No abstract

The emerging view of Nε-lysine acetylation in eukaryotes is of a relatively abundant post-translational modification (PTM) that has a major impact on the function, structure, stability and/or location of thousands of proteins involved in diverse cellular processes. This PTM is typically considered to arise by the donation of the acetyl group from acetyl-coenzyme A (acCoA) to the ε-amino group of a lysine residue that is reversibly catalyzed by lysine acetyltransferases and deacetylases. Here, we provide genetic, mass spectrometric, biochemical and structural evidence that Nε-lysine acetylation is an equally abundant and important PTM in bacteria. Applying a recently developed, label-free and global mass spectrometric approach to an isogenic set of mutants, we detected acetylation of thousands of lysine residues on hundreds of Escherichia coli proteins that participate in diverse and often essential cellular processes, including translation, transcription and central metabolism. Many of these acetylations were regulated in an acetyl phosphate (acP)-dependent manner, providing compelling evidence for a recently reported mechanism of bacterial Nε-lysine acetylation. These mass spectrometric data, coupled with observations made by crystallography, biochemistry, and additional mass spectrometry showed that this acP-dependent acetylation is both non-enzymatic and specific, with specificity determined by the accessibility, reactivity and three-dimensional microenvironment of the target lysine. Crystallographic evidence shows acP can bind to proteins in active sites and cofactor binding sites, but also potentially anywhere molecules with a phosphate moiety could bind. Finally, we provide evidence that acP-dependent acetylation can impact the function of critical enzymes, including glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase, and RNA polymerase.

The minor groove hydration spine is a key feature of the crystal structure of the B-DNA dodecamer duplex [d(CGCGAATTCGCG)]2. At the floor of the groove, water molecules bridge bases from opposite strands by hydrogen bonding to N3 and O2 atoms of adenine and thymine, respectively. However, the interpretation that the series of electron density peaks lining the groove represents indeed water molecules, while generally agreed upon, remains an assumption. The limited resolutions of dodecamer crystal structures have thus far made it impossible to reliably distinguish between water and monovalent metal cations, such as Na+, normally present in the crystallization buffer. Using X-ray diffraction data to near-atomic resolution of dodecamer crystals grown in the presence of either Rb+ or Cs+ cacodylate, we have tested the possibility of alkali metal ion coordination in the minor groove. The structural data are consistent with a single Rb+ intruding the hydration spine at the central ApT step. The ion has partial occupancy and replaces the water molecule that links the keto oxygens of thymines from opposite strands. The observed dimensions of the binding site suggest preferred binding of Rb+ or K+, while Na+ or Cs+ may be prevented from binding stably. Therefore, minor groove ion coordination appears to be an isolated event, highly sequence dependent and unlikely to significantly affect the particular geometry of the A-tract in the Dickerson−Drew dodecamer. In addition to allowing a distinction between water and alkali metal ions, the high-resolution crystal structures provide a more complete picture of the minor groove water structure:  four fused water hexagons dissect the central portion of the minor groove, with the inner corners of the hexagons coinciding with the original spine water positions. Thus, it may be more appropriate to refer to this arrangement as a ribbon of hydration instead of a spine of hydration.

Although TEM-1 beta-lactamase is among the best studied enzymes, its acylation mechanism remains controversial. To investigate this problem, the structure of TEM-1 in complex with an acylation transition-state analogue was determined at ultrahigh resolution (0.85 A) by X-ray crystallography. The quality of the data was such as to allow for refinement to an R-factor of 9.1% and an R(free) of 11.2%. In the resulting structure, the electron density features were clear enough to differentiate between single and double bonds in carboxylate groups, to identify multiple conformations that are occupied by residues and loops, and to assign 70% of the protons in the protein. Unexpectedly, even at pH 8.0 where the protein was crystallized, the active site residue Glu166 is clearly protonated. This supports the hypothesis that Glu166 is the general base in the acylation half of the reaction cycle. This structure suggests that Glu166 acts through the catalytic water to activate Ser70 for nucleophilic attack on the beta-lactam ring of the substrate. The hydrolytic mechanism of class A beta-lactamases, such as TEM-1, appears to be symmetrical, as are the serine proteases. Apart from its mechanistic implications, this atomic resolution structure affords an unusually detailed view of the structure, dynamics, and hydrogen-bonding networks of TEM-1, which may be useful for the design of inhibitors against this key antibiotic resistance target.

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