Two mutants of human heparin binding protein (CAP37): Toward the understanding of the nature of lipid A/LPS and BPTI binding

Heparin binding protein (HBP) is an inactive serine protease homologue with important implications in host defense during infections and inflammations . Two mutants of human HBP, [R23S,F25E]HBP and [G175@]HBP, have been produc ed to investigate structure-function relationships of residues in the putat ive lipid A/lipopolysaccharide (LPS) binding site and BPTI (bovine pancreat ic trypsin inhibitor) binding site. The X-ray structures have been determin ed at 1.9 Angstrom resolution for [G175Q]HBP and at 2.5 Angstrom resolution for the [R23S,F25E]HBP mutant, and the structures have been fully refined to R-factors of 18.2% and 20.7%, respectively. The G175Q mutation does not alter the overall structure of the protein, but the ability to bind BPTI ha s been eliminated, and the mutant mediates only a limited stimulation of th e LPS-induced cytokine release from human monocytes. The lipid A/LPS bindin g property of [G175Q]HBP is comparable with that of native HBP. The R23S,F2 5E mutations do not affect the binding of lipid A/LPS and BPTI or the LPS-i nduced cytokine release from human monocytes. This shows that two diverse l igands, lipid A/LPS and BPTI, do not share binding sites. Previously, there was convincing evidence for the proposed lipid A/LPS binding site of HBP. Unexpectedly, the extensive structural changes introduced by mutation of Ar g23 and Phe25 do not affect the binding of lipid A/LPS, indicating that ano ther not yet identified site on HBP is involved in the binding of lipid A/L PS. (C) 2001 Wiley-Liss, Inc.