C-type inactivation is a process by which ion flux through a voltage-gated K
+
(K
v
) channel is regulated at the selectivity filter. While prior studies have indicated that C-type inactivation involves structural changes at the selectivity filter, the nature of the changes has not been resolved. Here, we report the crystal structure of the K
v
1.2 channel in a C-type inactivated state. The structure shows that C-type inactivation involves changes in the selectivity filter that disrupt the outer two ion binding sites in the filter. The changes at the selectivity filter propagate to the extracellular mouth and the turret regions of the channel pore. The structural changes observed are consistent with the functional hallmarks of C-type inactivation. This study highlights the intricate interplay between K
+
occupancy at the ion binding sites and the interactions of the selectivity filter in determining the balance between the conductive and the inactivated conformations of the filter.
Methionine
aminopeptidases (MetAPs) are essential enzymes that
make them good drug targets in cancer and microbial infections. MetAPs
remove the initiator methionine from newly synthesized peptides in
every living cell. MetAPs are broadly divided into type I and type
II classes. Both prokaryotes and eukaryotes contain type I MetAPs,
while eukaryotes have additional type II MetAP enzyme. Although several
inhibitors have been reported against type I enzymes, subclass specificity
is scarce. Here, using the fine differences in the entrance of the
active sites of MetAPs from Mycobacterium tuberculosis, Enterococcus faecalis, and human,
three hotspots have been identified and pyridinylpyrimidine-based
molecules were selected from a commercial source to target these hotspots.
In the biochemical evaluation, many of the 38 compounds displayed
differential behavior against these three enzymes. Crystal structures
of four selected inhibitors in complex with human MetAP1b and molecular
modeling studies provided the basis for the binding specificity.
It is intriguing how nature attains recognition specificity between molecular interfaces where there is no apparent scope for classical hydrogen bonding or polar interactions. Methionine aminopeptidase (MetAP) is one such enzyme where this fascinating conundrum is at play. In this study, we demonstrate that a unique C-HS hydrogen bond exists between the enzyme methionine aminopeptidase (MetAP) and its N-terminal-methionine polypeptide substrate, which allows specific interaction between apparent apolar interfaces, imposing a strict substrate recognition specificity and efficient catalysis, a feature replicated in Type I MetAPs across all kingdoms of life. We evidence this evolutionarily conserved C-HS hydrogen bond through enzyme assays on wild-type and mutant MetAP proteins from Mycobacterium tuberculosis that show a drastic difference in catalytic efficiency. The X-ray crystallographic structure of the methionine bound protein revealed a conserved water bridge and short contacts involving the Met side-chain, a feature also observed in MetAPs from other organisms. Thermal shift assays showed a remarkable 3.3 °C increase in melting temperature for methionine bound protein compared to its norleucine homolog, where C-HS interaction is absent. The presence of C-HS hydrogen bonding was also corroborated by nuclear magnetic resonance spectroscopy through a change in chemical shift. Computational chemistry studies revealed the unique role of the electrostatic environment in facilitating the C-HS interaction. The significance of this atypical hydrogen bond is underscored by the fact that the function of MetAP is essential for any living cell.
Methionine aminopeptidase (MetAP) represents a unique class of proteases that is responsible for removing the N-terminal initiator methionine from newly synthesized proteins. The lone MetAP gene in prokaryotes is essential for the survival of the microorganism suggesting that it could be used as a drug target. Here, we describe the crystal structure of the Enterococcus faecalis MetAP in the apo-form, biochemical characterization, metal affinity and small molecule library screening. Enzyme inhibition and modeling studies of the best inhibitor, 2,2 0 -bipyridine, were performed. Employing the molecular modeling tools, 2,2 0 -bipyridine derivatives were generated that could specifically inhibit class specific MetAPs.
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