Crystal structure of an oxygen-binding heme domain related to soluble guanylate cyclases

Abstract

Soluble guanylate cyclases are nitric oxide-responsive signaling proteins in which the nitric oxide sensor is a heme-binding domain of unknown structure that we have termed the heme-NO and oxygen binding (H-NOX) domain. H-NOX domains are also found in bacteria, either as isolated domains, or are fused through a membrane-spanning region to methyl-accepting chemotaxis proteins. We have determined the crystal structure of an oxygen-binding H-NOX domain of one such signaling protein from the obligate anaerobe Thermoanaerobacter tengcongensis at 1.77-angstroms resolution, revealing a protein fold unrelated to known structures. Particularly striking is the structure of the protoporphyrin IX group, which is distorted from planarity to an extent not seen before in protein-bound heme groups. Comparison of the structure of the H-NOX domain in two different crystal forms suggests a mechanism whereby alteration in the degree of distortion of the heme group is coupled to changes on the molecular surface of the H-NOX domain and potentially to changes in intermolecular interactions.

Fig. 1.

Multiple sequence alignment of selected H-NOX domains. Secondary structure annotation, and numbering on top, correspond to the H-NOX domain from T. tengcongensis. α-helices are represented by spirals and β-strands by arrows. The position of Tyr-140 is indicated by a green box. Accession numbers are as follows: Ther_tengcongensis_gi|20807169|, Clos_acetobutylicum_gi|15896488|, Desu_desulfuricans_gi|23475919|, Rat_beta1_sGC_gi|27127318|, Rat_beta2_sGC_gi|21956635|, Nost_punctiforme_gi|23129606|, Nost_sp._gi|17229770|, Vibr_vulnificus_gi|27361734|, Caul_crescentus_gi|16127222|, Micr_degradans_gi|23027521|, Vibr_cholerae_gi|15601476|, and Shew_oneidensis_gi|24373702|. Alignments were carried out by using the program multalin (42). Fig. 1 was prepared by using the program espript (43).

Fig. 2.

Structural features of the H-NOX domain. A stereo side view of the H-NOX domain is shown. The protein fold is represented by ribbon diagrams. The heme, dioxygen ligand, and proximal histidine are shown as ball-and-stick models. Helices are labeled A-G according to the nomenclature shown in Fig. 1. β-strands are labeled 1–4.

Fig. 3.

Heme environment. (A) Ligand-binding pocket. Tyr-140 is shown hydrogen bonding (red dashes) to the heme-bound oxygen ligand. Trp-9 and Asn-74 interact with Tyr-140. The proximal ligand, His-102, is also shown. (B) The YxSxR motif, corresponding to residues Tyr-131, Ser-133, and Arg-135, coordinates heme propionates.

Fig. 5.

Structural changes associated with different heme conformations. (A) Ribbon diagram showing the superimposition of two molecules exhibiting different heme conformations. Molecule A (monoclinic) is green and molecule B (monoclinic) is white. (B) A 90° rotation of A about the y axis. (C and D) Diagram showing the major changes in networking interactions. Molecule A (monoclinic) with the more distorted heme is shown in C. Molecule B (monoclinic), which has the less distorted heme, is shown in D. The enlargements in C and D are of the boxed area in B.

Fig. 4.

Heme distortion. Schematic diagram of the heme group with pyrrole groups A–D labeled. Pyrrole groups B and C were used to superimpose the heme groups from different proteins with that of oxy-myoglobin. Heme propionates have been removed for clarity.