The amino-terminal residues in the crystal structure of connective tissue activating peptide-III (des10) block the ELR chemotactic sequence

Abstract

α-Chemokines comprise a family of cytokines that are chemotactic for neutrophils and have a structure similar to platelet factor 4 (PF4), in which the first two cysteine residues are separated by one residue (Cys-X-Cys). The two α-chemokines, connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2 (NAP-2), are carboxyl-terminal fragments of platelet basic protein (PBP) that are generated by monocyte-derived proteases. NAP-2 strongly stimulates neutrophils that are present during inflammation whereas its precursors, PBP and CTAP-III, are inactive, although they also possess the highly conserved, amino-terminal sequence, Glu-Leu-Arg (ELR), that is critical for receptor binding. To resolve this conundrum, we have determined the crystal structure of recombinant Asp-CTAP, which has ten fewer amino-terminal residues than CTAP-III but five more than NAP-2. The space group is P 21, with unit cell dimensions a=43.8 Å, b=76.8 Å, c=43.8 Å, and β =97.0°, and a tetramer in the asymmetric unit. The molecular replacement method, with the NAP-2 tetramer as a starting model, was used to determine the initial phase information. The final R -factor is 0.196 (Rfree=0.251) for 2σ data from 7.0 to 1.75 Å resolution. This high-resolution model of Asp-CTAP is the longest defined structure of an α-chemokine to date. The electron density map shows an over-all structure for Asp-CTAP that is very similar to that of NAP-2, but with the additional five amino-terminal residues folding back through a type-II turn, thereby stabilizing the oligomeric “inactive” state, and masking the critical ELR receptor binding region that is exposed in the structure of NAP-2.