Crystal structure of a light-harvesting protein C-phycocyanin from spirulina platensis and bioinformatics analysis of beta-bulge turns in proteins

Abstract

The conclusions for the crystal structure of light-harvesting phycobiliprotein, C-phycocyanin from an important cyanobacterium, Spirulina platensis, are summarized as follows:

We have solved the three-dimensional structure of S. platensis C-phycocyanin at 2.2 Å resolution. The crystal asymmetric unit contains two hexameric aggregates of S. platensis CPC (2 × 225 kDa) closely associated with each other. The structure has been refined without the application of non-crystallographic symmetry restraints to R_cryst = 19.2% (R_free = 23.8%) and deposited at the PDB with access code 1HA7.

The structure of CPC from S. platensis forms the first example of a hexameric phycobiliprotein occurring in the crystal asymmetric unit, with two such hexamers asymmetrically associated in relation to each other.

The monoclinic system in which CPC_SP crystallized forms another unique example not only among CPCs but also among all phycobiliproteins.

The study supplements existing structural knowledge on phycobiliproteins in terms of asymmetric association of a highly symmetric molecule. Further, the study of a hitherto uncharacterized microheterogeneity in the - and -subunits within all known structures of CPC should reflect the various modalities of functional associations, viz., face-to-face and side-to-side interactions among CPC associations in phycobilisomes.

Excitation transfer analysis based on the application of parameters obtained from the crystal structure of S. platensis CPC into the Förster mechanism takes the experimental spectroscopic parameters into consideration while calculating the transfer rate, thereby accounting for subtle differences in spectroscopic properties of each type of chromophore and the specific transitions. Transfer rates have been calculated for various forms of aggregates such as monomers, trimers, and hexamers and are found to be compatible with experimental results for CPCs from other organisms.

For the first time, we propose a quantitative model for inter-rod excitation transfer in the lateral direction between the light-harvesting antennae rods of cyanobacterial phycobilisomes, mediated through peripheral P155 chromophores. The model is significant considering the organization of light-harvesting antennae rods of phycobilisomes as seen in nature. We hope our work will stimulate spectroscopic experimental tests of the efficacy of our lateral energy transfer model.

Our analysis based on the Förster mechanism of energy transfer highlights an additional putative role for the P155 chromophores in terms of inter-rod energy transfer in the lateral direction. The analysis also emphasizes the importance of asymmetric association of ()₆-hexamers and lateral energy transfer between them, considering the association of light-harvesting antenna rods as relevant to its occurrence in nature.

The following conclusions are emphasized based on the analysis carried out to understand the conformational features associated with a series of 'reverse' patterned hydrogen-bonded rings, namely 13, 14, 15, 16 motifs. The emphasis of results obtained is mainly summarized for Type 1 (15 with a nested 41) and Type 2 (16 with a nested 51) 3-bulge hydrogen-bonded turn conformations using 731 protein crystal structures determined to better than 2.5 Å resolution by X-ray crystallography:

For the first time, we have characterized the conformation of a very rarely occurring, new type of tight turn as a hydrogen-bonded motif of 13 type (Cn ring). The tightness of the turn dictates a signature conformation, which we have characterized. At this stage, the rarity of their occurrence does not allow us to do a statistically meaningful analysis on them.

Type 1 3-bulge turns are quite common motifs that occur mostly between -hairpin motifs. From the frequency of their occurrence, we estimate that they form the second major class of turns next to the classical two-residue -turns (Winkatachalam, 1968) as observed in proteins (Wilmot & Thornton, 1988; Gunasekaran et al., 1997). The Type 1 -bulge is present in a ratio of an average one turn in two proteins.

Type 2 -bulge turns are not as common compared to Type 1 motifs. The Type 2 -bulge is present in a ratio of an average of one turn in 6–7 proteins.

Generally, both classes of -bulge turns occur at the ends of -hairpin motifs. However, they are also seen in a significant ratio (20–30%) between -, -, and - motifs. A few are also seen as ordered structures within long loops connecting different secondary structures in proteins. Hence, the present study particularly highlights the significant occurrence of such hydrogen-bonded turn conformations in a variety of contexts and not only at the edges of -strands as their name suggests. The analysis supports Milner-White (1987), who, based on a small sample, argued that -bulge turns should be regarded as independent turn motifs occurring in proteins.

Type 1 -bulge turn is characterized by pRRLp conformation for the five residues (1–5), stabilized by 15 and 41 hydrogen bonds.

Type 2 -bulge turn has pRRRLp conformation for residues 1–6, stabilized by 16 and 51 hydrogen bonds.

The amino acid preferences for Type 1 and Type 2 -bulge turns are different. In Type 1 -bulge turn, Pro, Asx, and remarkably Gly are preferred for the central three residue positions (2, 3, 4), respectively. Position 1 has a high preference for Asx residues, and position 5 can flexibly accommodate hydrophilic, polar, or charged residues.

In Type 2 -bulge turn, Pro, Asx/Glx/Ser/Thr, Thr, and Gly are preferred for the four central residues (2, 3, 4, 5), respectively. Position 1 has a remarkable preference for Asx residues, with Cys also accommodated with high preference. Position 6 is mostly occupied by polar or charged amino acids such as Lys, Arg, Asp, Trp, and Glu.

The well-defined sequence and conformational characteristics of both Type 1 and Type 2 -bulges indicate the local nature of interactions observed within the turns. It should be possible to incorporate these as 'design principles' in de novo design of protein super-secondary structural motifs. A few designed peptides with synthesized sequences of -bulge turns borrowed from protein structures have been characterized to adopt the desired Type 1 -bulge turns (Alba et al., 1997; Zerella et al., 2000). This supports the conclusion that local interactions alone suffice for the stability of the motif.

The results of the present analysis can be useful for protein structure analysis, structure prediction, modeling, de novo protein design, and particularly the design of super-secondary structural motifs.

Future plans include exploring in detail the functional contexts related to both Types of -bulges. The characteristic sequence patterns seen at different positions can be incorporated into artificial neural network (ANN)-based algorithms to predict -bulge turn structural motifs from sequence patterns alone. It is noteworthy that new ANN algorithms have remarkably enhanced the identification of putative sequence signatures in proteins and genomic sequences (Saleh et al., 2001).

The original design strategy of examining the role of a two-residue spacer between APhe residues in a smaller peptide was to examine the preference of such a sequence to adopt the 3₁₀ helical conformation. It is found that, as per the design strategy, the molecular structure assumes the 3₁₀ helical conformation in the solid state. This structure enunciates that the positioning of APhe residues at the i-th and i+3rd positions alone suffices for the formation of a 3₁₀-helix. The 3₁₀ helical conformation in the present structure results in stacking of APhe residues one above the other, promoting the formation of aromatic slabs in the crystal packing. More studies will certainly augment our understanding regarding the role of various natural amino acids in the spacer region and the consequences of their interactions on peptide conformation. Nevertheless, observations from the present structure can be incorporated during the design of longer peptides of the desired fold.