title: The elastase-PK101 structure: mechanism of an ultrasensitive activity-based probe revealed.
abstract: Human neutrophil elastase (HNE) plays a central role in neutrophil host defense, but its broad specificity makes HNE a difficult target for both inhibitor and probe development. Recently, we identified the unnatural amino acid containing activity-based probe PK101, which exhibits astounding sensitivity and selectivity for HNE, yet completely lacks mechanistic explanation for its unique characteristics. Here, we present the crystal structure of the HNE-PK101 complex which not only reveals the basis for PK101 ultrasensitivity but also uncovers so far unrecognized HNE features. Strikingly, the Nle(O-Bzl) function in the P4 position of PK101 reveals and leverages an "exo-pocket" on HNE as a critical factor for selectivity. Furthermore, the PK101 P3 position harbors a methionine dioxide function, which mimics a post-translationally oxidized methionine residue and forms a critical hydrogen bond to the backbone amide of Gly219 of HNE. Gly219 resides in a Gly-Gly motif that is unique to HNE, yet compulsory for this interaction. Consequently, this feature enables HNE to accommodate substrates that have undergone methionine oxidation, which constitutes a hallmark post-translational modification of neutrophil signaling.
authors.affiliation: †Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Bernhard C
authors.initials: BC
authors.lastname: Lechtenberg
authors.affiliation: ‡Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Kasperkiewicz
authors.affiliation: §Photon Sciences, Brookhaven National Laboratory, Upton, New York 11973, United States.
authors.forename: Howard
authors.initials: H
authors.lastname: Robinson
authors.affiliation: ‡Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drag
authors.affiliation: †Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Stefan J
authors.initials: SJ
authors.lastname: Riedl
mesh_terms: D018360:Crystallography, X-Ray; D006801:Humans; D006860:Hydrogen Bonding; D019272:Leukocyte Elastase / Q000737:chemistry* / Q000378:metabolism; D008715:Methionine / Q000737:chemistry; D015335:Molecular Probes / Q000737:chemistry*; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D015335:Molecular Probes; D008715:Methionine; D019272:Leukocyte Elastase