permission from Nguyen et al (2011b). …”

“…The last four residues of NAP-2 were not included in the X-ray crystal structure due to poorly defined electron density in this region (Malkowski et al, 1995). NMR relaxation experiments reveal that, although the last five residues are overall quite flexible compared to the rest of NAP-2, the final two residues become more motionally restricted than the residues immediately preceding them (Figure 2B) (Nguyen et al, 2011b). By comparison, the TC-1 backbone continually increases in flexibility toward the end of its sequence and the C-terminus was not seen to fold back over the α-helix in the corresponding peptide.…”