Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
The molecular mechanism of heme loss from oxidized soluble guanylate cyclase induced by conformational change
Introduction
Soluble guanylate cyclase (sGC) is a primary nitric oxide (NO)receptor in mammalian NO-sGC-cGMP signaling pathway [1], [2]. Binding of NO to the ferrous heme cofactor of sGC initiates the NO-dependent signaling cascade, and stimulates the cyclase activity by catalyzing the conversion of guanosine 5′-triphosphate (GTP) to 3′, 5′-cyclic guanosine monophosphate (cGMP). cGMP, as a second messenger, regulates the downstream physiological process, such as vasodilation, platelet aggregation and neurotransmission [3], [4]. Impaired NO-sGC-cGMP signaling has been implicated in various cardiovascular diseases such as arterial hypertension, neurotransmission, and heart failure [5], [6].
sGC is a heme-containing α/β heterodimeric enzyme and the most extensively studied isoform is α1β1 heterodimer. The prosthetic heme group is non-covalently bound to the β1 subunit via the His105. Each subunit contains four domains: an N-terminal heme-NO/O2-binding (H-NOX) domain, a Per/Arnt/Sim (PAS) domain, a helical (CC) domain and a C-terminal catalytic domain (Fig. 1). Although the structure of eukaryotic sGC holo-enzyme remains unknown, the crystal structures of truncated eukaryotic sGC domains (PAS, CC, and catalytic domains) and H-NOX domains from prokaryotic sGC have been reported [7], [8], [9], [10]. The high-order domain architecture of sGC also has been revealed from single-particle EM, indicating that sGC is assembled from two rigid modules: the catalytic domain and the clustered PAS and H-NOX domain flexibly connected by a parallel helical domain [11].
NO activates sGC through binding to the ferrous heme of H-NOX domain leading to the rupture of the Fe2 +–N (H105) coordination bond [12], [13]. The mechanism of the NO-induced sGC activation also has been proposed by diverse approaches [14], [15], [16], [17]. Haase et al. have revealed closed proximity between N and C-termini of sGC using fluorescent fusion protein based on fluorescence resonance energy transfer (FRET), supporting that the direct interaction between the H-NOX domain and the catalytic domain represses sGC activity [14]. Recently, Underbakke et al. have revealed the domains' motion in several discrete region upon NO binding using HDX-MS [16], suggesting that the NO-induced conformational changes involve in the heme-associated helix surface, an H-NOX-associated surface of the PAS domain, the PAS-helical domain linker, the helical domain and the active site of the catalytic domain. These domain interactions also have been observed previously [18]. A FRET study based on FRET between endogenous tryptophan and substrate analog 2′-Mant-3′-dGTP has recently provided a unique opportunity to directly detect the movement of the functional domain relative to the substrate binding catalytic region upon NO binding, which also supports the mechanism proposed by Underbakke et al. [17]. In general, it is an allosteric pathway transmitting the NO-signaling from the H-NOX domain to the active site catalytic domain.
The ferrous heme group plays a key role in sGC-mediated NO-signaling activation and stabilization of sGC. And NO is a poor ligand for ferric sGC and can't activate sGC [19], [20]. Previous studies have shown that sGC is prone to heme loss during isolation, particularly after oxidation [21]. This also occurs in vivo, as the reactive oxygen species (ROS) can oxidize heme group of sGC under conditions of oxidative stress, leading to the heme dissociation from the oxidized enzyme and the generation of NO-insensitive sGC in the diseased tissue [22]. The levels of oxidized or heme-free sGC are increased in certain cardiovascular diseases [23], [24]. Heme-free sGC is also prone to ubiquitin-mediated degradation [24], [25]. In addition, the oxidation of sGC heme is thought to be an important risk factor to the development of cardiovascular diseases [26], [27]. Thus, based on the oxidized or heme-free sGC, heme-independent sGC activators which can activate oxidized or heme-free sGC by acting as the heme group, such as cinaciguat (BAY 58-2667), are discovered [26], [27], [28]. Furthermore, Fritz et al. have found that the ferrous heme in sGC is very stable and resistant to heme loss both in the absence and presence of NO, but the ferric heme in sGC more readily loses its heme as observed using spectroelectrochemical titration and heme transfer experiments [29]. Surmeli et al. also have demonstrated that oxidized sGC loses heme more readily than the ferrous sGC and the activator cinaciguat activates sGC involving facilitation of heme loss from ferric sGC and subsequent replacement of heme in the heme pocket [30]. However, it has not been clearly why oxidized sGC loses its heme more readily compared to reduced sGC, and the molecular mechanism of heme dissociation from sGC is not understood clearly. To this end, we herein investigated the molecular mechanism of sGC heme oxidation and loss, based on the energy transfer between the heme and the fluorescein arsenical helix binder (FlAsH-EDT2) labeled at different domains of the sGC β1 subunit.
As an effective probe to monitor the protein conformational change, protein tertiary and quaternary structure formation in vitro and in vivo [31], [32], [33], [34], [35], FlAsH-EDT2 can bind specifically to a small tetracysteine (TC: CCPGCC) with rather small size, and can be introduced by mutagenesis. The emission spectrum of FlAsH-EDT2 has a good overlap with α and β absorbance bands of sGC heme group [22], [36], [37], [38]. Thus, the heme group can effectively quench the fluorescence of FlAsH-EDT2, which can be used to study the conformational change upon sGC heme oxidation by ODQ. The fluorophore FlAsH-EDT2 was also used to study the heme loss of sGC in vivo [22].
In the present work, we investigated systemically conformational change of sGC β1 upon heme oxidation by ODQ based on the energy transfer between heme and the fluorophore FlAsH-EDT2. The fluorophore FlAsH-EDT2 was labeled at different domains of sGC β1. The treatment of sGC by ODQ resulted in fast oxidation of the ferrous truncated sGC β1 to the ferric state, and subsequently initiated the slow conformational change of the truncated sGC β1. The synergistic effect of these conformational changes of the discrete region induced by heme oxidation contributed to the heme loss.
Section snippets
Materials
1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one (ODQ) and diethylammonium (Z)-1-(N, N-diethylamino) diazen-1-ium-1, 2-diolate (DEA/NO) were purchased from Cayman Chemical Company. Fluorescein arsenical helix binder (FlAsH-EDT2) was purchased from Toronto Research Chemicals Inc. (Toronto, Canada). KOD-Plus-Mutagenesis Kit was purchased from TOYOBO (Osaka, Japan). Hemin and aminolevulinic acid (5-ALA) were purchased from Sigma. Nickel nitrilotriacetic acid (Ni-NTA) resin and Sephadex G-25 resin
Preparation and characterization of sGC proteins
Since FlAsH-EDT2 could bind tightly to the TC (CCPGCC) motif, the TC motif was successfully introduced into the sGC proteins by mutagenesis, which was confirmed by DNA sequencing analysis, at C-terminus of [sGC β1(1–195)-196TC201 and sGC β1(1–385)-386TC391] or at PAS domains of [sGC β1(1–385)-243TC248 and sGC β1(1–619)-243TC248]. All the soluble human sGC proteins of sGC β1(1–195)-196TC201, sGC β1(1–385), sGC β1(1–385)-243TC248 and sGC β1(1–385)-386TC391 were effectively expressed in
Conclusions
Based on all the results above, the molecular mechanism for heme loss from the truncated sGC β1 by oxidation was proposed, which contained two processes shown in Fig. 12. The ferrous heme sGC was first oxidized by ODQ to form the ferric heme sGC. This was a fast process. Then, the conformational change was initiated which contained that the heme-associated αF helix moved away from the heme, the PAS domain labeled with FlAsH-EDT2 moved closer to the heme, and the helical domain labeled with
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Acknowledgment
This work was supported partly by the Natural Science Foundation of China (No. 21472027, No. 31270869, and No. 91013001), the Ph.D. Program of the Education Ministry of China (20100071110011), Shanghai & Beijing Synchrotron Radiation Facility/high magnetic field laboratory of Chinese Academy of Science, and Xiyuan/Wangdao Research Training Program for undergraduate students of Fudan University. We thank Prof. Pingwei Li and Dr. Chang Shu from the Department of Biochemistry & Biophysics, Texas
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