Structural and Functional Evidence Indicates Selective Oxygen Signaling in Caldanaerobacter subterraneus H-NOX

Abstract

Acute and specific sensing of diatomic gas molecules is an essential facet of biological signaling. Heme nitric oxide/oxygen binding (H-NOX) proteins are a family of gas sensors found in diverse classes of bacteria and eukaryotes. The most commonly characterized bacterial H-NOX domains are from facultative anaerobes and are activated through a conformational change caused by formation of a 5-coordinate Fe(II)-NO complex. Members of this H-NOX subfamily do not bind O2 and therefore can selectively ligate NO even under aerobic conditions. In contrast, H-NOX domains encoded by obligate anaerobes do form stable 6-coordinate Fe(II)-O2 complexes by utilizing a conserved H-bonding network in the ligand-binding pocket. The biological function of O2-binding H-NOX domains has not been characterized. In this work, the crystal structures of an O2-binding H-NOX domain from the thermophilic obligate anaerobe Caldanaerobacter subterraneus (Cs H-NOX) in the Fe(II)-NO, Fe(II)-CO, and Fe(II)-unliganded states are reported. The Fe(II)-unliganded structure displays a conformational shift distinct from the NO-, CO-, and previously reported O2-coordinated structures. In orthogonal signaling assays using Cs H-NOX and the H-NOX signaling effector histidine kinase from Vibrio cholerae (Vc HnoK), Cs H-NOX regulates Vc HnoK in an O2-dependent manner and requires the H-bonding network to distinguish O2 from other ligands. The crystal structures of Fe(II) unliganded and NO- and CO-bound Cs H-NOX combined with functional assays herein provide the first evidence that H-NOX proteins from obligate anaerobes can serve as O2 sensors.

Discovery of stimulator binding to a conserved pocket in the heme domain of soluble guanylyl cyclase

“…While a highresolution structure of an sGC H-NOX domain has not been reported, crystal structures for several bacterial homologues are known, including those from Caldanaerobacter subterraneus (30), also known as Thermoanaerobacter tengcongensis (22,31), Nostoc sp. 7120 (32,33) and Shewanella oneidensis (34).…”

“…The overall H-NOX fold is ~180 residues long and displays an N-terminal sub-domain encompassing residues 1-60, which is dominated by a 3-helix bundle, followed by a larger mixed helix/sheet sub-domain that contains the heme pocket. Alignment of the larger subdomains of several H-NOX structures indicate the smaller and larger domains can move independently of one another, altering the orientation of the two domains, which has been proposed to be key for signal transduction by H-NOX domains and proteins (29,30,32).…”

Bacterial Haemoprotein Sensors of NO

“…4). This distal pocket H-bonding network has been shown to play a role in O 2 binding by this H-NOX (Boon, Huang, & Marletta, 2005; Hespen, Bruegger, Phillips-Piro, & Marletta, 2016) and is hypothesized to contribute to NO/O 2 ligand discrimination in the H-NOX family (Boon et al, 2005; Boon & Marletta, 2005a, 2005b), as will be further discussed in Section 5.2.…”

Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase

“…PCC7120 ( Ns H-NOX) [7,8,9,10]. On the other hand, H-NOXs found in obligate anaerobes are domains of methyl-accepting chemotaxis proteins (MCP) and often bind O 2 to form an oxyferrous complex, such as the ones from firmicutes Clostridium botulinum ( Cb H-NOX) and thermophilic Thermoanaerobacter tengcongensis ( Tt H-NOX, renamed Caldanaerobacter subterraneus H-NOX recently) [7,11,12,13]. …”