Design principles for chlorophyll-binding sites in helical proteins

Abstract

The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, and hemes are ubiquitous cofactors of biological catalysis that are involved in a multitude of reactions. One systematic approach for understanding how Nature achieves functional diversity with only this handful of cofactors is by designing de novo simple and robust protein scaffolds with heme and/or (bacterio)chlorophyll [(B)Chls]-binding sites. This strategy is currently mostly implemented for heme-binding proteins. To gain more insight into the factors that determine heme-/(B)Chl-binding selectivity, we explored the geometric parameters of (B)Chl-binding sites in a nonredundant subset of natural (B)Chl protein structures. Comparing our analysis to the study of a nonredundant database of heme-binding helical histidines by Negron et al. (Proteins 2009;74:400-416), we found a preference for the m-rotamer in (B)Chl-binding helical histidines, in contrast to the preferred t-rotamer in heme-binding helical histidines. This may be used for the design of specific heme- or (B)Chl-binding sites in water-soluble helical bundles, because the rotamer type defines the positioning of the bound cofactor with respect to the helix interface and thus the protein-binding site. Consensus sequences for (B)Chl binding were identified by combining a computational and database-derived approach and shown to be significantly different from the consensus sequences recommended by Negron et al. (Proteins 2009;74:400-416) for heme-binding helical proteins. The insights gained in this work on helix- (B)Chls-binding pockets provide useful guidelines for the construction of reasonable (B)Chl-binding protein templates that can be optimized by computational tools.

Figure 1.

A) Distribution of the side chain torsion angles, χ₁ and χ₂, in the non-redundant dataset of (B)Chl-bound helical histidines. B) Angular and radial positions of histidine ligands, and the Mg atoms of (B)Chls, color coded according to their respective rotamers. The coordinating Nε and Nδ nitrogens of each histidine, and the Mg atoms of the (B)Chls are marked by squares, triangles, and circles respectively.

Figure 2.

Macrocycle topology of m- and t-type rotamer ligated (B)Chls. Angular and radial positions of m- (A) and t- (B) rotamer histidines, and their bound (B)Chls shaded according to the tilt angle, φ, of the (B)Chl plane. Solid lines are used for Nε-bound, and dotted lines for Nδ-bound (B)Chls. Distributions of φ absolute values in m- (C, light grey bars) and t- (D, dark grey bars represent Nδ-bound, dotted bars Nε-bound (B)Chls) rotamers are centered about |φ| = 0, and |φ| = 19 degrees, respectively.

Figure 3.

Rotational angle (ω) distribution of (B)Chls are distinctly discontinuous and depend on their (B)Chl α- or β-ligation mode (top histogram, dotted and dark grey bars, respectively) but not on the m- or t-rotamers of binding histidines (bottom histogram, striped and light grey bars, respectively). Notably, the 95-degrees rotation of (B)Chl is found only in α conformers.

Figure 4.

Contact maps of (B)Chl and aa residues along the binding helix (positions ±8 relative to ligating histidine: A) m-rotamers, B) t-rotamers with Nδ as ligand, C) t-rotamers with Nε as ligand. The color coding indicates contact frequency.

Figure 5.

Relative energy (ΔΔE) maps versus position and amino acid type: A) m-rotamers, B) t-rotamers with Nδ as ligand, C) t-rotamers with Nε as ligand. ΔΔE values larger than 10 kcal/mol are colored dark blue.

Figure 6.

Amino acids frequencies (bottom) and energies calculated by protCAD (top) at positions P-4 (light grey), P-1 (striped), and P+3 (dotted) where P0 is the position of m-rotamer Chl-binding histidine. Empty bars represent occurrence frequency of residues at a given position, full bars represent interaction frequency, i.e. only residues that interact with the bound (B)Chl. Black, and red lines indicate relative amino acids frequencies in TM core of a random data set, and those of (B)Chl binding proteins of the data set used in this study, respectively. Probability values are marked for amino acids that scored below the 5% significance level in a two-sided binomial test vs. the TM core frequencies.

Figure 7.

Interactions of helical histidine-bound (B)Chls with pigment, protein and solvent environment. Bound (B)Chls are aligned as described in Methods. Views and statistics are shown for interacting residues on the same side of the binding helix (top panels), and across it (bottom panels). (B)Chls α- and β-ligated to m-rotamer histidines (m-α, and m-β, respectively), and (B)Chls α-ligated to t-rotamer histidines (t-α) are considered. Protein-, (B)Chl, and carotenoid residues, within 4 Å from the bound (B)Chl are shown in red, green, and yellow stick and small spheres representation, respectively, except ligating histidines shown in pink. Water, lipid, and quinone atoms are shown as blue, pink, and golden spheres, respectively. The respective distributions of contacts per bound (B)Chl are shown to the right of each panel. Residue types are amino acids (AAs), neighboring (B)Chls (Chl), carotenes (CRT), lipids or detergent molecules (LPD), and water (HOH). The distribution for all (B)Chls in the database is shown as grey bars in the background.

Scheme 1:

Structural parameters of (B)Chl-bound helical histidines. A) The Cartesian PDB coordinates of (B)Chls and proteins were transformed into an internal cylindrical coordinate system (r, θ, z) whereby the z axis is aligned with the helix axis such that the helical axis is crossing r = 0, and Cα of the binding histidine is at z = 0 and θ = 0. The helix is represented as an ideal heptad repeat with seven distinct position labeled a – e, and the binding histidine at position a. The angular positions of residue −8 to +8 relative to the binding histidine are also noted based on 18 points helical wheel representation. The (B)Chl is represented by the central Mg atom (solid circle), and a projection of a line drawn on the (B)Chl plane between points p₁ and p₂ (see Methods for a detailed definition). The histidine’s nitrogen ligand is indicated by the solid square. B) The tilt angle φ is the angle between the helix axis and the (B)Chl plane. C) The rotational angle ω is the counter-clockwise angle between a vector connecting the projection of (B)Chl atoms N₂₄ and N₂₂, and the helical axis. The Chl atoms considered in this work are shown labeled according IUPAC conventions. BChl atoms are identical except for O32 replacing C32 and the reduced ring B. The phytyl chains (R) are not considered. In α- and β-ligated (B)Chls, the histidine ligand and C171 of the propionic acid side chain are either on opposite, or the same sides of the (B)Chl plane, respectively.

Scheme 2.

Comparison between consensus sequences of (B)Chl binding helices that were computationally derived by the protCAD software, and those derived from non-redundant databases of (B)Chl- and heme-binding structural motifs.

Scheme 3.

Organization schemes for single-chain four-helix bundles incorporating a dimer of (B)Chls, assuming (B)Chls are bound to the m80 rotamer of histidine. Possible configurations include (B)Chls interacting at their non-ligated sides and bound between anti-parallel (A), or parallel (B) helices, as well as (B)Chls interacting at their ligated sides and bound between anti-parallel helices (C). Helices are shown as ideal heptade repeats with seven distinct position labeled a – e, and the binding histidine at position a. The angular positions of residue −8 to +8 with the binding histidine at position 0 are also noted based on 18 points helical wheel representation. The notations “C” or “N” indicating whether the carboxy- or amino-end is closer to the viewer. Positions with hydrophilic and hydrophobic residues are marked by dark and light grey background, respectively. Intermediate positions are shown with black to white color gradient.