The Rational Design of Therapeutic Peptides for Aminopeptidase N using a Substrate-Based Approach

Joshi, Shilvi; Chen, Lang; Winter, Michael B.; Lin, Yi-Lun; Yang, Yang; Shapovalova, Mariya; Smith, Paige M.; Liu, Chang; Li, Fang; LeBeau, Aaron M.

Porcine APN ectodomain was crystallized as previously described 35 . Briefly, pAPN at 10 mg/mL concentration in buffer containing 20 mM Tris pH7.2 and 200 mM NaCl was crystallized by itself and then amino acid was soaked into the pAPN crystal. Crystallization of pAPN was set up with 1 µL protein solution and 1 µL well solution containing 18% PEG3350, 200 mM Li 2 SO4, and 100 mM HEPES pH7.2 mixed together in sitting drops. Crystals of pAPN were grown at 4 °C for two weeks, and were then transferred to amino acid-soaking solution containing 5 mM amino acid, 20% ethylene glycol, 25% PEG3350, 200 mM Li 2 SO4, and 100 mM HEPES pH7.2 for another two days. The crystals were flash frozen in liquid N2 and used for data collection. Data were collected at APS beamline 24-ID and ALS beamline 4.2.2. X-ray diffraction data were processed using HKL2000 36 . The structures of pAPN complexed different amino acids were determined by directly refining the model of the unliganded pAPN structure (PDB 4FKE) against the amino acid-soaked crystal data. Programs CNS 37 and CCP4 refmac 38 were used for structure refinement. Program COOT was used for model building and structural superposition 39 .