The Structure of a Cyanobacterial Bicarbonate Transport Protein, CmpA

Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO2 over O2, cyanobacteria have developed highly efficient CO2-concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here, we have described the structure of the bicarbonate-binding protein CmpA in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein NrtA. CmpA binds carbonic acid at the entrance to the ligand-binding pocket, whereas bicarbonate binds in nearly an identical location compared with nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca2+ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal appears to be highly cooperative and suggests that CmpA may co-transport bicarbonate and calcium or that calcium acts a cofactor in bicarbonate transport. Cyanobacteria, blue-green algae, are the most abundant autotrophs in aquatic environments and form the base of the food chain by fixing carbon and nitrogen into cellular biomass. To compensate for the low selectivity of Rubisco for CO2 over O2, cyanobacteria have developed highly efficient CO2-concentrating machinery of which the ABC transport system CmpABCD from Synechocystis PCC 6803 is one component. Here, we have described the structure of the bicarbonate-binding protein CmpA in the absence and presence of bicarbonate and carbonic acid. CmpA is highly homologous to the nitrate transport protein NrtA. CmpA binds carbonic acid at the entrance to the ligand-binding pocket, whereas bicarbonate binds in nearly an identical location compared with nitrate binding to NrtA. Unexpectedly, bicarbonate binding is accompanied by a metal ion, identified as Ca2+ via inductively coupled plasma optical emission spectrometry. The binding of bicarbonate and metal appears to be highly cooperative and suggests that CmpA may co-transport bicarbonate and calcium or that calcium acts a cofactor in bicarbonate transport. Cyanobacteria are the most abundant microorganisms in aquatic environments and play a key role in the global carbon cycle (1Whitton B.A. Potts M. The Ecology of Cyanobacteria: Their Diversity in Time and Space. Kluwer Academic Publishers, Norwell, MA2000Google Scholar). It is estimated that these photosynthetic microbes are responsible for ∼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 CO2 declined and O2 increased (2Gould S.J. The Book of Life: An Illustrated History of the Evolution of Life on Earth, 2nd Ed.. W. W. Norton & Co., New York2001Google Scholar). Because O2 can compete with CO2 for binding to the carbon-fixing enzyme Rubisco (3Badger M.R. Price G.D. J. Exp. Bot. 2003; 54: 609-622Crossref PubMed Scopus (610) Google Scholar), cyanobacteria evolved the most effective CO2-concentrating mechanism (CCM) 2The abbreviations used are: CCM, CO2-concentrating mechanism; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase; rTEV, recombinant tobacco etch virus; HEPPS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; ICP-OES, inductively coupled plasma optical emission spectrometry; ICP-MS, inductively coupled plasma mass spectrometry. that allows them to concentrate CO2 levels around Rubisco up to 1000-fold. The CCM involves the import and accumulation of inorganic carbon as HCO3− in the cytoplasm and subsequent conversion to CO2 in the protein microcompartment called the “carboxysome” via carbonic anhydrase. 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 (4Omata T. Price G.D. Badger M.R. Okamura M. Gohta S. Ogawa T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13571-13576Crossref PubMed Scopus (196) Google Scholar). This transporter is composed of four polypeptides, a high affinity solute-binding lipoprotein (CmpA), an integral membrane permease (CmpB), a cytoplasmic ATPase (CmpD), and an ATPase/solute-binding fusion protein (CmpC) that regulates transport (Fig. 1). The CmpABCD transporter is the highest affinity bicarbonate transporter of cyanobacteria (4Omata T. Price G.D. Badger M.R. Okamura M. Gohta S. Ogawa T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13571-13576Crossref PubMed Scopus (196) Google Scholar). This affinity is predominantly conferred by the binding of bicarbonate to CmpA (Kd = 5 μm) (5Maeda S. Price G.D. Badger M.R. Enomoto C. Omata T. J. Biol. Chem. 2000; 275: 20551-20555Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). CmpA is anchored to the periplasmic face of the cytoplasmic membrane via a lipid anchor attached to a conserved cysteine (6Omata T. Ogawa T. Plant Physiol. 1986; 80: 525-530Crossref PubMed Google Scholar). 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 (7Omata T. Plant Cell Physiol. 1995; 36: 207-213Crossref PubMed Scopus (84) Google Scholar). We recently published an analysis (8Koropatkin N.M. Pakrasi H.B. Smith T.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9820-9825Crossref PubMed Scopus (63) Google Scholar) 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. To better compare and contrast these two important ABC transport systems, the x-ray structure of CmpA has been determined in three different states, complexed with H2CO3 (carbonic acid) at pH 5.0, bound with HCO3− (bicarbonate) at pH 8.0, and in the absence of ligands at pH 8.0. The “C-clamp” structure of CmpA is remarkably homologous to that of NrtA (8Koropatkin N.M. Pakrasi H.B. Smith T.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9820-9825Crossref PubMed Scopus (63) Google Scholar) with ligands binding in the cleft between the two domains. At pH 5.0, nearly all of the bicarbonate is in the fully protonated, carbonic acid form. Under these conditions, the ligand is not bound deep inside the cleft, as was observed in NrtA, but rather at the entrance to the ligand-binding region. At pH 8.0, almost all of the dissolved inorganic carbon is in the bicarbonate form. In this case, the ligand is found deep inside the cleft of the C-clamp and, unexpectedly, is bound concomitantly with a calcium ion. Indeed, bicarbonate binds to CmpA if (and only if) calcium is also present. These and other results suggest that calcium and bicarbonate bind in a strongly cooperative manner and CmpA may transport calcium and bicarbonate simultaneously or that calcium acts as a cofactor in the bicarbonate transport. Cloning of cmpA from Synechocystis PCC 6803—The solute-binding domain of CmpA (residues 27–452) from Synechocystis PCC 6803 was cloned from genomic DNA. The cmpA gene was PCR-amplified with Platinum Pfx DNA polymerase (Invitrogen) according to the manufacturer's instructions and standard cycling conditions. The forward primer 5′-GGGAATTCCATATGGCCGGCAATCCCCCCGAT-3′ included an NdeI restriction site, and the reverse primer 5′-CCGCTCGAGTTAGACTTTTTTGATTGCCAAACTTTGCAG-3′ included an XhoI restriction site for cloning into pET-28a. The PCR product was purified with the QIAquick PCR purification kit (Qiagen) followed by digestion with both NdeI and XhoI at 37 °C overnight. The gene was separated from digestion by-products on a 1.0% agarose gel and purified by the QIAquick Gel purification kit (Qiagen). The purified cmpA fragment was then ligated into the expression vector pET-28a (Novagen). The pET-28a (Novagen) vector was previously modified such that the thrombin cleavage site was replaced with a recombinant tobacco etch virus (rTEV) cleavage site. Escherichia coli DH5α cells were transformed with the ligation mixture and then plated onto LB medium supplemented with 30 μg/ml kanamycin. Individual colonies were selected and cultured overnight, and plasmid DNA was extracted with the QIAprep Spin Miniprep kit (Qiagen). Positive clones were sequenced by Lark Technologies (Houston, TX). Protein Expression—For protein expression, E. coli Rosetta(DE3)pLysS cells (Novagen) were transformed with the pET28a-cmpA plasmid and plated onto LB medium supplemented with 30 μg/ml kanamycin. After ∼16 h growth at 37 °C, the colonies were harvested from the plates and used for the inoculation of 2 × 4-liter baffled flasks containing TB (Terrific Broth) medium supplemented with 30 μg/ml kanamycin and 30 μg/ml chloramphenicol. The cells were grown at 37 °C with aeration to an A600 of ∼0.4, at which time the temperature was lowered to 22.5 °C for the remainder of the experiment. Thirty minutes after lowering the temperature, protein expression was induced by the addition of 0.5 mm isopropyl 1-thio-β-d-galactopyranoside. The cells were allowed to grow for an additional 16 h before harvesting by centrifugation. The cell paste was frozen in liquid nitrogen and stored at -80 °C. Expression of the Selenomethionine-labeled Protein in E. coli—Selenomethionine-substituted protein was expressed via the methionine inhibitory pathway (9Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1091) Google Scholar). Rosetta(DE3)pLysS (Novagen) cells were transformed with the pET28a-CmpA plasmid and plated onto LB medium supplemented with kanamycin. After ∼16 h growth at 37 °C, the colonies were harvested from the plates and used for the inoculation of 2 × 4-liter baffled flasks containing M9 (42 mm Na2HPO4, 136 mm KH2PO4, 58 mm NaCl, 53.5 mm NHyCl) medium supplemented with 0.015 mm thiamine, 30 μg/ml kanamycin, and 30 μg/ml chloramphenicol. Cultures were grown at 37 °C to an A600 of ∼0.4 before the temperature was adjusted to 22.5 °C for the remainder of the growth. Cultures were grown to an A600 of ∼0.8 before each flask was supplemented with 200 mg each of l-lysine, l-threonine, and l-phenylalanine and 100 mg each of l-leucine, l-isoleucine, l-valine, and l-selenomethionine. After 30 additional min of growth, the cells were induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside and allowed to grow for 16 h. Cultures were harvested by centrifugation, and the cell paste was frozen in liquid nitrogen for storage at -80 °C. Protein Purification—Native and selenomethionine-substituted CmpA were purified in an identical manner. Approximately 10 g of frozen cells were thawed in 50 ml of cold buffer A (25 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole, pH 8.0) with one Complete EDTA-free protease inhibitor tablet (Roche Applied were on by four of separated by min of was by at °C for 30 min at × The was onto a with and with buffer A. After the was with ml of buffer buffer (25 mm NaH2PO4, 300 mm NaCl, 300 mm at pH 8.0) followed by of the protein from 10 to buffer were on and mm with 200 mm at °C. of the protease was to CmpA at a of of and the mixture was for h at °C and then 16 h at °C. The protease an which allowed the of CmpA from both CmpA and via affinity as described CmpA was at °C mm pH with mm and then onto a with the After the was with buffer the and then the protein was with a of mm in mm pH were on and mm with 100 mm CmpA was to on an of as by the of and Selenomethionine-labeled the of mm was to CmpA to conditions were the at both temperature and °C via the of were observed at temperature from 2 conditions were and of CmpA and complexed with were by of the into plates containing 50 mm pH 5.0, and mm additional of CmpA were determined at pH the of and These were by the into plates containing 50 mm HEPPS, pH 8.0, and 5 mm mm where to of × × mm in of the CmpA were with similar cell as in 1 and The of the was with one in the for is in CmpA Cell in a and is in pH pH pH pH Cell or in a the and selenomethionine-substituted protein were in the manner. were harvested from the plates and for in a composed of 100 mm or 100 mm and 300 mm The were included in all and at 5 mm the used for were to a containing 100 mm pH 5.0, or 100 mm HEPPS, pH 8.0, 300 mm NaCl, and the of The were then by in liquid from the selenomethionine-substituted and CmpA complexed with were on a at the The x-ray were with and with W. PubMed Scopus Google Scholar). x-ray are in on the CmpA grown in the presence of additional were from a x-ray at and The x-ray were and a on the three and and were by at in The were on a at with The was a was the and from the structure of CmpA was via with x-ray from the selenomethionine-substituted protein The J. Biol. 1999; PubMed Scopus Google Scholar) was used to and the of the and the Biol. 2000; PubMed Scopus Google Scholar) was then used to and protein of for and the were the A. PubMed Scopus Google Scholar). of with and the to for all and are in similar of were in the most in the allowed in the allowed and in the In all three are in the allowed of the and the high for these is and with the The of to or and both are in Ca2+ is in Ca2+ binding and is in a two that the of the solute-binding is of a that two for of the calcium and in the protein was inductively coupled plasma optical emission A was were at of and and was at The was after with were also for both calcium and were A for other was inductively coupled plasma mass A was This only the presence of and at The molecular structure of CmpA with carbonic acid is in CmpA is an protein and to the protein Biol. PubMed Scopus Google Scholar). to the protein NrtA, CmpA is composed of two and in a C-clamp The of each domain of a by which is domain is composed of three different of the and This domain and as a by and domain is composed of of the and The of domain is similar to that of domain with a by and In domain two and that are to the The two of CmpA are by and on of the solute-binding cleft by on the of the in each domain CmpA to of the protein Biol. PubMed Scopus Google Scholar), which also the for and A. J. Biol. Chem. Full Text PDF PubMed Google 2003; Full Text Full Text PDF PubMed Scopus Google J. Mol. Biol. PubMed Scopus Google Full Text Full Text PDF PubMed Scopus Google S. Biol. PubMed Scopus Google Scholar). similar to NrtA, is amino these The 100 of CmpA are in an with the of the in the These form that around the of the structure and the two domains. observed in NrtA, the on the face to the ligand-binding cleft of CmpA is almost composed of solute-binding and have a similar and, analysis of the NrtA structure, to suggest that such a may of to the by of the solute-binding protein with the The between the CmpA and NrtA is that the CmpA is in a that of NrtA. This suggests that at pH = = is in the form. with a was observed but not the solute-binding cleft At pH 5.0, nearly all of the is in the protonated, carbonic acid form. it appears that carbonic acid not bind in the ligand-binding and, in not of the the of carbonic acid is of the of but not with other in the is the carbon in the aquatic environment of Synechocystis and binds with a of it is that this carbonic in it is also that the of carbonic acid an between bicarbonate and the of the domain to of to the binding This of of ligand to of the transport protein was in the of the protein S. M. Pakrasi H.B. Smith T.J. J. Mol. Biol. 2003; PubMed Scopus Google of the CmpA and NrtA of the of CmpA and NrtA in and The two with a of over of bicarbonate and Ca2+ in the of nitrate in the for all and are as that the for in and are identical and were from the of the CmpA and NrtA amino To the structure of CmpA with the pH of the medium was increased to pH = = At this nearly all of the dissolved CO2 is in the bicarbonate form. It is important to at this bicarbonate is observed in the ligand-binding cleft or not it was to the In both bicarbonate is in the binding cleft by the of the two of CmpA and the C-clamp to over the bound ligands Unexpectedly, a metal is also observed to the bicarbonate and bound by from both the and domains. is that bicarbonate binding and transport by CmpA is The metal is by amino acid and one bicarbonate in a The ligands are by chain of and a from The of the metal coordination is by chain from and which are and of the The a at the of the coordination sphere and was highly of a the and of the coordination sphere that the bound metal was Ca2+ or Biol. 2000; PubMed Scopus Google Biol. PubMed Scopus Google Scholar). It is important to that this metal was not observed in the structure at pH 5.0, and the of the are to be by the from pH to 8.0. To which is the likely metal bound to both were into the and was in Ca2+ to a of nearly identical to the of the In was at this the as a of The that the bound metal was a Ca2+ was by analysis the and both and Ca2+ are observed to have similar it is not as to by and it the are In Ca2+ is most observed by from such as and Indeed, that the only metal in the CmpA was calcium at a of for 1 of Because the purification and not the of the metal is It is that CmpA the Ca2+ from E. coli expression and it the purification Indeed, this has been observed in a of high affinity ABC the S. M. Pakrasi H.B. Smith T.J. J. Mol. Biol. 2003; PubMed Scopus Google Scholar) and the M. and T. J. is bound to Ca2+ in a in the binding The of bicarbonate is at the and is by the Ca2+ that is The of bicarbonate is from the of that is The is also of the of The of O2, that the of the binding cleft is of the and of the chain of and to two To the role of calcium on bicarbonate 10 mm was to the CmpA medium at pH 8.0. This structure was to a to with and of and In this the C-clamp of CmpA is in the and is of both metal and bicarbonate not This structure of at pH is identical to that of CmpA complexed with carbonic with a of for all both Ca2+ and bicarbonate suggests that binding is bicarbonate or calcium to the bicarbonate and metal are found in the binding pocket, but both are by the addition of It be that compared with other solute-binding a The between the of the and of CmpA is only The between these two are found in that a to the binding (Fig. This that with bicarbonate and into the ligand-binding cleft This of a to the of bicarbonate (Fig. To this CmpA were grown at pH in the presence of 10 mm and an additional mm The structure of CmpA with an and of and Unexpectedly, CmpA was observed in the with both Ca2+ and bicarbonate in the binding The from this structure and of CmpA with bicarbonate and are nearly with a of over all these results suggest that metal and bicarbonate bind to CmpA in a strongly cooperative manner. CmpA is a of the periplasmic binding protein and of two as a C-clamp with the cleft between the two domains. other of this CmpA not a This suggests that the binding is and that binding not of the The closest homologue of CmpA is the protein NrtA that and 61% amino acid sequence and We previously the 1.6-Å structure of NrtA from PCC 6803 complexed with nitrate (8Koropatkin N.M. Pakrasi H.B. Smith T.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9820-9825Crossref PubMed Scopus (63) Google Scholar). The of CmpA and NrtA can be with a of for (Fig. The most in the of these in with a in In the structure of NrtA, this to the In the solute-binding of CmpA with bicarbonate to The in CmpA that the bound bicarbonate are from sequence between CmpA and NrtA (8Koropatkin N.M. Pakrasi H.B. Smith T.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 9820-9825Crossref PubMed Scopus (63) Google Scholar). in CmpA and NrtA bind their and bicarbonate the but nitrate is in the cleft and the by the Ca2+ in CmpA. In both the on bicarbonate and nitrate is at the of each but this is in each In NrtA, the of is of the the (Fig. In this is replaced by (Fig. In analysis of the NrtA structure, we that the of for was to a to the of This in the in CmpA with also to the Ca2+ coordination the of lysine in NrtA is replaced by which with the on the of the To Ca2+ in other compared with NrtA. In and the Ca2+ coordination whereas in NrtA, these are replaced by and and are for the and O2 of the of in is in NrtA. The chain of is from the of and likely in the of the lysine a metal in the CmpA structure was Ca2+ or were for the bound metal of the of in the coordination sphere and the This was by analysis that identified calcium as the metal with the protein with of Ca2+ of CmpA. Because of the purification or of it is that CmpA Ca2+ expression in E. coli and it subsequent purification This has been observed for periplasmic that bind their ligand with a of binding of both metal and has been observed with other binding with or has been observed for both the and periplasmic P.A. PubMed Scopus Google M. W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of these complexed with and that the not only to the coordination sphere of the metal but and with the protein as 2003; PubMed Scopus Google Biol. PubMed Scopus Google Scholar). In these the the of the protein and binds with the PubMed Scopus Google Scholar). A similar with the binding of calcium and bicarbonate in CmpA. The of and bicarbonate on ligand binding are but suggest that metal and bicarbonate bind in a strongly cooperative manner. The that both metal and bicarbonate are found in the binding at pH 8.0, were purification and that these at bind with high is to grown in the absence of bicarbonate and is to the metal in This is to both bicarbonate and calcium from the solute-binding the are grown at pH 5.0, the dissolved CO2 is nearly all in the carbonic acid form and to bind to the solute-binding Under these conditions, metal is also to bind the that of the protein are to to a the pH is to These results strongly suggest that of metal or bicarbonate of the other This is by the that bicarbonate the of metal is to the This suggests that high of bicarbonate the to the bound and metal from CmpA. it that Ca2+ and bicarbonate binding and are highly Ca2+ is an metal in a of the and of Ca2+ in has not been nearly as as it has in In Ca2+ is an of the of photosynthetic and may also be a for PubMed Scopus Google W. J. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). In the of calcium may be used to the binding affinity or selectivity of bicarbonate over the other such as In this calcium be a that transport an important It is to that only the of Synechocystis have the operon and that has levels of bicarbonate and calcium and compared with and M. of The of the and & New Scholar). it is to that the calcium bicarbonate is into the is that suggests that CmpABCD is in such a Indeed, is that calcium is in via a of to the cmpABCD operon T. J. PubMed Scopus Google Scholar). are to the role of calcium in the transport of We the of the at the at for with and We also for the of these for the expression and M. of for and