NDM-1 Zn1-binding residue His116 plays critical roles in antibiotic hydrolysis.

Bacteria expressing NDM-1 have been labeled as superbugs because it confers upon them resistance to a broad range of ß-lactam antibiotics. The enzyme has a di­zinc active centre, with the Zn2 site extensively studied. The roles of active-site Zn1 ligand residues are, however, still not fully understood. We carried out structure-function studies using the mutants, H116A, H116N, and H116Q. Zinc content analysis showed that Zn1 binding was weakened by 40 to 60% in the H116 mutants. The enzymatic-activity studies showed that the lower hydrolysis rates were mainly caused by their weaker substrate binding. The catalytic efficiency (kcat/Km) of the mutants followed the order: WT > > H116Q (decreased by 4-20 fold) > H116A (decreased by 20-700 fold) ≥ H116N (decreased by 6-800 fold). The maximum effect was observed on H116N against penicillin G, whereas ampicillin was not hydrolyzed at all. The fold-increase of Km values, which informs the weakening of substrate binding, were: H116A by 5-45 fold; H116N by 6-100 fold; H116Q by 2-10 fold. Molecular dynamics simulations suggested that the Zn1 site mutations affected the positions of Zn2 and the bridging hydroxide, by 0.8 to 1.2 Å, with the largest changes of ~1.5 Å observed on Zn2 ligand C221. A native hydrogen bond between H118 and D236 was disrupted in the H116N and H116Q mutants, which led to increased flexibility of loop 10. Consequently, residue N233 was no longer maintained at an optimal position for substrate binding. H116 connected loop 7 across Zn1 to loop 10, thereby contributed to the overall integrity. This work revealed that the H116-Zn1 interaction plays a critical role in defining the substrate-binding site. From these results, it can be inferred that inhibition strategies targeting the zinc ions may be a new direction for drug development.

Discovery of FtsZ inhibitors by virtual screening as antibacterial agents and study of the inhibition mechanism.

Inhibition of bacterial cell division is a novel mechanistic action in the development of new antimicrobial agents. The FtsZ protein is an important antimicrobial drug target because of its essential role in bacterial cell division. In the present study, potential inhibitors of FtsZ were identified by virtual screening followed by in vivo and in vitro bioassays. One of the candidates, Dacomitinib (S2727), shows for the first time its potent inhibitory activity against the MRSA strains. The binding mode of Dacomitinib in FtsZ was analyzed by docking, and Asp199 and Thr265 are thought to be essential residues involved in the interactions.

'Light up' protein-protein interaction through bioorthogonal incorporation of a turn-on fluorescent probe into ß-lactamase.

Fluorescent labeling of biomacromolecules to 'light up' biological events through non-invasive methods is of great importance, but is still challenging in terms of fluorophore properties and the labeling methods used. Herein, we designed and synthesized a biocompatible and conformation sensitive tetraphenylethene derivative EPB with aggregation induced emission (AIE) properties. By introducing EPB into TEM-1 ß-lactamase (TEM-1 Bla) through a two-step approach, a conformation-dependent fluorescent sensor EPB104-Bla was genetically engineered, which was applied to monitor the protein-protein interaction (PPI) with ß-lactamase inhibitor protein (BLIP). The fluorescence signal of EPB104-Bla increases by an approximately 5-fold upon binding to BLIP, indicating that EPB-104 Bla is capable of lighting up the PPI. The dissociation constant (Kd) between EPB104-Bla and BLIP was estimated to be 0.6 µM, which is consistent with that derived from the kinetic inhibition assay. This study demonstrates that genetic modification of proteins with AIE probes might open up new opportunities to develop biosensors in PPI analysis.

Caught in the act: covalent cross-linking captures activator-coactivator interactions in vivo.

Currently there are few methods suitable for the discovery and characterization of transient, moderate affinity protein-protein interactions in their native environment, despite their prominent role in a host of cellular functions including protein folding, signal transduction, and transcriptional activation. Here we demonstrate that a genetically encoded photoactivatable amino acid, p-benzoyl-l-phenylalanine, can be used to capture transient and/or low affinity binding partners in an in vivo setting. In this study, we focused on ensnaring the coactivator binding partners of the transcriptional activator VP16 in S. cerevisiae. The interactions between transcriptional activators and coactivators in eukaryotes are moderate in affinity and short-lived, and due in part to these characteristics, identification of the direct binding partners of activators in vivo has met with only limited success. We find through in vivo photo-cross-linking that VP16 contacts the Swi/Snf chromatin-remodeling complex through the ATPase Snf2(BRG1/BRM) and the subunit Snf5 with two distinct regions of the activation domain. An analogous experiment with Gal4 reveals that Snf2 is also a target of this activator. These results suggest that Snf2 may be a valuable target for small molecule probe discovery given the prominent role the Swi/Snf complex family plays in development and in disease. More significantly, the successful implementation of the in vivo cross-linking methodology in this setting demonstrates that it can be applied to the discovery and characterization of a broad range of transient and/or modest affinity protein-protein interactions.