Heme Ligand Binding Properties and Intradimer Interactions in the Full-length Sensor Protein Dos from Escherichia coli and Its Isolated Heme Domain

Abstract

Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nM) is more stable than the dimeric heme domain (association constant ϳ1 M), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O 2 ) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated ϳ10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins. Dos from Escherichia coli (EcDos)4 is a modular gas sensor protein in which phosphodiesterase activity is coupled with the binding and release of external ligands in an associated hemebinding sensor domain (1-3). Cyclic AMP (4) and cyclic diGMP act as substrates of this enzyme, the latter with much higher activity (5). EcDos has been found to display 6 -7-fold enhanced catalytic activity toward cyclic diGMP after binding of gaseous molecules such as O 2 , CO, and NO to the heme.Although the heme-containing PAS sensor domains of EcDos and the rhizobial sensor protein FixL (6) share a conserved structural fold, the ligand-induced regulation of both proteins differs considerably. In contrast to EcDos, FixL is an oxygen-specific sensor that couples the status of its sensor domain to the activity of a histidine kinase. This enzymatic activity is decreased to a great extent upon O 2 binding (2). Furthermore, whereas in the ferrous deoxy form of FixL the heme is pentacoordinate, in EcDos the heme iron is coordinated to a proximal histidine (His-77) and a distal methionine (Met-95). Met-95 can be replaced by small gaseous molecules and plays a crucial role in the regulation of the catalytic activity.The three-dimensional structures of the Fe(III), Fe(II), and Fe(II)-O 2 complexes of the isolated heme sensor domain of EcDosH (EcDosH) have been determined (7,8). These structures have revealed the importance of se...

Structures of the catalytic EAL domain of theEscherichia colidirect oxygen sensor

“…DosP EAL was found to be dimeric by gel-filtration chromatographic analysis ( Supplementary Fig. S1), which is in line with the previously reported dimerization of full-length DosP (Lechauve et al, 2009) and other full-length EAL-domain proteins such as YkuI (Minasov et al, 2009) and BlrP1 (Barends et al, 2009). In the crystal structure of the native DosP EAL domain the dimer interface is formed mainly by interactions between a pair of antiparallel -helices (5 and 6b), which were named the compound helix and the dimerization helix, respectively, in BlrP1 (Barends et al, 2009).…”

Environmental Heme-Based Sensor Proteins: Implications for Understanding Bacterial Pathogenesis

“…In these proteins, oxidation, reduction, or modification of heme generally results in an alteration in protein activity. Many microbial sensor proteins have been shown to bind O 2 , NO, and/or CO via their heme group (65), which causes structural changes to the heme that ultimately regulate protein activity (13,50). NO and CO are particularly important as they are physiologically relevant and generated by human iNOS (inducible nitric oxide synthase) and HO-1 (heme oxygenase-1), respectively, as part of the protective response against microbial invasion (44) or oxidative stress (67).…”

Characterization and Structure of the Vaccinia Virus NF-κB Antagonist A46

“…Yields were between 0.5 and 1 mg/liter of culture. In order not to have to rely on standard proteins and an assumption of their conformation, we coupled SEC to multi-angle laser light scattering analysis to enable absolute molecular masses to be calculated (47). Such measurements revealed that the full-length A46 had an apparent molecular mass of 110 kDa ( Fig.…”