Novel Anion-independent Iron Coordination by Members of a Third Class of Bacterial Periplasmic Ferric Ion-binding Proteins

The uptake of the element iron is vital for the survival of most organisms. Numerous pathogenic Gram-negative bacteria utilize a periplasm-to-cytosol ATP-binding cassette transport pathway to transport this essential atom in to the cell. In this study, we investigated the Yersinia enterocolitica (YfuA) and Serratia marcescens (SfuA) iron-binding periplasmic proteins. We have determined the 1.8-Å structures of iron-loaded (YfuA) and iron-free (SfuA) forms of this class of proteins. Although the sequence of these proteins varies considerably from the other members of the transferrin structural superfamily, they adopt the same three-dimensional fold. The iron-loaded YfuA structure illustrates the unique nature of this new class of proteins in that they are able to octahedrally coordinate the ferric ion in the absence of a bound anion. The iron-free SfuA structure contains a bound citrate anion in the iron-binding cleft that tethers the N- and C-terminal domains of the apo protein and stabilizes the partially open structure. The uptake of the element iron is vital for the survival of most organisms. Numerous pathogenic Gram-negative bacteria utilize a periplasm-to-cytosol ATP-binding cassette transport pathway to transport this essential atom in to the cell. In this study, we investigated the Yersinia enterocolitica (YfuA) and Serratia marcescens (SfuA) iron-binding periplasmic proteins. We have determined the 1.8-Å structures of iron-loaded (YfuA) and iron-free (SfuA) forms of this class of proteins. Although the sequence of these proteins varies considerably from the other members of the transferrin structural superfamily, they adopt the same three-dimensional fold. The iron-loaded YfuA structure illustrates the unique nature of this new class of proteins in that they are able to octahedrally coordinate the ferric ion in the absence of a bound anion. The iron-free SfuA structure contains a bound citrate anion in the iron-binding cleft that tethers the N- and C-terminal domains of the apo protein and stabilizes the partially open structure. Metal ions are indispensable components of biological systems. Iron is required for the growth and survival of nearly all living organisms (1Guerinot M.L. Annu. Rev. Microbiol. 1994; 48: 743-772Crossref PubMed Scopus (538) Google Scholar). Although iron is the most abundant transition element and the fourth most plentiful element in the Earth's crust, this important atom remains a limiting factor for growth for the vast majority of organisms. Because of the extremely low solubility of Fe3+ (∼10-18m) at neutral pH in aerobic environments most organisms are faced with the problem of obtaining enough iron from their environment (2Braun V. Killmann H. Trends Biochem. Sci. 1999; 24: 104-109Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Although iron is not readily available to most organisms, in vivo iron is a key component of a number of essential metabolic enzymes, including ribonucleotide reductase and cytochromes. Its biological function is almost entirely dependent on its incorporation into protein molecules. Through the variation of the coordinating ligands surrounding iron, its redox potential can be altered between -300 and +700 mV, making it ideally suited to participate in a wide range of electron transfer reactions involved in intermediary metabolism (3Andrews S.C. Robinson A.K. Rodriguez-Quinones F. FEMS Microbiol. Rev. 2003; 27: 215-237Crossref PubMed Scopus (1851) Google Scholar). Although the reactivity of iron makes it uniquely suited for numerous biological applications, it also contributes to a variety of undesirable effects. Free iron has the ability to generate toxic derivatives within the body (4Wessling-Resnick M. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 285-314Crossref PubMed Scopus (60) Google Scholar). The highly reactive hydroxyl radical, a product of Fenton chemistry, can damage lipids by inducing the formation of unsaturated bonds, decrease membrane fluidity, and cause cell lysis. Thus, it is vital that biological systems maintain control of the chemical environment of iron. Most organisms have developed specialized systems for retrieval, transport, and storage of this vital element to maintain it in a non-toxic state. In higher organisms such as vertebrates, there is little free iron present in the extracellular compartment because of the presence of the monomeric glycoproteins transferrin (in sera) and lactoferrin (on mucosal surfaces), which are high affinity bilobed iron-binding proteins. Lactoferrin and transferrin are each composed of a single polypeptide chain of ∼650 amino acid residues that are arranged in two homologous globular lobes, the N- and C-lobes, representing the N- and C-terminal halves of the molecules (5Baker E.N. Anderson B.F. Baker H.M. MacGillivray R.T.A. Moore S.A. Peterson N.A. Shewry S.C. Tweedie J.W. Adv. Exp. Med. Biol. 1998; 443: 1-14Crossref PubMed Scopus (47) Google Scholar). Each lobe is further subdivided into two domains with the specific iron binding sites in the intradomain cleft of each lobe. The iron atom is bound by identical iron-binding ligands in each lobe, two tyrosines, a histidine, and an aspartic acid residue (6Anderson B.F. Baker H.M. Norris G.E. Rice D.W. Baker E.N. J. Mol. Biol. 1989; 209: 711-734Crossref PubMed Scopus (548) Google Scholar, 7Bailey S. Evans R.W. Garrat R.C. Gorinsky B. Hasnain S. Horsburgh C. Jhoti H. Lindley P.F. Mydin A. Sarra R. Watson J.L. Biochemistry. 1988; 27: 5804-5812Crossref PubMed Scopus (369) Google Scholar). The ability of transferrin and lactoferrin to bind iron also depends on the presence of a carbonate anion. The presence of these glycoproteins throughout the body of the host organism reduces the levels of free ferric iron below that required to support bacterial growth. In response to the problem of iron scarcity, extracellular Gram-negative pathogenic bacteria have developed a variety of different high affinity iron acquisition systems to survive. One strategy that is effective in a variety of environments involves the synthesis and secretion of small iron chelating molecules, termed siderophores (8Neilands J.B. J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1231) Google Scholar). Siderophores function by complexing and removing iron from the host proteins or by scavenging it from precipitates of ferric hydroxide, as they possess affinity constants in excess of host proteins. An alternate iron uptake system utilized by Gram-negative bacteria has also been discovered in a number of human and veterinary pathogens (9Gray-Owen S.D. Schryvers A.B. Trends Microbiol. 1996; 4: 185-191Abstract Full Text PDF PubMed Scopus (257) Google Scholar, 10Schryvers A.B. Stojiljkovic I. Mol. Microbiol. 1999; 32: 1117-1123Crossref PubMed Scopus (206) Google Scholar). These bacteria possess outer membrane surface receptors that are used for acquiring iron directly from transferrin and/or lactoferrin. The bacteria use this alternative mechanism for iron acquisition as they are unable to produce siderophores. Even though the specific lactoferrin and transferrin outer membrane receptors are distinct complexes, the mechanism of iron removal is thought to occur through similar mechanisms (9Gray-Owen S.D. Schryvers A.B. Trends Microbiol. 1996; 4: 185-191Abstract Full Text PDF PubMed Scopus (257) Google Scholar). Following transport across the outer membrane, the ensuing transport of iron into the cell is mediated by an ATP-binding cassette transport system consisting of a periplasmic-binding protein and an inner membrane transport complex (11Koster W. Res. Microbiol. 2001; 152: 291-301Crossref PubMed Scopus (213) Google Scholar). This transport system belongs to the superfamily of ATP-binding cassette transporters that includes a broad and diverse group of import and export systems found in prokaryotes and eukaryotes (12Schmitt L. Tampe R. Curr. Opin. Struct. Biol. 2002; 12: 754-760Crossref PubMed Scopus (276) Google Scholar). The periplasmic-binding protein is required to transport the complex across the periplasm and release it at the inner membrane permease complex. This inner membrane complex consists of at least two proteins, one to span the membrane and transport the substrate across the inner membrane and another that contains an ATP-binding cassette and that can hydrolyze ATP to provide the energy required for transport (13Higgins C.F. Res. Microbiol. 2001; 152: 205-210Crossref PubMed Scopus (482) Google Scholar). The iron uptake pathways from host transferrin in pathogenic Neisseria species and Haemophilus influenzae have received considerable attention because of their predicted importance in vivo, which has been established experimentally for gonococcal infection in humans (14Cornelissen C.N. Kelley M. Hobbs M.M. Anderson J.E. Cannon J.G. Cohen M.S. Sparling P.F. Mol. Microbiol. 1998; 27: 611-616Crossref PubMed Scopus (180) Google Scholar). This pathway is dependant upon the periplasmic FbpA (ferric-binding protein A) (15Khun H.H. Kirby S.D. Lee B.C. Infect. Immun. 1998; 66: 2330-2336Crossref PubMed Google Scholar, 16Kirby S.D. Gray-Owen S.D. Schryvers A.B. Mol. Microbiol. 1997; 25: 979-987Crossref PubMed Scopus (20) Google Scholar). FbpA was shown to possess similar properties to the transferrins (17Nowalk A.J. Tencza S.B. Mietzner T.A. Biochemistry. 1994; 33: 12769-12775Crossref PubMed Scopus (49) Google Scholar, 18Adhikari P. Kirby S.D. Nowalk A.J. Veraldi K.L. Schryvers A.B. Mietzner T.A. J. Biol. Chem. 1995; 42: 25142-25149Abstract Full Text Full Text PDF Scopus (79) Google Scholar) to the extent that they have been called bacterial transferrins (19Taboy C.H. Vaughan K.G. Mietzner T.A. Aisen P. Crumbliss A.L. J. Biol. Chem. 2001; 276: 2719-2724Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Structural studies revealed a nearly identical set of amino acid residues involved in iron coordination (20Bruns C.M. Norwalk A.J. Avrai A.S. McTigue M.A. Vaughan K.A. Mietzner T.A. McRee D.E. Nat. Struct. Biol. 1997; 4: 919-924Crossref PubMed Scopus (162) Google Scholar) that led to the suggestion that the forces of convergent evolution had selected for an optimal coordination mode. However, sequence and spectral differences observed with a from the S.D. W. A. M. Schryvers A.B. 1998; PubMed Scopus Google Scholar) that the with transferrin not be a of this class of proteins. structural studies with the M. protein revealed a of iron coordination residues and a carbonate anion McRee D.E. Schryvers A.B. J. PubMed Scopus Google Scholar). SfuA of Serratia marcescens L. A. V. J. 1989; PubMed Google Scholar) and YfuA of Yersinia enterocolitica A. J. J. Med. Microbiol. PubMed Scopus Google Scholar) are also of because they are of binding and the Fe3+ ion S. Infect. Immun. 2001; PubMed Scopus Google Scholar, A. B. V. J. PubMed Google Scholar). The sequence of YfuA and SfuA with the from Haemophilus and Neisseria a considerable of sequence and of key iron amino However, the spectral properties of the iron-loaded forms of these proteins from transferrins and from the Haemophilus and Neisseria that they utilize a of iron In this study, we present the structure of iron-loaded YfuA and the iron-free SfuA structure. between the iron-loaded and iron-free structures has to the of iron and the of the ferric ion in protein that YfuA and SfuA coordinate iron in a unique YfuA and SfuA not a anion to bind a ferric This new class of bacterial ferric proteins is the of the transferrin structural superfamily able to bind and transport iron in the absence of a anion. and was from a of S. marcescens from the with a an at the by a and with a a the The product was with and and into a of the with and The has a between the binding and similar strategy was utilized to the from enterocolitica from that the a in of a The and the with the for YfuA and SfuA The into for of SfuA and and with their that the into the proteins be and to the The proteins and the periplasmic a McRee D.E. Schryvers A.B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The protein pH at and to anion as a The pH at and with a of for SfuA and for The of the was by utilized in or at on a consisting of pH as the for the from to Each was in and for SfuA and YfuA determined by high single was for and two forms of YfuA used for at for SfuA and at for YfuA by the with of protein and of and an The for SfuA was pH and for YfuA was and pH of SfuA to the group and YfuA to the group in The by from the with a The SfuA was into a and pH for it was in The YfuA was into with The of proteins at on a on a with a and the J.W. Biol. 1999; PubMed Scopus Google Scholar). for the proteins are in and for the YfuA and SfuA cell in is the for the set of and set of of for range of of protein of from in is the for the set of and set of of in a new and iron-loaded YfuA structure was and used as a to the SfuA structure. the YfuA single the Biol. 2002; PubMed Scopus Google Scholar) to the atom the from the iron, and bound to from these atom by use of the 2002; Scopus Google Scholar). The by and with the P. Biol. 1998; PubMed Scopus Google Scholar). The of to of almost the protein structure in electron with the A. Biol. 2002; PubMed Scopus Google Scholar). The structure and electron the D.E. J. Struct. Biol. 1999; PubMed Scopus Google Scholar). of and of the the D.E. J. Struct. Biol. 1999; PubMed Scopus Google Scholar) and with 1997; PubMed Scopus Google Scholar) to and the a function and for was by the of used to the bound iron, and molecules. The apo SfuA structure was determined by a the from the of 1994; PubMed Scopus Google Scholar). Because the structure of YfuA was in a with the SfuA two the N- and C-terminal domains of the protein had to be to the structure. of of the the D.E. J. Struct. Biol. 1999; PubMed Scopus Google Scholar) use of with A. Biol. 2002; PubMed Scopus Google and with 1997; PubMed Scopus Google Scholar) was to and the a function and for was the of used to the bound citrate and molecules. The for each structure are in I. of SfuA and YfuA in complex with citrate and iron, have each been in the and of the two structures the of all residues in of The was used to similar proteins in the on of the proteins L. C. Res. 1997; 25: PubMed Scopus Google Scholar). and SfuA and YfuA from and into a The of the proteins with amino acid to YfuA and SfuA in The presence of the in and export of the proteins into the periplasm of of to of The SfuA and YfuA proteins from the periplasm of and by Following and the protein to to The protein had a in to iron-loaded transferrins and from Neisseria and Haemophilus that are at these of the protein that at and for YfuA and the at for the H. influenzae FbpA used H. influenzae M. Following an high was a high of The structure of the iron-loaded YfuA was by single An set of was for the SfuA protein The structure of YfuA as a in to the SfuA structure. Structural and are in I. almost all of the protein the electron is and In the SfuA structure the two residues of the not in the electron and the residue of the YfuA was not by electron and was from the structure. The and the for each which that all of the residues are in as by the 1994; PubMed Scopus Google that these structures are of high SfuA and YfuA sequence at the amino acid and identical polypeptide structures of all of the periplasmic ferric proteins to in the an almost identical of SfuA and YfuA have a similar structure to the H. influenzae periplasmic the L. C. Res. 1997; 25: PubMed Scopus Google Scholar) for proteins with three-dimensional similar to SfuA and YfuA in the that these proteins possess the structural with C.M. Norwalk A.J. Avrai A.S. McTigue M.A. Vaughan K.A. Mietzner T.A. McRee D.E. Nat. Struct. Biol. 1997; 4: 919-924Crossref PubMed Scopus (162) Google Scholar). SfuA and YfuA sequence with However, the of the and YfuA structures with a of of the to most other periplasmic-binding protein structures and other members of the transferrin structural superfamily, the SfuA and YfuA molecules are composed of two domains termed the N- and The polypeptide chain of SfuA and YfuA between the N- and these two domains the iron-binding Each is composed of a by The two domains are by two that as a between the two YfuA is able to coordinate a single Fe3+ ion in an amino acid residues and a and The coordination observed in this structure is because of the presence of an residue and the absence of a coordinating anion. The of the is composed of a and the coordination and are at the The iron coordination is from with from to The YfuA structure a class of ferric periplasmic proteins that not a anion to the iron the iron binding residues of YfuA and that in YfuA is able to the anion in iron of the iron binding in in a new a of the high in the used for there are bound to the structure. Although these ions have in the function of the they to of the of the for and utilize amino acid residues from two molecules, which in the was shown that important for the of the protein C.H. H. A.K. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). In to molecules and an aspartic acid is also by a anion in the YfuA structure. The coordination range from to coordinating ligands surrounding each and coordination for ions in proteins have been in other protein structures L. Biochemistry. 1996; PubMed Scopus Google Scholar, S. M. M. M. H. Biochemistry. 1999; PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar, W. J. Mol. Biol. 1994; PubMed Scopus Google Scholar). SfuA ferric periplasmic protein structures determined to in the they from the iron-loaded to the iron-free In a structural of the apo (SfuA) and (YfuA) forms a the The and are similar with a in the of This of the two domains with other FbpA structures is because of the presence of the citrate observed in the iron-binding cleft of the SfuA structure that tethers the domains The high electron and the of the citrate in the binding cleft and on used the and they because of their of with the electron The citrate anion is able to the two domains by with two residues of each in the cleft the residue forms a with of and forms two with and through a from the C-terminal forms a with the of from the is able to two with and of the citrate anion. The citrate anion is not bound in a at the of the binding cleft in an to the observed in other members of the transferrin structural In the citrate anion is bound at the surface of the iron-binding cleft and in iron In the citrate have been for a bound ferric anion was observed in the iron-loaded YfuA further of the SfuA and YfuA structures that the citrate anion the of the N- and C-terminal domains as and not the of key residues in the of the iron-binding that the ferric ion from The in YfuA and SfuA are by residues and are similar to observed in and Thus, it is to that YfuA and SfuA are of similar in to observed in the and structures C.M. Anderson Vaughan K.G. Nowalk A.J. McRee D.E. Mietzner T.A. Biochemistry. 2001; PubMed Scopus Google Scholar). on structural it that is in the SfuA structure by the bound citrate and the SfuA structure not to a open The SfuA and YfuA structures adopt the periplasmic protein to most periplasmic proteins Mol. Microbiol. 1996; PubMed Scopus Google Scholar). These proteins possess structures in which the two domains are by two with periplasmic ferric proteins (20Bruns C.M. Norwalk A.J. Avrai A.S. McTigue M.A. Vaughan K.A. Mietzner T.A. McRee D.E. Nat. Struct. Biol. 1997; 4: 919-924Crossref PubMed Scopus (162) Google Scholar, McRee D.E. Schryvers A.B. J. PubMed Scopus Google they to the periplasmic-binding superfamily A. C. Res. 32: PubMed Google Scholar). Although the of these proteins are that YfuA and SfuA a new class in this iron-binding of proteins on coordination in with of the and ligands by structural studies that of periplasmic iron-binding proteins can be into at least The by H. influenzae FbpA (20Bruns C.M. Norwalk A.J. Avrai A.S. McTigue M.A. Vaughan K.A. Mietzner T.A. McRee D.E. Nat. Struct. Biol. 1997; 4: 919-924Crossref PubMed Scopus (162) Google a set of amino similar to transferrin and amino required for coordinating the bound anion that the coordination complex with a Most of these proteins are from human pathogens a pathway for acquiring iron directly from transferrin and/or lactoferrin and the to produce siderophores. Because transferrin and lactoferrin are the of iron for the pathway in these these be for this The from the species not an amino acid to the of that is involved in coordinating the anion and have been in the The in this that YfuA and SfuA possess all of the amino found in the and transferrin the of iron coordination is and a class of periplasmic ferric binding proteins the proteins possess two residues in the with a and acid residue from the that coordinate the ferric However, of a anion in the coordination of the iron YfuA an residue these two structures are this chain the same as the anion and is able to coordinate the ferric ion in a similar the anion by a protein this class of proteins the of the anion that is to be a for Fe3+ release for Neisseria and human transferrin H. Anderson Mietzner T.A. Crumbliss A.L. Biol. Chem. 2003; PubMed Scopus Google Scholar). the of iron for YfuA and its is and the potential of an iron strategy that not a anion is In Yersinia the that the required for of the and the that another uptake shown to be essential for infection in Mol. Microbiol. 1999; 32: PubMed Scopus Google because of an acquisition of iron at different of The protein is not to the periplasmic iron-binding proteins in this belongs to a of and proteins by the protein from Nat. Struct. Biol. 1999; PubMed Scopus Google that has a it from most periplasmic proteins. The ATP-binding cassette pathway is not required for infection in A. J. J. Med. Microbiol. PubMed Scopus Google Scholar, S. Infect. Immun. 2001; PubMed Scopus Google Scholar) for growth in that have been potential for survival in the has been not The two of ferric proteins have been from a because they residues and into the same on sequence Although the of the proteins that to the class and the shown to be vital for iron there is to is that these proteins of ferric proteins on alternate coordination or periplasmic proteins for transport of other such as The FbpA from M. McRee D.E. Schryvers A.B. J. PubMed Scopus Google Scholar) is a of a class that is by a unique iron coordination and a bound carbonate anion that coordinating members of this class have an residue at that is utilized to coordinate the carbonate anion in The residue at involved in coordinating the carbonate anion in varies members of this which differences in anion Although the of are found in other pathogens that possess uptake systems of acquiring iron directly from there is considerable in the of bacteria that possess to this protein Kirby McRee D.E. Schryvers A.B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, it is that this class of FbpA is involved in acquiring iron from a diverse range of iron is from the in that the between the ferric proteins not the the species these proteins. In this it is also to that the host human pathogens that utilize uptake from transferrin Neisseria have class the pathogens Haemophilus possess class The presence of two distinct of within members of the that the have been by or that of proteins present in an of the Although the different of iron coordination in FbpA specific for the they there is to support this The pathways are all of the uptake of iron in systems in a of in P. Kirby S.D. Nowalk A.J. Veraldi K.L. Schryvers A.B. Mietzner T.A. J. Biol. Chem. 1995; 42: 25142-25149Abstract Full Text Full Text PDF Scopus (79) Google Scholar, S.D. W. A. M. Schryvers A.B. 1998; PubMed Scopus Google Scholar, A. J. J. Med. Microbiol. PubMed Scopus Google Scholar, A. B. V. J. PubMed Google Scholar). However, this system on a of iron by the of the iron that is of of iron across the outer Thus, the different of be of acquiring ferric ions from different remains an open of the different of iron coordination also provide into the mechanism of iron removal and transport across the inner membrane, the mechanism was the However, at this little is the and there is the that the mechanism is the In we have determined the two structures of a new class of bacterial iron-binding proteins. protein are utilized with a for iron is also that this class of proteins has the ability to bind such as the citrate present in the SfuA the iron release by this class of proteins, structural have to be with and

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