ABSTRACT
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.
Subject(s)
Inhibitor of Apoptosis Proteins/chemistry , Neuronal Apoptosis-Inhibitory Protein/chemistry , Protein Structure, Tertiary , Amino Acid Sequence , Animals , Baculoviral IAP Repeat-Containing 3 Protein , Crystallography, X-Ray , Humans , Inhibitor of Apoptosis Proteins/genetics , Models, Molecular , Molecular Sequence Data , Neuronal Apoptosis-Inhibitory Protein/genetics , Protein Structure, Tertiary/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Ubiquitin-Protein LigasesABSTRACT
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.
Subject(s)
Apoptosis Regulatory Proteins/chemistry , Membrane Proteins/chemistry , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins/chemistry , Amino Acid Sequence , Apoptosis , Bcl-2-Like Protein 11 , Crystallography, X-Ray , Humans , Minor Histocompatibility Antigens , Molecular Sequence Data , Myeloid Cell Leukemia Sequence 1 Protein , Protein Structure, SecondaryABSTRACT
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.
Subject(s)
Crystallization/methods , Crystallography/methods , Peptide Hydrolases/chemistry , Proteins/chemistry , Proteins/ultrastructure , Protein ConformationABSTRACT
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.
Subject(s)
Diacylglycerol Kinase/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Amino Acid Sequence , Animals , Binding Sites , Catalytic Domain , Cell Differentiation/physiology , Cell Proliferation , Crystallography, X-Ray , Humans , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Protein Structure, Tertiary , Structural Homology, ProteinABSTRACT
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.
