Article

In situ proteolysis for protein crystallization and structure determination.

Structural Genomics Consortium, University of Toronto, 100 College Street, Toronto, Ontario M5G 1L5, Canada.
Nature Methods (Impact Factor: 25.95). 01/2008; 4(12):1019-21. DOI: 10.1038/nmeth1118
Source: PubMed

ABSTRACT 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.

Full-text

Available from: Wael Rabeh, May 30, 2015
1 Follower
 · 
442 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s: zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the 2-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1, but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and, for P450 17A2, with Prog. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ~50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternate ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 12/2014; 290(6). DOI:10.1074/jbc.M114.627265 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Gram-negative bacterium enteropathogenic Escherichia coli (EPEC) uses a syringe-like type III secretion system (T3SS) to inject virulence or effector proteins into the cytoplasm of host intestinal epithelial cells. In order to assemble, the T3SS must traverse both bacterial membranes, as well as the peptidoglycan layer. Peptidoglycan is made of repeating N-acetylmuramic acid and N-acetyl glucosamine disaccharides crosslinked by pentapeptides to form a tight mesh barrier. Assembly of many macromolecular machines requires a dedicated peptidoglycan lytic enzyme (PG-lytic enzyme) to locally clear peptidoglycan. Here we have solved the first structure of a T3SS-associated PG-lytic enzyme, EtgA from EPEC. Unexpectedly, the active site of EtgA has features in common with both lytic transglycosylases and hen egg-white lysozyme (HEWL). Most notably, the β-hairpin region resembles that of lysozyme, and contains an aspartate that aligns with lysozyme Asp52 (a residue critical for catalysis), a conservation not observed in other previously characterized lytic transglycosylase families which the conserved T3SS enzymes had been presumed to belong to. Mutation of the EtgA catalytic glutamate, Glu 42, conserved across lytic transglycosylases and HEWL, and this differentiating aspartate diminishes type III secretion in vivo, supporting its essential role in clearing the peptidoglycan for T3SS assembly. Finally, we show that EtgA forms a 1:1 complex with the building block of the polymerized T3SS inner rod component, EscI, and that this interaction enhances PG-lytic activity of EtgA in vitro, collectively providing the necessary strict localization and regulation of the lytic activity to prevent overall cell lysis. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 02/2015; 290(16). DOI:10.1074/jbc.M115.639013 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A major bottleneck in structural, biochemical and biophysical studies of proteins is the need for large amounts of pure homogenous material, which is generally obtained by recombinant overexpression. Here we introduce a vector collection, the pCri System, for cytoplasmic and periplasmic/extracellular expression of heterologous proteins that allows the simultaneous assessment of prokaryotic and eukaryotic host cells (Escherichia coli, Bacillus subtilis, and Pichia pastoris). By using a single polymerase chain reaction product, genes of interest can be directionally cloned in all vectors within four different rare restriction sites at the 5'end and multiple cloning sites at the 3'end. In this way, a number of different fusion tags but also signal peptides can be incorporated at the N- and C-terminus of proteins, facilitating their expression, solubility and subsequent detection and purification. Fusion tags can be efficiently removed by treatment with site-specific peptidases, such as tobacco etch virus proteinase, thrombin, or sentrin specific peptidase 1, which leave only a few extra residues at the N-terminus of the protein. The combination of different expression systems in concert with the cloning approach in vectors that can fuse various tags makes the pCri System a valuable tool for high throughput studies.
    PLoS ONE 11/2014; 9(11):e112643. DOI:10.1371/journal.pone.0112643 · 3.53 Impact Factor