RESUMO
We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.
Assuntos
Cristalização/métodos , Cristalografia/métodos , Peptídeo Hidrolases/química , Proteínas/química , Proteínas/ultraestrutura , Conformação ProteicaRESUMO
The inhibitor of apoptosis (IAP) family of proteins contains key modulators of apoptosis and inflammation that interact with caspases through baculovirus IAP-repeat (BIR) domains. Overexpression of IAP proteins frequently occurs in cancer cells, thus counteracting the activated apoptotic program. The IAP proteins have therefore emerged as promising targets for cancer therapy. In this work, X-ray crystallography was used to determine the first structures of BIR domains from human NAIP and cIAP2. Both structures harbour an N-terminal tetrapeptide in the conserved peptide-binding groove. The structures reveal that these two proteins bind the tetrapeptides in a similar mode as do other BIR domains. Detailed interactions are described for the P1'-P4' side chains of the peptide, providing a structural basis for peptide-specific recognition. An arginine side chain in the P3' position reveals favourable interactions with its hydrophobic moiety in the binding pocket, while hydrophobic residues in the P2' and P4' pockets make similar interactions to those seen in other BIR domain-peptide complexes. The structures also reveal how a serine in the P1' position is accommodated in the binding pockets of NAIP and cIAP2. In addition to shedding light on the specificity determinants of these two proteins, the structures should now also provide a framework for future structure-based work targeting these proteins.
Assuntos
Proteínas Inibidoras de Apoptose/química , Proteína Inibidora de Apoptose Neuronal/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Proteína 3 com Repetições IAP de Baculovírus , Cristalografia por Raios X , Humanos , Proteínas Inibidoras de Apoptose/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteína Inibidora de Apoptose Neuronal/genética , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Ubiquitina-Proteína LigasesRESUMO
Evasion of apoptosis is recognized as a characteristic of malignant growth. Anti-apoptotic B-cell lymphoma-2 (Bcl-2) family members have therefore emerged as potential therapeutic targets due to their critical role in proliferating cancer cells. Here, we present the crystal structure of Bfl-1, the last anti-apoptotic Bcl-2 family member to be structurally characterized, in complex with a peptide corresponding to the BH3 region of the pro-apoptotic protein Bim. The structure reveals distinct features at the peptide-binding site, likely to define the binding specificity for pro-apoptotic proteins. Superposition of the Bfl-1:Bim complex with that of Mcl-1:Bim reveals a significant local plasticity of hydrophobic interactions contributed by the Bim peptide, likely to be the basis for the multi specificity of Bim for anti-apoptotic proteins.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Membrana/química , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas/química , Sequência de Aminoácidos , Apoptose , Proteína 11 Semelhante a Bcl-2 , Cristalografia por Raios X , Humanos , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Estrutura Secundária de ProteínaRESUMO
The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.
Assuntos
Diacilglicerol Quinase/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Diferenciação Celular/fisiologia , Proliferação de Células , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Fosfotransferases (Aceptor do Grupo Álcool)/química , Estrutura Terciária de Proteína , Homologia Estrutural de ProteínaRESUMO
The implementation of efficient technologies for the production of recombinant mammalian proteins remains an outstanding challenge in many structural and functional genomics programs. We have developed a new method for rapid identification of soluble protein expression in E. coli, based on a separation of soluble protein from inclusion bodies by a filtration step at the colony level. The colony filtration (CoFi) blot is very well suited to screen libraries, and in the present work we used it to screen a deletion mutagenesis library.