title: Structure of wild-type Plk-1 kinase domain in complex with a selective DARPin.
abstract: As a key regulator of mitosis, the Ser/Thr protein polo-like kinase-1 (Plk-1) is a well validated drug target in cancer therapy. In order to enable structure-guided drug design, determination of the crystal structure of the kinase domain of Plk-1 was attempted. Using a multi-parallel cloning and expression approach, a set of length variants were identified which could be expressed in large amounts from insect cells and which could be purified to high purity. However, all attempts to crystallize these constructs failed. Crystals were ultimately obtained by generating designed ankyrin-repeat proteins (DARPins) selective for Plk-1 and using them for cocrystallization. Here, the first crystal structure of the kinase domain of wild-type apo Plk-1, in complex with DARPin 3H10, is presented, underlining the power of selective DARPins as crystallization tools. The structure was refined to 2.3 A resolution and shows the active conformation of Plk-1. It broadens the basis for modelling and cocrystallization studies for drug design. The binding epitope of 3H10 is rich in arginine, glutamine and lysine residues, suggesting that the DARPin enabled crystallization by masking a surface patch which is unfavourable for crystal contact formation. Based on the packing observed in the crystal, a truncated DARPin variant was designed which showed improved binding characteristics.
authors.affiliation: Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780 Oeiras, Portugal.
authors.forename: Tiago M
authors.initials: TM
authors.lastname: Bandeiras
authors.forename: Roman Christian
authors.initials: RC
authors.lastname: Hillig
authors.forename: Pedro M
authors.initials: PM
authors.lastname: Matias
authors.forename: Uwe
authors.initials: U
authors.lastname: Eberspaecher
authors.forename: Jörg
authors.initials: J
authors.lastname: Fanghänel
authors.forename: Mónica
authors.initials: M
authors.lastname: Thomaz
authors.forename: Sandra
authors.initials: S
authors.lastname: Miranda
authors.forename: Kerstin
authors.initials: K
authors.lastname: Crusius
authors.forename: Vera
authors.initials: V
authors.lastname: Pütter
authors.forename: Patrick
authors.initials: P
authors.lastname: Amstutz
authors.forename: Maya
authors.initials: M
authors.lastname: Gulotti-Georgieva
authors.forename: H Kaspar
authors.initials: HK
authors.lastname: Binz
authors.forename: Caterina
authors.initials: C
authors.lastname: Holz
authors.forename: Arndt A P
authors.initials: AA
authors.lastname: Schmitz
authors.forename: Christine
authors.initials: C
authors.lastname: Lang
authors.forename: Peter
authors.initials: P
authors.lastname: Donner
authors.forename: Ursula
authors.initials: U
authors.lastname: Egner
authors.forename: Maria A
authors.initials: MA
authors.lastname: Carrondo
authors.forename: Beate
authors.initials: B
authors.lastname: Müller-Tiemann
mesh_terms: D000595:Amino Acid Sequence; D017487:Ankyrins / Q000737:chemistry*; D002151:Calorimetry; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D003460:Crystallization; D003625:Data Collection; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D000097869:Polo-Like Kinase 1
chemical_list: D017487:Ankyrins; D018797:Cell Cycle Proteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D000097869:Polo-Like Kinase 1