title: Structural basis for bivalent Smac-mimetics recognition in the IAP protein family.
abstract: XIAP is an apoptotic regulator protein that binds to the effector caspases -3 and -7 through its BIR2 domain, and to initiator caspase-9 through its BIR3 domain. Molecular docking studies suggested that Smac-DIABLO may antagonize XIAP by concurrently targeting both BIR2 and BIR3 domains; on this basis bivalent Smac-mimetic compounds have been proposed and characterized. Here, we report the X-ray crystal structure of XIAP-BIR3 domain in complex with a two-headed compound (compound 3) with improved efficacy relative to its monomeric form. A small-angle X-ray scattering study of XIAP-BIR2BIR3, together with fluorescence polarization binding assays and compound 3 cytotoxicity tests on HL60 leukemia cell line are also reported. The crystal structure analysis reveals a network of interactions supporting XIAP-BIR3/compound 3 recognition; moreover, analytical gel-filtration chromatography shows that compound 3 forms a 1:1 stoichiometric complex with a XIAP protein construct containing both BIR2 and BIR3 domains. On the basis of the crystal structure and small-angle X-ray scattering, a model of the same BIR2-BIR3 construct bound to compound 3 is proposed, shedding light on the ability of compound 3 to relieve XIAP inhibitory effects on caspase-9 as well as caspases -3 and -7. A molecular modeling/docking analysis of compound 3 bound to cIAP1-BIR3 domain is presented, considering that Smac-mimetics have been shown to kill tumor cells by inducing cIAP1 and cIAP2 ubiquitination and degradation. Taken together, the results reported here provide a rationale for further development of compound 3 as a lead in the design of dimeric Smac mimetics for cancer treatment.
authors.affiliation: Department of Biomolecular Sciences and Biotechnology, University of Milano, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Cossu
authors.forename: Mario
authors.initials: M
authors.lastname: Milani
authors.forename: Eloise
authors.initials: E
authors.lastname: Mastrangelo
authors.forename: Patrice
authors.initials: P
authors.lastname: Vachette
authors.forename: Federica
authors.initials: F
authors.lastname: Servida
authors.forename: Daniele
authors.initials: D
authors.lastname: Lecis
authors.forename: Giulia
authors.initials: G
authors.lastname: Canevari
authors.forename: Domenico
authors.initials: D
authors.lastname: Delia
authors.forename: Carmelo
authors.initials: C
authors.lastname: Drago
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Rizzo
authors.forename: Leonardo
authors.initials: L
authors.lastname: Manzoni
authors.forename: Pierfausto
authors.initials: P
authors.lastname: Seneci
authors.forename: Carlo
authors.initials: C
authors.lastname: Scolastico
authors.forename: Martino
authors.initials: M
authors.lastname: Bolognesi
mesh_terms: D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins; D002460:Cell Line; D018360:Crystallography, X-Ray; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D024101:Mitochondrial Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human