Structural analysis of heme proteins: implications for design and prediction

Abstract

Background: Heme is an essential molecule and plays vital roles in many biological processes. The structural determination of a large number of heme proteins has made it possible to study the detailed chemical and structural properties of heme binding environment. Knowledge of these characteristics can provide valuable guidelines in the design of novel heme proteins and help us predict unknown heme binding proteins.

Results: In this paper, we constructed a non-redundant dataset of 125 heme-binding protein chains and found that these heme proteins encompass at least 31 different structural folds with all-α class as the dominating scaffold. Heme binding pockets are enriched in aromatic and non-polar amino acids with fewer charged residues. The differences between apo and holo forms of heme proteins in terms of the structure and the binding pockets have been investigated. In most cases the proteins undergo small conformational changes upon heme binding. We also examined the CP (cysteine-proline) heme regulatory motifs and demonstrated that the conserved dipeptide has structural implications in protein-heme interactions.

Conclusions: Our analysis revealed that heme binding pockets show special features and that most of the heme proteins undergo small conformational changes after heme binding, suggesting the apo structures can be used for structure-based heme protein prediction and as scaffolds for future heme protein design.

Figure 1

Chemical structures of heme b and heme c.

Figure 2

Examples of three-dimensional structure of multi-heme proteins and identification of heme-binding environment. (A) Cytochrome c nitrite reductase of Wolinella succinogenes (PDB chain: 1FS7A) with 5 heme b molecules; (B) Thioalkalivibrio nitratireducens cytochrome c nitrite reductase (PDB chain: 3F29A) with 8 heme c molecules; (C) globin domain of globin-coupled sensor in Geobacter sulfurreducens (PDB chain: 2W31A). The red sticks are axial ligands of the heme iron and the blue sticks represent other heme interacting residues. For better visualization, the neighboring heme residues in A and B are colored yellow and green respectively. The heme molecules are shown as spacefill. The images were generated using Pymol http://www.pymol.org.

Figure 3

Distribution of the axial ligands for heme b (HEM) and heme c (HEC).

Figure 4

Relative frequency of the heme interacting amino acids. (A) Relative frequency of residues in heme b (HEM), heme c (HEC), and heme b and c (ALL); (B) the relative frequency of the 5 residues with or without them as axial heme ligands.

Figure 5

Frequencies of secondary structure types for heme interacting residues.

Figure 6

Sequence motifs surrounding the axial ligands. (A) Sequence logo from 32 heme c proteins with histidine as axial ligand shows the classic CXXCH heme c binding motif; (B) Sequence logo from 18 heme b proteins with cysteine as axial ligand. The sequence logos were created with WebLogo [74]; (C) Arginine-334 (red sticks) of 1N97A interacts with heme propionates (red spheres); and (D) Interactions between histidine-353 (red sticks) of 1GWIA and heme propionate groups (red spheres).

Figure 7

Three-dimensional structures of heme proteins with "CP" motifs. (A) 3CQVA; (B) 2CIWA; (C) 1GIWA; and (D) 2PBJA. The CP dipeptides are shown as red sticks. The immediate downstream structures of the CP dipeptides are shown in blue.

Figure 8

Structural comparison of apo-holo heme protein pairs. (A,B) 2ZDOA-1XBWD; (C,D) 3CQVA-2V7CA.

Figure 9

Examples of heme proteins with multiple apo structures. (A) RMSD distribution of apo structure of three heme protein chains, 1KBIA, 1N45A, and 1N5UA. The 28 apo structures of 1N5UA form two clusters (red ovals). (B) Structure of 1N5UA with one heme and five myristic acid molecules (MYR, red spacefill). (C) Structure of 1AO6A with no ligands. (D) Structure of 3CX9A with five myristic acid molecules (MYR, red spacefill) and one LPX ligand (orange spacefill).