title: Structural and biophysical characterization of XIAP BIR3 G306E mutant: insights in protein dynamics and application for fragment-based drug design.
abstract: Previous reports describe modulators of X-linked inhibitor of apoptosis (XIAP)-caspase interaction designed from the AVPI N-terminal peptide sequence of second mitochondria-derived activator of caspase. A fragment-based drug design strategy was initiated to identify therapeutic non-peptidomimetic antagonists of X-linked inhibitor of apoptosis protein-protein interactions. Fragments that bind to the AVPI binding site of BIR3 (bacculoviral inhibitory repeat) were identified, and to further localize the fragment binding within the AVPI binding site, a point mutation was designed which alters the dynamics of flexible loops and blocks PI region of the binding cleft, thus enabling definition of weakly bound small molecules in the AV portion of the binding cleft. Nuclear magnetic resonance analysis confirmed the G306E mutation stabilizes the AV pocket. Biophysical characterization of the mutant confirms conformation change within the PI sub-pocket as evidenced by a significant diminishment in binding affinity of AVPI mimetics, yet the binding affinity of the smaller AV mimetics is maintained or slightly improved in the mutant compared with wild-type. Additional data from non-covalent mass spectrometry analysis shows enhanced binding of AV mimetics to the G306E mutant over the wild-type. The presented data outline a protein engineering strategy that allowed mapping of AV-replacements with better sensitivity and precision.
authors: Department of Chemistry, Pfizer Global Research and Development, La Jolla Laboratories, San Diego, CA 92121, USA. cathy.d.moore@pfizer.com
authors: Cathy D
authors: CD
authors: Moore
authors: Haihong
authors: H
authors: Wu
authors: Ben
authors: B
authors: Bolaños
authors: Simon
authors: S
authors: Bergqvist
authors: Alexei
authors: A
authors: Brooun
authors: Tom
authors: T
authors: Pauly
authors: Dawn
authors: D
authors: Nowlin
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D020169:Caspases / Q000378:metabolism; D003198:Computer Simulation; D015195:Drug Design; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013816:Thermodynamics; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; D020169:Caspases