How do cell-surface receptors transmit signals into cells? This study resolves how signal relay occurs through the HAMP domains of bacterial chemoreceptors by causing them to switch between two conformational states.
Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS and poly-HAMP domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state, identifies conformational changes induced by ligand binding that are likely essential for the signaling mechanism. Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS, but propagate to the Iβ-strand in a manner predicted to alter PAS-PAS associations and the downstream HAMP junction within full-length Aer2. SAXS of PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initioSAXS molecular envelopes and pulsed-dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the E. coliAer paradigm, where side-on PAS-HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.
ACCESSION NUMBERSThe coordinates and structure factors for the ferric Aer2 PAS domain have been deposited in the protein data bank with PDB ID 4HI4.
We present an approach for preparing cryoEM grids to study short-lived molecular states. Using piezo electric dispensing, two independent streams of ~50 pL sample drops are deposited within 10 ms of each other onto a nanowire EM grid surface, and the mixing reaction stops when the grid is vitrified in liquid ethane, ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.
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The diarrheal pathogen Vibrio cholerae navigates complex environments using three chemosensory systems and 44-45 chemoreceptors. Chemosensory cluster II modulates chemotaxis, whereas clusters I and III have unknown functions. Ligands have been identified for only five V. cholerae chemoreceptors. Here we report that the cluster III receptor, VcAer2, binds and responds to O . VcAer2 is an ortholog of Pseudomonas aeruginosa Aer2 (PaAer2), but differs in that VcAer2 has two, rather than one, N-terminal PAS domain. We have determined that both PAS1 and PAS2 form homodimers and bind penta-coordinate b-type heme via an Eη-His residue. Heme binding to PAS1 required the entire PAS core, but receptor function also required the N-terminal cap. PAS2 functioned as an O -sensor [K , 19 μM], utilizing the same Iβ Trp (W276) as PaAer2 to stabilize O . The crystal structure of PAS2-W276L was similar to that of PaAer2-PAS, but resided in an active conformation mimicking the ligand-bound state, consistent with its signal-on phenotype. PAS1 also bound O [K 12 μM], although O binding was stabilized by either a Trp or Tyr residue. Moreover, PAS1 appeared to function as a signal modulator, regulating O -mediated signaling from PAS2, and resulting in activation of the cluster III chemosensory pathway. This article is protected by copyright. All rights reserved.
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