Structural, functional, and mutational analysis of the NblA protein provides insight into possible modes of interaction with the phycobilisome

Abstract

The enormous macromolecular phycobilisome antenna complex (>4 MDa) in cyanobacteria and red algae undergoes controlled degradation during certain forms of nutrient starvation. The NblA protein (approximately 6 kDa) has been identified as an essential component in this process. We have used structural, biochemical, and genetic methods to obtain molecular details on the mode of action of the NblA protein. We have determined the three-dimensional structure of the NblA protein from both the thermophilic cyanobacterium Thermosynechococcus vulcanus and the mesophilic cyanobacterium Synechococcus elongatus sp. PCC 7942. The NblA monomer has a helix-loop-helix motif which dimerizes into an open, four-helical bundle, identical to the previously determined NblA structure from Anabaena. Previous studies indicated that mutations to NblA residues near the C terminus impaired its binding to phycobilisome proteins in vitro, whereas the only mutation known to affect NblA function in vivo is located near the protein N terminus. We performed random mutagenesis of the S. elongatus nblA gene which enabled the identification of four additional amino acids crucial for NblA function in vivo. This data shows that essential amino acids are not confined to the protein termini. We also show that expression of the Anabaena nblA gene complements phycobilisome degradation in an S. elongatus NblA-null mutant despite the low homology between NblAs of these cyanobacteria. We propose that the NblA interacts with the phycobilisome via "structural mimicry" due to similarity in structural motifs found in all phycobiliproteins. This suggestion leads to a new model for the mode of NblA action which involves the entire NblA protein.

FIGURE 1.

Amino acid alignment of NblA from various organisms. S. elongatus (Syn7942), Anabaena sp. PCC 7120 (Anab7120), Nostoc punctiforme (Nostoc73102), Synechococcus sp. JA-2-3B (SynJA-2-3B), Synechococcus sp. JA-3-3Ab (SynJA-3-3Ab), Synechocystis sp. PCC 6803 (Syn6803), Thermosynechococcus elongatus BP-1 (Thermosyn elo), T. vulcanus (Thermosyn vul), Crocosphaera watsonii WH 8501 (Croco8501), Cyanidioschyzon merolae strain 10D (Cyanidioschyzon), Porphyra purpurea (Porphyra), Cyanidium caldarium (Cyanidium), Gracilaria tenuistipitata var. liui (Gracilaria), Aglaothamnion neglectum (Aglaothamnion), Galdieria sulphuria (Galdieria), and F. diplosiphon (Fremyella). a and b denote chromosomal and plasmid encoded NblAs, respectively. Synechocystis sp. PCC 6803 possesses two chromosomally encoded NblAs. The black background indicates amino acids identical in at least 18 out of the 19 sequences. Similar amino acids that are present in at least 12 sequences are indicated by gray background. Stars indicate the positions of mutations in non-bleaching mutants of S. elongatus described below. Numbering is according to NblA of S. elongatus. Sequences selected for this alignment represent NblA proteins from a verity of organisms including cyanobacteria and red algae as well as mesophilic and thermophilic cyanobacteria.

FIGURE 2.

NblA crystal structures. Monomers A and B are depicted in cyan and magenta schematics, whereas side chains are colored according to the CPK system. A, Tv-NblA+urea dimer; arrows indicate urea molecules bound to structure, and white circles show dimer stabilizing contacts as described under “Results.” B, Tv-NblA asymmetric unit; circles show dimer-dimer stabilizing interactions. C, dimer of Se-NblA; circles show two areas with polar interactions which stabilize the dimer.

FIGURE 3.

Effects of manipulations of NblA on phycobilisome degradation. A-E, cultures of the relevant strains (round plates at the left of the figure) and schematic presentation of native nblA gene loci and the relevant molecular modifications. A, nblA of S. elongatus (WT). B, a strain possessing insertional inactivated nblA (AΩ). C, AΩ containing native nblA of S. elongatus in a neutral site (NS) in the genome (AΩ/Se-nblA). D, AΩ containing the L18P mutated NblA in a neutral site in the genome. E, AΩ containing the promoter region of nblA of S. elongatus fused to the coding region of the chromosomally located NblA of Anabaena PCC 7120 inserted in a shuttle vector (AΩ/An-nblA). km^R and spc^R stand for kanamycin and spectinomycin resistance cassettes, respectively. F, absorbance spectra of cultures of wild type (black), AΩ (blue), AΩ/Se-nblA (green), AΩ/Se-nblA L18P (red), and AΩ/An-nblA (purple). Red and blue curves overlap. Cultures were starved for nitrogen for 48 h before spectral analysis. Absorbance maxima at 620 nm and 680 nm represent PC and chlorophyll a (chl), respectively.

FIGURE 4.

Positions of mutations inducing the non-bleaching phenotype on the S. elongatus NblA dimer. Stars indicate the approximate position of the S9F mutation identified previously (10) but could not be identified in the electron density map.

FIGURE 5.

Calculated surface electrostatic potentials of PC and PEC monomers and molecular structures of dimers of NblA from three organisms: T. vulcanus (Tv), S. elongatus (Se), and Anabena (An). Surface potentials were calculated using the in vacuo approximation of the Pymol system. The distribution of patches of positive or negative potentials are shown in blue and red, respectively. The Tv-PC (1KTP crystal structure), Se-PC, An-PC, and An-PEC (molecular models) are shown from the faces that point outward from the PBS rod substructure. The black ovals show the position of the α-subunit Y-A helices indicated by Bienert et al. (18) as interacting with the An-NblA protein. The round circles indicate the positions of the N and C termini on the crystal structures of the NblA proteins.

FIGURE 6.

Comparison between Nbla and PC helix-loop-helix fragments. NblA is shown as both monomer (red) and dimer (green). The Tv-PC structure (1KTP) was carved to show five structures which show the same motif; helices X-Y-A (blue), helices G-H (α-subunit in magenta, and β-subunit in wheat), helices E-F-F′ (α-subunit in yellow, and β-subunit in orange). Notice that the two long helices of the NblA dimer form a non-covalently bonded helix-loop-helix motif as well (black circle).

FIGURE 7.

Hypothetical models for the mode of NblA-PBS interaction. A rod structure was built using the Tv-PC 1KTP crystal structure and its symmetry-related monomers. The interface between two (αβ)₆ hexamers is shown, with an orifice formed between two PC monomers (blue and yellow schematic representation). Other PC monomers are shown in white ribbon for clarity. Panel A, a NblA dimer (red) has wedged itself into the orifice parallel to helix H of both PC monomers (white circles). The rod assembly and the NblA dimer were treated as rigid bodies. Panel B shows how the two long helices of the NblA dimer can mimic the G-H helices of a PC monomer, with the small helices disrupting the interface contacts.