Bicarbonate transport in Synechocystis 6803
Cyanobacteria are the most abundant microorganisms in aquatic environments and play a key role in the global carbon cycle. It is estimated that these photosynthetic microbes are responsible for about 50% of carbon fixation in the oceans. Over their 2.7-billion-year existence, cyanobacteria had to adapt to a changing gaseous environment where the levels of carbon dioxide declined and gaseous oxygen increased. Because O2 can compete with CO2 for binding to the carbon-fixing enzyme Rubisco, cyanobacteria evolved the most effective CO2-concentrating mechanism (CCM) that allows them to concentrate CO2 levels around Rubisco up to 1000-fold. The CCM involves the import and accumulation of inorganic carbon as bicarbonate in the cytoplasm and subsequent conversion to CO2 in the protein microcompartment called the “carboxysome” via carbonic anhydrase.
Bicarbonate transport
One component of this CCM machinery in Synechocystis PCC 6803 is the cmpABCD operon that encodes a high affinity bicarbonate ABC transporter that is induced under low CO2 conditions. This transporter is composed of four polypeptides, a high affinity solute-binding lipoprotein (CmpA), anintegral membrane permease (CmpB), a cytoplasmic ATPase (CmpD), and an ATPase/solute-binding fusion protein(CmpC) that regulates transport. The CmpABCD transporter is the highest affinity bicarbonate transporter of cyanobacteria. This affinity is predominantly conferred by the binding of bicarbonate to CmpA. CmpA is anchored to the periplasmic face of the cytoplasmic membrane via a lipid anchor attached to a conserved cysteine. The closest known homologue of CmpA is NrtA, the solute-binding protein of the nitrate-specific NrtABCD transporter that is 48% identical and 61% similar in amino acid sequence. We recently published an analysis of the 1.6-Å structure of NrtA complexed with nitrate to elucidate the molecular determinants of nitrate specificity. From this structure, it seemed likely that the nitrate versus bicarbonate specificity was mainly due to the replacement of a lysine in the nitrate coordination sphere in NrtA with a glutamate in CmpA.
The cmp operon
Schematic representation of the assembled CmpABCD bicarbonate transporter. CmpA is tethered to the periplasmic membrane by a flexible linker and captures bicarbonate in the periplasm for delivery to the transmembrane complex created by CmpB. In many ABC transporters, the transmembrane pore is created by a dimer of two transmembrane-spanning polypeptides. CmpC and CmpD are ATPases that couple ATP hydrolysis to bicarbonate transport through the pore. CmpC is unique in that it contains a C-terminal solute-binding domain homologous to CmpA.
CmpA structure
The structure of CmpA. Left: ribbon diagram of the CmpA crystal structure at pH 5.0 with carbonic acid. The protein is gradiently colored blue to red as the chain extends from the N to the C terminus. The view is of the front of the C-clamp, which opens to the ligand-binding cleft. Carbonic acid is depicted as spheres. Right: ribbon diagram of the CmpA crystal structure at pH 8.0 with bicarbonate, in an identical view and coloring as in the left figure. Bicarbonate is also depicted as spheres. Note the difference in the position of carbonic acid and bicarbonate in the ligand-binding cleft.
Bicarbonate versus cabonic acid binding
Representative electron density in the ligand-binding cleft. Left: representative electron density of carbonic acid in CmpA. The map shown was calculated with the coefficients (2Fo-Fc), where Fo is the experimentally observed structure factor amplitude and Fc is the calculated structure factor amplitude from the model. The map is contoured at 1.5 sigma. Right: representative electron density of bicarbonate and Ca2&. The map shown was calculated with the coefficients (2Fo-Fc), also contoured at 1.5 sigma.
NrtA versus CmpA ligand binding
Comparison of the CmpA and NrtA structures. Left: ball-and-stick representation of bicarbonate and calcium in the CmpA-binding cleft. Right: ball-and-stick representation of nitrate in the NrtA-binding cleft. Protein-ligand interactions for all potential hydrogen-bonding and electrostatic interactions are depicted as dashed lines. Note that the view for ball-and-stick representations are identical and were obtained from the overlay of the CmpA and NrtA amino acids.
The unexpected result was that calcium and bicarbonate bind in a synergistic fashion - calcium is not found bound in the absence of bicarbonate and bicarbonate does not bind without calcium. Further, the carbonic acid does bind, but at the entrance to the mouth rather than deep into the proper binding site.
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