Two ligand-binding sites in the O ₂ -sensing signal transducer HemAT: Implications for ligand recognition/discrimination and signaling

Abstract

We have identified a ligand (CO) accommodation cavity in the signal transducer sensor protein HemAT (heme-based aerotactic transducer) that allows us to gain single-molecule insights into the mechanism of gas sensor proteins. Specific mutations that are distal and proximal to the heme were designed to perturb the electrostatic field near the ligand that is bound to the heme and near the accommodated ligand in the cavity. We report the detection of a second site in heme proteins in which the exogenous ligand is accommodated in an internal cavity. The conformational gate that directs the ligand-migration pathway from the distal to the proximal site of the heme, where the ligand is trapped, has been identified. The data provide evidence that the heme pocket is the specific ligand trap and suggest that the regulatory mechanism may be tackled starting from more than one position in the protein. Based on the results, we propose a dynamic coupling between the two distinct binding sites as the underlying allosteric mechanism for gas recognition͞discrimination that triggers a conformational switch for signaling by the oxygen sensor protein HemAT.docking site ͉ FTIR spectroscopy ͉ gas sensor proteins

Mechanism and Role of Globin-Coupled Sensor Signalling

“…Biochemical and spectroscopic characterization of purified HemAT- Bs and the isolated HemAT- Bs globin domain have provided insights into protein structure and ligand binding of GCS proteins (Aono et al, 2002; El-Mashtoly et al, 2008; El-Mashtoly et al, 2012; Hou, Belisle, et al, 2001; Hou, Freitas, et al, 2001; Ohta, Yoshimura, Yoshioka, Aono, & Kitagawa, 2004; Pinakoulaki, Yoshimura, Daskalakis, et al, 2006; Pinakoulaki, Yoshimura, Yoshioka, Aono, & Varotsis, 2006; Yoshida, Ishikawa, Aono, & Mizutani, 2012; Yoshimura et al, 2006; Yoshimura, Yoshioka, Mizutani, & Aono, 2007; Zhang et al, 2005; Zhang & Phillips, 2003b). Full-length HemAT- Bs forms a dimer in solution with an elongated, asymmetric shape, while the globin domain forms a monomer in solution but has been crystallized as a dimer (Zhang et al, 2005; Zhang & Phillips, 2003b).…”

“…In addition to the distal hydrogen bonding residues, a heme pocket leucine (L92; Figure 3B) serves as a conformational gate that both helps to maintain protein conformations following ligand binding and serves to direct ligand migration. Within the proximal pocket, a cavity near Y133 serves to bind and/or store gas molecules that also can be accessed through the L92 conformational gate once ligands have dissociated from the heme iron (Pinakoulaki, Yoshimura, Daskalakis, et al, 2006; Pinakoulaki, Yoshimura, Yoshioka, et al, 2006). …”

Kinetic studies of the reactions of O2 and NO with reduced Thermus thermophilus ba3 and bovine aa3 using photolabile carriers

“…This raises questions whether the photodissociated CO does indeed bind to ${\text{Cu}}_{\text{B}}^{+}$ and whether the infrared frequency observed at 2053 cm −1 following photolysis of the CO-bound ba 3 arises from CO bound to ${\text{Cu}}_{\text{B}}^{+}$ [25,26] or from CO being trapped at “docking” site [42]. Interestingly, FTIR studies of the signal transducer O 2 -sensing protein HemAT, a heme protein lacking copper, revealed a well-defined peak at 2065 cm −1 in the spectrum of the equilibrium CO-bound sensor and in its light-minus-dark FTIR difference spectrum [43]. A recent crystal structure of the bovine aa 3 -CO shows that CO binds to ${\text{Cu}}_{\text{B}}^{+}$ in a side-on fashion after photolysis of CO from heme ${\text{italica}}_3^{2+}$.…”

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

“…This assignment was originally evidenced by shifts of the IR spectral line associated with the CO stretch from ~ 1975 cm −1 , arising from the CO-stretch mode of Fe-bound CO, to ~ 2050 cm −1 , thought to arise from the CO-stretch of Cu B -bound CO [14, 16, 17] as suggested in Scheme 1. Recent work by Varotsis and co-workers [18] indicates, however, that the stretching frequency of non-metal bound CO may show similar values for CO bound in polar protein cavities. If proven to occur in the cytochrome c oxidases, such a finding would complicate the now venerable interpretation that the observed ~ 2050 cm −1 band represents the CO stretching frequency of the Cu B -CO complex.…”