Unique structure of Ascaris suum b5-type cytochrome: an additional alpha-helix and positively charged residues on the surface domain interact with redox partners

Abstract

Cytochrome b5 of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a soluble protein that lacks a C-terminal membrane-anchoring domain, but possesses an N-terminal pre-sequence of 30 amino acids. During the maturation of cytochrome b5, the N-terminal pre-sequence is proteolytically cleaved to form the mature protein of 82 amino acid residues. A. suum cytochrome b5 is a basic protein containing more lysine residues and exhibiting a higher midpoint redox potential than its mammalian counterparts. We developed an expression system for the production of the recombinant nematode cytochrome b5, which is chemically and functionally identical with the native protein. Using this recombinant protein, we have determined the X-ray crystal structure of A. suum cytochrome b5 at 1.8 A (1 A=0.1 nm) resolution, and we have shown that this protein is involved in the reduction of nematode body-wall metmyoglobin. The crystal structure of A. suum cytochrome b5 consists of six alpha-helices and five beta-strands. It differs from its mammalian counterparts by having a head-to-tail disulphide bridge, as well as a four-residue insertion in the vicinity of the sixth ligating histidine, which forms an additional alpha-helix, alpha4A, between helices alpha4 and alpha5. A. suum cytochrome b5 exists predominantly as a haem-orientation B isomer. Furthermore, the haem plane is rotated approx. 80 degrees relative to the axis formed by haem-Fe and N atoms of the two histidine residues that are ligated to haem-Fe. The charge distribution around the haem crevice of A. suum cytochrome b5 is remarkably different from that of mammalian cytochrome b5 in that the nematode protein bears positively charged lysine residues surrounding the haem crevice. Using immunohistochemistry, we found that A. suum cytochrome b5 is present in the nematode hypodermis. Based on this histochemical and structural information, the physiological function of A. suum cytochrome b5 and its interaction with nematode metmyoglobin can be hypothesized.

Figure 1. Overall structure (A) and topology diagram (B) of A. suum cytochrome b₅

The protein is folded in six helices and five β strands, comprising a β-pleated sheet. (A) Ribbon and ball-and-stick drawing of the overall structure. The six helices are numbered 1, 2, 3, 4, 4A and 5 in grey, while the five β strands are denoted A–E in dark grey. The haem and His³⁸, His⁶², Cys¹ and Cys⁷⁷ are drawn as ball-and-stick models. (B) α-Helices and β-strands are represented as rectangles and broad arrows respectively, with residue numbers showing their starting and ending positions. Two histidine residues (His³⁸ and His⁶²) ligating the haem iron and the disulphide bond between Cys¹ and Cys⁷⁷ are also shown. The nomenclature of α-helices and β-strands is the same as in (A).

Figure 2. Structure-based alignment of the amino acid sequence of A. suum cytochrome b₅ with the catalytic domains of several mammalian cytochromes b₅

Residues composing the α-helices and β-strands are represented as letters in grey and black boxes respectively. Sequence 1, A. suum cytochrome b₅ (mature, 82 residues) [8]. Sequence 2, rat microsomal cytochrome b₅ (97 residues) [48]. Sequence 3, rat mitochondrial outer membrane cytochrome b₅ (92 residues) [49]. Sequence 4, bovine microsomal cytochrome b₅ (97 residues) [27]; the underlined residues, 1′–93′, are those identified by X-ray crystallographic analyses of the lipase-treated protein. Sequence 5, porcine microsomal cytochrome b₅ (97 residues) [48]. Sequence 6, human cytochrome b₅ (97 residues) [48]. Sequence 7, human erythrocyte cytochrome b₅ (97 residues) [50]. Except for sequence 5, the first residues of alanine are acetylated. For comparison, the residue numbers for mammalian cytochromes b₅ are shown beneath as numbers with a prime, based on the numbering system for bovine cytochrome b₅. The two histidine residues that are ligated to the haem iron are denoted by an asterisk. Up arrows indicate the acidic residues of mammalian cytochrome b₅, which electrostatically interact with the lysine residues of their reaction partner proteins. The acidic residues of mammalian cytochrome b₅ that are replaced by other residues in A. suum cytochrome b₅ are marked by +, and those replaced by lysine residues are indicated by down arrows. Hyphens indicate the positions of deletions or insertions.

Figure 3. Haem conformation in A. suum cytochrome b₅

(A) Two haem conformations about the α,γ-meso axis are denoted as isomers A and B. The haem-Fe atom is bound to His³⁸ and His⁶² from the front and back sides respectively. Three residues, Ile²², Met³¹ and Asn⁴⁶, which are in close contact with the haem group, are shown in the respective Figure. (B) Stereo view of an F_o-F_c omit map for haem at 1.8 Å resolution. The haem group and residues within 10 Å of the haem are excluded from structure factor calculations and difference electron density is contoured at the 3σ level. Isomer B, the predominant haem form of A. suum cytochrome b₅, is drawn as a ball-and-stick model.

Figure 4. Stereo diagram of the haem-binding site

The haem moiety in isomer B is drawn as a ball-and-stick model. Met³¹, Phe³⁴, Leu⁵⁶, Val⁶⁹, Lys⁷² and Leu⁷³ form a hydrophobic pocket, which accommodates the vinyl group of haem pyrrole I.

Figure 5. Stereo diagram showing an overlap of the α-carbon backbone and the haem group of A. suum (heavy line) and bovine (pale line) cytochrome b₅ structures

The α-carbon atom of every fifth residue of the A. suum protein is labelled with a sequence number. Both structures are well superimposed with their β-sheet cores, while the A. suum helical bundle is twisted by 10° from its position in the bovine protein.

Figure 6. Stereo view of ribbon-and-stick model for A. suum (A) and bovine (B) cytochrome b₅

Although the haem-binding motifs in the two proteins are similar, the porphyrin ring in the A. suum protein is rotated by 80° about a bond between the haem-Fe and two histidines, His³⁸ and His⁶².

Figure 7. Immunohistochemical localization of A. suum cytochrome b₅ in the body wall of adult nematode

(A) Cross-section of adult A. suum reacted with anti-(A. suum cytochrome b₅) antibody showing the staining in the hypodermis and muscle fibre. Note that the ventral nerve (shown by an arrow), which projects into the body cavity from the hypodermis, was also heavily stained. (B) Control section reacted with normal rabbit serum showing no significant staining.

Figure 8. Stimulation by A. suum cytochrome b₅ of nematode cytochrome b₅ reductase-catalysed reduction of nematode body-wall metmyoglobin

(A) Difference spectra obtained by repeated scanning show the time course of metmyoglobin reduction in the presence of 0.384 μM cytochrome b₅. Time-dependent increase in absorptions at 543 and 578 nm was observed during 13 cycle scanning. (B) Dose-dependent relationship between reduction rate and concentration of added cytochrome b₅. Experimental details are described in the Experimental section.

Figure 9. Comparison of the electrostatic molecular surfaces around the haem crevices of A. suum (A) and bovine (B) cytochrome b₅, and A. suum model (C) and horse metmyoglobins (D) (PDB code 1WLA)

The electrostatic potential mapped on the protein surface was calculated using APBS [51]. A positive potential is indicated in blue, and a negative one is indicated in red.