Structural, Biochemical, and In Vivo Characterization of MtrR-Mediated Resistance to Innate Antimicrobials by the Human Pathogen Neisseria gonorrhoeae.

Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE-encoded multidrug efflux pump, which expels microbicides, such as bile salts, fatty acids, and multiple extrinsically administered drugs, from the cell. In the absence of these cytotoxins, the TetR family member MtrR represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observed electron density that we hypothesized was N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interactions, both in vitro and in vivo, revealed that these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate but not taurodeoxycholate, suggesting different binding modes of the bile salts. These data provide insight into a crucial mechanism utilized by the pathogen to overcome innate human defenses.IMPORTANCENeisseria gonorrhoeae causes a significant disease burden worldwide, and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. The multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage on the pathogen within the host. Transcription of the pump is repressed by MtrR but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials.

Co-crystal structures of PKG Iß (92-227) with cGMP and cAMP reveal the molecular details of cyclic-nucleotide binding.

BACKGROUND: Cyclic GMP-dependent protein kinases (PKGs) are central mediators of the NO-cGMP signaling pathway and phosphorylate downstream substrates that are crucial for regulating smooth muscle tone, platelet activation, nociception and memory formation. As one of the main receptors for cGMP, PKGs mediate most of the effects of cGMP elevating drugs, such as nitric oxide-releasing agents and phosphodiesterase inhibitors which are used for the treatment of angina pectoris and erectile dysfunction, respectively. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the mechanism of cyclic nucleotide binding to PKG by determining crystal structures of the amino-terminal cyclic nucleotide-binding domain (CNBD-A) of human PKG I bound to either cGMP or cAMP. We also determined the structure of CNBD-A in the absence of bound nucleotide. The crystal structures of CNBD-A with bound cAMP or cGMP reveal that cAMP binds in either syn or anti configurations whereas cGMP binds only in a syn configuration, with a conserved threonine residue anchoring both cyclic phosphate and guanine moieties. The structure of CNBD-A in the absence of bound cyclic nucleotide was similar to that of the cyclic nucleotide bound structures. Surprisingly, isothermal titration calorimetry experiments demonstrated that CNBD-A binds both cGMP and cAMP with a relatively high affinity, showing an approximately two-fold preference for cGMP. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that CNBD-A binds cGMP in the syn conformation through its interaction with Thr193 and an unusual cis-peptide forming residues Leu172 and Cys173. Although these studies provide the first structural insights into cyclic nucleotide binding to PKG, our ITC results show only a two-fold preference for cGMP, indicating that other domains are required for the previously reported cyclic nucleotide selectivity.