The Enolization Chemistry of a Thioester-Dependent Racemase: The 1.4 Å Crystal Structure of a Reaction Intermediate Complex Characterized by Detailed QM/MM Calculations
In the active site of the bacterial α-methylacyl-CoA racemase of Mycobacterium tuberculosis (MCR), the chirality of the 2-methyl branched C2-atom is interconverted between (S) and (R) isomers. Protein crystallographic data and quantum mechanics/molecular mechanics (QM/MM) computational approaches show that this interconversion is achieved via a planar enolate intermediate. The crystal structure, at 1.4 Å, shows the mode of binding of a reaction intermediate analog, 2-methylacetoacetyl-CoA, in a well-defined planar enolate form. Computational studies have confirmed that in the conversion from (S) to (R), first a proton is abstracted by Nδ1 (His126), and subsequently the planar enolate form is reprotonated by Oδ2 (Asp156). The calculations also show that the negatively charged thioester oxygen of the enolate intermediate is stabilized by an oxyanion hole formed by N (Asp127), as well as by the side-chain atoms of the catalytic residues, Asp156 and His126, both being protonated simultaneously, at the intermediate stage of the catalytic cycle. Computational analysis also revealed that the conversion of (S)- to (R)-chirality is achieved by a movement of 1.7 Å of the chiral C2-carbon, with smaller shifts (approximately 1 Å) of the carbon atom of the 2-methyl group, the C3-atom of the fatty acid tail, and the C1-carbon and O1-oxygen atoms of the thioester moiety (Sharma et al., 2012).
Proteome-wide analysis of lysine acetylation suggests its broad regulatory scope in Saccharomyces cerevisiae
Posttranslational modification of proteins by lysine acetylation plays important regulatory roles in living cells. The budding yeast Saccharomyces cerevisiae is a widely used unicellular eukaryotic model organism in biomedical research. S. cerevisiae contains several evolutionarily conserved lysine acetyltransferases and deacetylases. However, only a few dozen acetylation sites in S. cerevisiae are known, presenting a major obstacle for further understanding the regulatory roles of acetylation in this organism. Here we used high resolution mass spectrometry to identify about 4,000 lysine acetylation sites in S. cerevisiae. Acetylated proteins are implicated in the regulation of diverse cytoplasmic and nuclear processes including chromatin organization, mitochondrial metabolism, and protein synthesis. Bioinformatic analysis of yeast acetylation sites shows that acetylated lysines are significantly more conserved compared with non-acetylated lysines. A large fraction of the conserved acetylation sites are present on proteins involved in cellular metabolism, protein synthesis, and protein folding. Furthermore, quantification of the Rpd3-regulated acetylation sites allowed us to identify several previously known, as well as new putative substrates of this deacetylase. Rpd3 deficiency increased acetylation of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex subunit Sgf73 on K33. This acetylation site is located within a critical regulatory domain in Sgf73 that interacts with Ubp8 and is involved in the activation of the Ubp8-containing histone H2B deubiquitylase complex. Our data represent the first global survey of acetylation in budding yeast, and suggest a wide-ranging regulatory scope of this modification. The provided dataset may serve as an important resource for functional analysis of lysine acetylation in eukaryotes (Henriksen et al., 2012).
An Atomistic Model for Assembly of the Transmembrane Domain of the T-cell Receptor Complex
The T-cell receptor (TCR), together with accessory Cluster of differentiation 3 (CD3) molecules (TCR-CD3 complex), is a key component in the primary function of T-cells. The nature of association of the transmembrane domains is of central importance to the assembly of the complex and is largely unknown. Using multi-scale molecular modeling and simulations, we have investigated the structure and assembly of the TCRa-CD3e-CD3d transmembrane domains both in membrane and micelle environments. We demonstrate that in a membrane environment the transmembrane basic residue of the TCR closely interacts with both of the transmembrane acidic residues of the CD3 dimer. In contrast, in a micelle the basic residue interacts with only one of the acidic residues. Simulations of a recent micellar nuclear magnetic resonance structure of the natural killer (NK) cell-activating NKG2C-DAP12-DAP12 trimer in a membrane further indicate that the environment significantly affects the way these trimers associate. Since the currently accepted model for transmembrane association is entirely based on a micellar structure, we propose a revised model for the association of transmembrane domains of activating immune receptors in a membrane environment (Sharma and Juffer, 2013).
Last updated: 28.10.2016