Three Dimensional Structure of the MqsR:MqsA Complex: A Novel TA Pair Comprised of a Toxin Homologous to RelE and an Antitoxin with Unique Properties

One mechanism by which bacteria survive environmental stress is through the formation of bacterial persisters, a sub-population of genetically identical quiescent cells that exhibit multidrug tolerance and are highly enriched in bacterial toxins. Recently, the Escherichia coli gene mqsR (b3022) was identified as the gene most highly upregulated in persisters. Here, we report multiple individual and complex three-dimensional structures of MqsR and its antitoxin MqsA (B3021), which reveal that MqsR:MqsA form a novel toxin:antitoxin (TA) pair. MqsR adopts an α/β fold that is homologous with the RelE/YoeB family of bacterial ribonuclease toxins. MqsA is an elongated dimer that neutralizes MqsR toxicity. As expected for a TA pair, MqsA binds its own promoter. Unexpectedly, it also binds the promoters of genes important for E. coli physiology (e.g., mcbR, spy). Unlike canonical antitoxins, MqsA is also structured throughout its entire sequence, binds zinc and coordinates DNA via its C- and not N-terminal domain. These studies reveal that TA systems, especially the antitoxins, are significantly more diverse than previously recognized and provide new insights into the role of toxins in maintaining the persister state

The structure of the RCAN1:CN complex explains the inhibition of and substrate recruitment by calcineurin

Regulator of calcineurin 1 (RCAN1) is an endogenous inhibitor of the Ser/Thr phosphatase calcineurin (CN). It has been shown that excessive inhibition of CN is a critical factor for Down syndrome and Alzheimer's disease. Here, we determined RCAN1's mode of action. Using a combination of structural, biophysical, and biochemical studies, we show that RCAN1 inhibits CN via multiple routes: first, by blocking essential substrate recruitment sites and, second, by blocking the CN active site using two distinct mechanisms. We also show that phosphorylation either inhibits RCAN1-CN assembly or converts RCAN1 into a weak inhibitor, which can be reversed by CN via dephosphorylation. This highlights the interplay between posttranslational modifications in regulating CN activity. Last, this work advances our understanding of how active site inhibition of CN can be achieved in a highly specific manner. Together, these data provide the necessary road map for targeting multiple neurological disorders.National Institute of General Medical SciencesOpen access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape

Structural and functional analysis of the NLRP4 pyrin domain

NLRP4 is a member of the nucleotide-binding and leucine-rich repeat receptor (NLR) family of cytosolic receptors and a member of an inflammation signaling cascade. Here, we present the crystal structure of the NLRP4 pyrin domain (PYD) at 2.3 Å resolution. The NLRP4 PYD is a member of the death domain (DD) superfamily and adopts a DD fold consisting of six α-helices tightly packed around a hydrophobic core, with a highly charged surface that is typical of PYDs. Importantly, however, we identified several differences between the NLRP4 PYD crystal structure and other PYD structures that are significant enough to affect NLRP4 function and its interactions with binding partners. Notably, the length of helix α3 and the α2−α3 connecting loop in the NLRP4 PYD are unique among PYDs. The apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein whose interactions with a number of distinct PYDs are believed to be critical for activation of the inflammatory response. Here, we use co-immunoprecipitation, yeast two-hybrid, and nuclear magnetic resonance chemical shift perturbation analysis to demonstrate that, despite being important for activation of the inflammatory response and sharing several similarities with other known ASC-interacting PYDs (i.e., ASC2), NLRP4 does not interact with the adaptor protein ASC. Thus, we propose that the factors governing homotypic PYD interactions are more complex than the currently accepted model, which states that complementary charged surfaces are the main determinants of PYD–PYD interaction specificity

The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin

<div><p>Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca²⁺/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, “LxVP.” The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that “LxVP” sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent “LxVP”-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.</p> </div

Potential interaction modes of CN substrates/regulators with CN.

<p>(A) The CN-RII peptide complex obtained by MD. Colors as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio-1001492-g002" target="_blank">Figure 2A, C</a>. CN is shown in surface representation and the RII peptide in dark green with the LxVP motif (LDVP) and phospho-Ser95 as green sticks. LDVP is bound to the LxVP binding pocket (light green), and phospho-Ser95 is bound in the CN active site (cyan). (B) Electrostatic interactions between CN and the RII peptide. The CN electrostatic surface has positively and negatively charged areas colored blue and red, respectively. The LxVP motif and residues in RII that participate in polar interactions with CN are shown as green sticks. (C) Features of selected CN substrates and regulators, including substrates tested in this work (NFAT, Crz1, and the RII peptide). PxIxIT and LxVP motifs are highlighted in yellow and green, respectively, with intervening residues in grey. Regions containing S-T residues that are dephosphorylated by CN are pink. (D) Potential modes of interaction of CN with various binding partners. CN is shown in grey, with the active site in cyan, the PxIxIT docking site in yellow, and the LxVP docking site in green. CN binding partners are shown in blue, with PxIxIT and LxVP motifs in purple and phosphorylated regions shown as red circles. The residues between the two CN docking motifs, or between one docking motif and regions dephosphorylated by CN, are represented as coils, as they are predicted to be unstructured in solution. A238L is the CN-A238L crystal structure.</p

A238L interacts with CN via an LxVP and a PxIxIT motif.

<p>(A) C-terminal residues (200–239) of A238L showing putative docking site FLCVK (aa 228–232). Underlined residues were fused to GST. (B) Recombinant CN was incubated with GST fused to 15 amino acids encoding the LxVP motif of NFATc1 or the FLCVK sequence in A238L. CN co-purifies with both motifs; this interaction is disrupted by incubation with excess peptide LxVPc1 encoding the LxVP motif from NFATc1, but not LxVPmut. CN fails to co-purify with GST fused to mutated FLCVK sequence (FLCVK mutated to AACAA). (C) β-galactosidase activity of extracts from yeast strains that harbor 2xCDRE-lacZ, a CN-dependent reporter gene, and GST or GST-A238L truncations are shown. We added 50 mM CaCl₂ to the cell culture 2.5 h before harvesting to induce CN-dependent activation of the Crz1 transcription factor (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492-Stathopoulos1" target="_blank">[22]</a>; see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492.s007" target="_blank">Text S1</a>). Error bars indicate ± s.d. from three independent experiments. (D) Secondary plot of K<i>_i</i>^app as a function of [RII] for A238L_200–239 inhibition of CN. Data show a linear dependence characteristic of competitive inhibition, with K<i>_i</i> = 0.37 nM. K<i>_i</i>^app values were obtained from the nonlinear fit of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492.s001" target="_blank">Figure S1B</a>. Points represent averages ± s.e.m. (E) Isothermal titration calorimetry confirming that purified A238L_200–239 binds to CN.</p

Thermodynamic parameters and dissociation (K_D) and inhibition (K_i) constants for CN_{A1–391/B1–170} with A238L_200–239 wild-type, and A238L PxIxIT and LxVP mutants, derived from ITC experiments at 25°C or enzyme assays performed with RII at 37°C.

<p>Thermodynamic and dissociation constant data represent mean values ± one s.d. for triplicate measurements except A238L_WT, which was performed 5 times. Inhibition constants are mean values ± one s.e.m. from three independent experiments.</p

CN-A238L interactions: The LxVP substrate binding site.

<p>(A) Close-up view of the CN ΦLxVP substrate binding site. (B) Electrostatic surface potential of the A238L-CN complex, highlighting the hydrophobic nature of the ΦLxVP binding groove. (C) CN residues that make up the Leu229 hydrophobic binding pocket (left) and the Val231 hydrophobic binding pocket (right). (D) Electrostatic intermolecular interactions between CN and A238L and intramolecular interactions at the A238L kink, which coordinate A238L for ΦLxVP binding. (E) Secondary plot of <i>K</i>_i^app as a function of [RII] for inhibition of CN by A238L_PKIIITmut, which retains the FLCVK site. Data show a linear dependence characteristic of competitive inhibition, with <i>K</i>_i = 15 nM. Points represent averages ± s.e.m. from three independent experiments.</p