Architecture of the Soluble Receptor Aer2 Indicates an In-Line Mechanism for PAS and HAMP Domain Signaling

Abstract

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.

Proposed Role for KaiC-Like ATPases as Major Signal Transduction Hubs in Archaea

“…3). Many of these contain sensory and signal-transducing domains that have been thoroughly studied in the context of bacterial two-component signal transduction systems (53,54). This connection suggests that DUF835 proteins are involved in signal transduction pathways.…”

Differential Role of HAMP-like Linkers in Regulating the Functionality of the Group III Histidine Kinase DhNik1p

“…The P. aeruginosa chemoreceptor, Aer-2, is the only other system where functioning of the poly-HAMP module has been studied. In Aer-2, the PAS-sensing domain appeared to regulate the alternative HAMP structure of the successive C-terminal HAMP domains through an in-line mechanism (40). In the case of DhNik1p, the absence of any input sensor domain made the system more interesting and intriguing.…”

Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor

“…1c) (14). The heme itself appears to shift ϳ2.0 Å upon ligand binding, and the heme pocket adjusts accordingly (14).…”

“…In the absence of ligand, the I␤ Trp residue W283 appears to rotate ϳ90°, and an adjacent Leu residue, L264 on H␤, contracts toward the heme iron center to occupy the position where CN Ϫ was bound ( Fig. 1c) (14). The heme itself appears to shift ϳ2.0 Å upon ligand binding, and the heme pocket adjusts accordingly (14).…”

“…There are currently two structures for the Aer2 PAS domain: one contains cyanide bound to ferric heme (cyanomet, or Fe 3ϩ -CN; PDB entry 3VOL) (13) (Fig. 1b), and the other contains unliganded ferric heme (Fe 3ϩ ; PDB entry 4HI4) (14). These structures revealed several unusual PAS features, including an extended C␣/D␣ helix, a short 3 10 helix, called E (that replaces E␣), heme coordination via a His residue on E, and potential O 2 stabilization via the indole group of a Trp residue on I␤ (Fig.…”