RESUMO
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.
Assuntos
Anquirinas/química , Proteínas de Ciclo Celular/química , Proteínas Serina-Treonina Quinases/química , Proteínas Proto-Oncogênicas/química , Sequência de Aminoácidos , Calorimetria , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/isolamento & purificação , Clonagem Molecular , Cristalização , Coleta de Dados , Humanos , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/isolamento & purificação , Proteínas Recombinantes/química , Quinase 1 Polo-LikeRESUMO
This study describes an efficient multiparallel automated workflow of cloning, expression, purification, and crystallization of a large set of construct variants for isolated protein domains aimed at structure determination by X-ray crystallography. This methodology is applied to MAPKAP kinase 2, a key enzyme in the inflammation pathway and thus an attractive drug target. The study reveals a distinct subset of truncation variants with improved crystallization properties. These constructs distinguish themselves by increased solubility and stability during a parallel automated multistep purification process including removal of the recombinant tag. High-throughput protein melting point analysis characterizes this subset of constructs as particularly thermostable. Both parallel purification screening and melting point determination clearly identify residue 364 as the optimal C terminus for the kinase domain. Moreover, all three constructs that ultimately crystallized feature this C terminus. At the N terminus, only three amino acids differentiate a noncrystallizing from a crystallizing construct. This study addresses the very common issues associated with difficult to crystallize proteins, those of solubility and stability, and the crucial importance of particular residues in the formation of crystal contacts. A methodology is suggested that includes biophysical measurements to efficiently identify and produce construct variants of isolated protein domains which exhibit higher crystallization propensity.
Assuntos
Cristalização/métodos , Variação Genética/fisiologia , Proteínas Quinases/química , Proteínas Quinases/genética , Clonagem Molecular , Estabilidade Enzimática , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Moleculares , Proteínas Mutantes/química , Conformação Proteica , Desnaturação Proteica , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes/química , TemperaturaRESUMO
The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.
Assuntos
Cromatografia de Afinidade/métodos , Epitopos/química , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/isolamento & purificação , Fator de Crescimento Transformador alfa/genética , Motivos de Aminoácidos/genética , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Células Cultivadas , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Vetores Genéticos/síntese química , Humanos , Imunoprecipitação/métodos , Insetos , Fragmentos de Peptídeos/genética , Fosfotransferases/genética , Fosfotransferases/isolamento & purificação , Fosfotransferases/metabolismo , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/imunologia , Fator de Crescimento Transformador alfa/química , Fator de Crescimento Transformador alfa/metabolismoRESUMO
The crystal structure of a mutant androgen receptor (AR) ligand-binding domain (LBD) in complex with the agonist 9alpha-fluorocortisol has been determined at 1.95 A resolution. This mutant AR contains two mutations (L701H and T877A) and was previously reported as a high-affinity cortisol/cortisone responsive AR (AR(ccr)) isolated from the androgen-independent human prostate cancer cell lines MDA PCa 2a and 2b (Zhao et al. Nature Med. 2000, 6, 703-6). The three-dimensional structure of the AR(ccr) LBD complexed with 9alpha-fluorocortisol shows the typical conformation of an agonist-bound nuclear receptor in which helix 12 is precisely positioned as a "lid" for the ligand-binding pocket. Binding of 9alpha-fluorocortisol to the AR(ccr) involves favorable hydrogen bond patterns on the C17 and C21 substituents of the ligand due to the mutations at 701 and 877 in the AR(ccr). Our studies provide the first structural explanation for the glucocorticoid activation of AR(ccr), which is important for the development of new therapeutic treatments for androgen-independent prostate cancer.