NMR structure and dynamics of monomeric neutrophil-activating peptide 2

Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proi nflammatory activities, is a 72-residue protein belonging to the alpha-chem okine family. Although NAP-2, like other alpha-chemokines, is known to self -associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional H-1-NMR and N-15-NMR spectroscopy a nd computational modelling. The NAP-2 monomer consists of an amphipathic, t riple-stranded, antiparallel beta-sheet on which is folded a C-terminal alp ha-helix and an aperiodic N-terminal segment. The backbone fold is essentia lly the same as that found in other alpha-chemokines. N-15 T-1, T-2 and nuc lear Overhauser effects (NOEs) have been measured for backbone NH groups an d used in a model free approach to calculate order parameters and conformat ional exchange terms that map out motions of the backbone. N-terminal resid ues 1 to 17 and the C-terminus are relatively highly flexible, whereas the beta-sheet domain forms the most motionally restricted part of the fold. Co nformational exchange occurring on the millisecond time scale is noted at t he top of the C-terminal helix and at proximal residues from beta-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is appa rently responsible for increased internal mobility in NAP-2 compared with d imeric and tetrameric states in other alpha-chemokines.