We target the gatekeeper MET146 of c-Jun N-terminal kinase 3 (JNK3) to exemplify the applicability of X···S halogen bonds in molecular design using computational, synthetic, structural and biophysical techniques. In a designed series of aminopyrimidine-based inhibitors, we unexpectedly encounter a plateau of affinity. Compared to their QM-calculated interaction energies, particularly bromine and iodine fail to reach the full potential according to the size of their σ-hole. Instead, mutation of the gatekeeper residue into leucine, alanine, or threonine reveals that the heavier halides can significantly influence selectivity in the human kinome. Thus, we demonstrate that, although the choice of halogen may not always increase affinity, it can still be relevant for inducing selectivity. Determining the crystal structure of the iodine derivative in complex with JNK3 (4X21) reveals an unusual bivalent halogen/chalcogen bond donated by the ligand and the back-pocket residue MET115. Incipient repulsion from the too short halogen bond increases the flexibility of Cε of MET146, whereas the rest of the residue fails to adapt being fixed by the chalcogen bond. This effect can be useful to induce selectivity, as the necessary combination of methionine residues only occurs in 9.3% of human kinases, while methionine is the predominant gatekeeper (39%).

Background: Autolysins ensure the plasticity of bacterial cell walls, and deletion leads to impaired cell clusters. Results: High resolution structures of Staphylococcus aureus amidase AmiA shed light on peptidoglycan binding and cleavage. Conclusion: AmiA distinguishes peptidoglycan mostly by the peptide, and cleavage is facilitated by a zinc-activated water molecule. Significance: These structures will inform strategies to develop new therapeutics against MRSA.

To orchestrate a complex life style in changing environments, the filamentous cyanobacterium Nostoc punctiforme facilitates communication between neighboring cells through septal junction complexes. This is achieved by nanopores that perforate the peptidoglycan (PGN) layer and traverse the cell septa. The N‐acetylmuramoyl‐l‐alanine amidase AmiC2 (Npun_F1846; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/5/1/28.html) in N. punctiforme generates arrays of such nanopores in the septal PGN, in contrast to homologous amidases that mediate daughter cell separation after cell division in unicellular bacteria. Nanopore formation is therefore a novel property of AmiC homologs. Immunofluorescence shows that native AmiC2 localizes to the maturing septum. The high‐resolution crystal structure (1.12 Å) of its catalytic domain (AmiC2‐cat) differs significantly from known structures of cell splitting and PGN recycling amidases. A wide and shallow binding cavity allows easy access of the substrate to the active site, which harbors an essential zinc ion. AmiC2‐cat exhibits strong hydrolytic activity in vitro. A single point mutation of a conserved glutamate near the zinc ion results in total loss of activity, whereas zinc removal leads to instability of AmiC2‐cat. An inhibitory α‐helix, as found in the Escherichia coli AmiCE. coli structure, is absent. Taken together, our data provide insight into the cell‐biological, biochemical and structural properties of an unusual cell wall lytic enzyme that generates nanopores for cell–cell communication in multicellular cyanobacteria. The novel structural features of the catalytic domain and the unique biological function of AmiC2 hint at mechanisms of action and regulation that are distinct from other amidases. Database The AmiC2‐cat structure has been deposited in the Protein Data Bank under accession number http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5EMI.

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