Crystal Structure of Pasteurella haemolytica Ferric Ion-binding Protein A Reveals a Novel Class of Bacterial Iron-binding Proteins

Pasteurellosis caused by the Gram-negative pathogen Pasteurella haemolytica is a serious disease leading to death in cattle. To scavenge growth-limiting iron from the host, the pathogen utilizes the periplasmic ferric ion-binding protein A (PhFbpA) as a component of an ATP-binding cassette transport pathway. We report the 1.2-Å structure of the iron-free (apo) form of PhFbpA, which is a member of the transferrin structural superfamily. The protein structure adopts a closed conformation, allowing us to reliably assign putative iron-coordinating residues. Based on our analysis, PhFbpA utilizes a unique constellation of binding site residues and anions to octahedrally coordinate an iron atom. A surprising finding in the structure is the presence of two formate anions on opposite sides of the iron-binding pocket. The formate ions tether the N- and C-terminal domains of the protein and stabilize the closed structure, also providing clues as to probable candidates for synergistic anions in the iron-loaded state. PhFbpA represents a new class of bacterial iron-binding proteins. Pasteurellosis caused by the Gram-negative pathogen Pasteurella haemolytica is a serious disease leading to death in cattle. To scavenge growth-limiting iron from the host, the pathogen utilizes the periplasmic ferric ion-binding protein A (PhFbpA) as a component of an ATP-binding cassette transport pathway. We report the 1.2-Å structure of the iron-free (apo) form of PhFbpA, which is a member of the transferrin structural superfamily. The protein structure adopts a closed conformation, allowing us to reliably assign putative iron-coordinating residues. Based on our analysis, PhFbpA utilizes a unique constellation of binding site residues and anions to octahedrally coordinate an iron atom. A surprising finding in the structure is the presence of two formate anions on opposite sides of the iron-binding pocket. The formate ions tether the N- and C-terminal domains of the protein and stabilize the closed structure, also providing clues as to probable candidates for synergistic anions in the iron-loaded state. PhFbpA represents a new class of bacterial iron-binding proteins. Pasteurella haemolytica is the causative agent of the bovine respiratory disease called shipping fever or pneumonic pasteurellosis (1Whiteley L.O. Maheswaran S.K. Weiss D.J. Ames T.R. Kannan M.S. J. Vet. Intern. Med. 1992; 6: 11-22Crossref PubMed Scopus (139) Google Scholar). Bovine pneumonic pasteurellosis is the final stage of a complex multifactorial process typically involving stress and infectious organisms leading to pneumonia in cattle. Respiratory disease is a major cause of morbidity in the cattle industry resulting in a significant economic loss. In healthy cattle, P. haemolytica is present in low numbers in the nasal passages. However, following stress conditions such as shipment, commingling cattle from different sources, and overcrowding, respiratory disease may result in which the opportunistic pathogen P. haemolytica is the predominant bacteria isolated and responsible for acute lung injury (1Whiteley L.O. Maheswaran S.K. Weiss D.J. Ames T.R. Kannan M.S. J. Vet. Intern. Med. 1992; 6: 11-22Crossref PubMed Scopus (139) Google Scholar, 2Ames T.R. Markham R.J. Opuda-Asibo J. Leininger J.R. Maheswaran S.K. Can. J. Comp. Med. 1985; 49: 395-400PubMed Google Scholar). While under stress, proliferation of P. haemolytica is facilitated, leading to infection of the upper respiratory tract and eventual colonization of the lower airway (3Marchart J. Rehagen M. Dropmann G. Szostak M.P. Alldinger S. Lechleitner S. Schlapp T. Resch S. Lubitz W. Vaccine. 2003; 21: 1415-1422Crossref PubMed Scopus (38) Google Scholar). In the past, vaccine trials aimed at preventing disease due to P. haemolytica have met with difficulties and limited success (4Conlon J.A. Shewen P.E. Lo R.Y.C. Infect. Immun. 1991; 59: 587-591Crossref PubMed Google Scholar, 5Gilmour N.J.L. Martin W.B. Sharp J.M. Thompson D.A. Wells P.W. Donachie W. Res. Vet. Sci. 1983; 35: 80-86Crossref PubMed Google Scholar). Investigators have noted that for many Gram-negative pathogenic bacteria, iron acquisition directly corresponds to virulence (6Crosa J.H. Annu. Rev. Microbiol. 1984; 38: 69-89Crossref PubMed Scopus (93) Google Scholar, 7Weinberg E.D. Physiol. Rev. 1984; 64: 65-102Crossref PubMed Scopus (632) Google Scholar). Iron is an integral nutrient for the survival of nearly all organisms and is an essential cofactor of numerous metabolic and enzymatic processes (7Weinberg E.D. Physiol. Rev. 1984; 64: 65-102Crossref PubMed Scopus (632) Google Scholar, 8Schryvers A.B. Stojiljkovic I. Mol. Microbiol. 1999; 32: 1117-1123Crossref PubMed Scopus (206) Google Scholar). Therefore, researchers believe that proteins expressed under iron-limited conditions may be essential for bacterial survival and may ultimately serve as therapeutic targets. Iron is abundant in the environment and should not be a limiting factor for growth. However, under aerobic conditions and at neutral or alkaline pH, aqueous iron precipitates as an insoluble Fe3+ hydroxide. Furthermore, free iron can generate toxic derivatives within the body (9Wessling-Resnick M. Crit. Rev. Biochem. Mol. Biol. 1999; 34: 285-314Crossref PubMed Scopus (60) Google Scholar). Thus, it is vital that biological systems maintain control of the chemical environment of iron. The mammalian host utilizes the monomeric, bilobal, and iron-binding glycoproteins transferrin (Tf) 1The abbreviations used are: Tf, transferrin; ABC, ATP-binding cassette; TbpA, Tf-binding protein A; TbpB, transferrin-binding protein B; Fbp, ferric ion-binding protein; PhFbpA, P. haemolytica ferric ion-binding protein; PEG, polyethylene glycol; HiFbpA, H. influenzae periplasmic FbpA; PDB, Protein Data Bank; PLBP, periplasmic ligand-binding protein. (in sera) and lactoferrin (on mucosal surfaces) to limit the amount of free iron available to pathogens and transport iron throughout the body. In response to the problem of iron scarcity, the extracellular Gram-negative pathogens of the Pasteurellaceae and Neisseriaceae families have developed high affinity iron acquisition systems to obtain this essential nutrient (10Mietzner T.A. Morse S.A. Annu. Rev. Nutr. 1994; 14: 471-493Crossref PubMed Scopus (110) Google Scholar). Three components are involved in the successful uptake of iron from the eukaryotic host Tf as follows: (i) an outer membrane Tf receptor complex; (ii) an inner membrane-associated TonB complex; and (iii) an ATP-binding cassette (ABC)-type ferric ion transporter system. The host-specific bacterial outer membrane Tf receptor is composed of two proteins, Tf-binding proteins A (TbpA) and B (TbpB) (11Gray-Owen S.D. Schryvers A.B. Trends Microbiol. 1996; 4: 185-191Abstract Full Text PDF PubMed Scopus (257) Google Scholar). The TbpB receptor component is largely an extracellular hydrophilic macromolecule, which is anchored to the outer membrane through a lipidated tail (11Gray-Owen S.D. Schryvers A.B. Trends Microbiol. 1996; 4: 185-191Abstract Full Text PDF PubMed Scopus (257) Google Scholar). TbpA is a TonB-dependent integral membrane protein that is proposed to mediate transport of iron across the outer membrane. The transport of iron across the outer membrane requires the presence of a functional TonB protein (12Jarosik G.P. Maciver I. Hansen E.J. Infect. Immun. 1995; 63: 710-713Crossref PubMed Google Scholar). Following the removal of iron from transferrin and translocation across the outer membrane, the ferric ion is complexed by a periplasmic ferric ionbinding protein (FbpA). Studies have revealed the function of FbpA in complexing and transporting iron across the periplas mic space (13Chen C.-Y. Berish S.A. Morse S.A. Mietzner T.A. Mol. Microbiol. 1993; 10: 311-318Crossref PubMed Scopus (95) Google Scholar, 14Kirby S.D. Gray-Owen S.D. Schryvers A.B. Mol. Microbiol. 1997; 25: 979-987Crossref PubMed Scopus (20) Google Scholar, 15Kirby S.D. Lainson F.A. Donachie W. Okabe A. Tokuda M. Hatase O. Schryvers A.B. Microbiology. 1998; 144: 3425-3436Crossref PubMed Scopus (27) Google Scholar) as the periplasmic component of the iron ABC transport system (16Tam R. Saier M.H. Microbiol. Rev. 1993; 57: 320-346Crossref PubMed Google Scholar). The ABC family of transporters includes a broad and diverse group of import systems found in prokaryotes. ABC transporters generally consist of separate polypeptides that form an A1B2C2 complex at the inner membrane of the cell during transport. Thus, in order for the ferric ion to reach the cytoplasm, it is donated to an inner membrane complex consisting of the inner transmembrane protein FbpB and the cytoplasmic ATPase FbpC. This high affinity bacterial iron uptake system results in iron from host transferrin being liberated at the cell surface, translocated through the periplasmic space, and deposited into the cytoplasm for use or storage without the incorporation of the transferrin protein into the bacterial cell. Because the FbpABC pathway appears to be a common route for ferric iron uptake from several different sources, it is a particularly attractive therapeutic target. Identification of the gene encoding PhFbpA was based on complementation of an entA Escherichia coli strain transformed with a P. haemolytica λZAP II library-derived plasmid (15Kirby S.D. Lainson F.A. Donachie W. Okabe A. Tokuda M. Hatase O. Schryvers A.B. Microbiology. 1998; 144: 3425-3436Crossref PubMed Scopus (27) Google Scholar). Nucleotide sequence analysis of the fbpA gene confirmed homology with the cluster I group of the periplasmic binding protein family (15Kirby S.D. Lainson F.A. Donachie W. Okabe A. Tokuda M. Hatase O. Schryvers A.B. Microbiology. 1998; 144: 3425-3436Crossref PubMed Scopus (27) Google Scholar, 16Tam R. Saier M.H. Microbiol. Rev. 1993; 57: 320-346Crossref PubMed Google Scholar). Despite its homology to FbpA molecules of other organisms, PhFbpA has a peak-visible absorbance at 419 nm, which is significantly blue-shifted relative to the peak absorbance for other FbpAs (13Chen C.-Y. Berish S.A. Morse S.A. Mietzner T.A. Mol. Microbiol. 1993; 10: 311-318Crossref PubMed Scopus (95) Google Scholar, 17Adhikari 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). PhFbpA shares approximately the same iron affinity as transferrin and other FbpA molecules based on citrate competition assays (15Kirby S.D. Lainson F.A. Donachie W. Okabe A. Tokuda M. Hatase O. Schryvers A.B. Microbiology. 1998; 144: 3425-3436Crossref PubMed Scopus (27) Google Scholar). Thus, current evidence indicates that PhFbpA binds and transports Fe3+ ion, utilizing a unique iron-coordinating environment when compared with transferrin or any other biochemically characterized FbpA molecules. In this study, we describe the 1.2-Å resolution structure of PhFbpA. This is the first example of a closed apoFbpA molecule. Although the structure does not contain a metal atom, the putative metal coordinating residues are clearly evident. The current structure explains how this protein may coordinate iron and the possible locations and identities of synergistic anions. Purification and Crystallization Procedures—E. coli BL21(DE3)/pLysS harboring the pT7–7 plasmid encoding FbpA was utilized to express the native P. haemolytica 35-kDa ferric ion-binding protein A in the periplasm under isopropyl-1-thio-β-d-galactopyranoside induction. A selenomethionine version was also produced using the same plasmid in an E. coli strain that is a methionine auxotroph. This strain was grown overnight at 37 °C on M9 minimal media supplemented with ampicillin to 100 μg/ml and methionine to 40 μg/ml. The following day, the culture was inoculated to LeMaster's media. The medium was supplemented with ampicillin (100 μg/ml), selenomethionine (40 μg/ml), and Isovitalex to a 1% v/v final concentration. The culture was grown overnight with shaking at 37 °C and the following day inoculated to fresh media to an A 600 of 0.05 and induced with isopropyl-1-thio-β-d-galactopyranoside once the culture reached an A 600 of 0.5. The culture was grown overnight with shaking at 37 °C, and cells were harvested the following day. Both versions of protein were then purified using a modified osmotic shock procedure (18Shouldice S.R. Dougan D.R. Skene R.J. Tari L.W. McRee D.E. Yu R.-H. Schryvers A.B. J. Biol. Chem. 2003; 278: 11513-11519Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The supernatant was carefully removed without disrupting the pellet because the supernatant contains the periplasmic osmotic shock fraction. Finally, the sample was extensively dialyzed against 20 mm ethanolamine buffer, pH 9.0, at 4 °C. Following dialysis, the protein sample was subjected to anion-exchange chromatography on the Akta fast protein liquid chromatography system as a final purification step. The column was washed with 20 volumes of 20 mm ethanolamine buffer, pH 9.0, to remove unbound proteins and eluted with a gradient of 0–1.6 m The from the column was to a final of and using a this the protein in the was into mm buffer, pH the were to a final of The resulting were based on analysis not Protein were at °C were used for The was for of the proteins. The used of and of against a of the protein at 20 °C in from a protein and m pH that was against a and m pH to the space group of native PhFbpA were grown at 4 °C from using the high protein developed at used of PhFbpA and of against a of the native protein in from a protein and m pH that was against a and m pH were for and to the space group and Data were harvested by with a The native was then into a and m pH for were in liquid the selenomethionine was by the in the supplemented with were at 100 during by use of a were from a of the PhFbpA sample using an 4 at at the using a of The were at the on The for all of the were and using the W. 1997; Scopus Google Scholar). The for all of the are in of high cell in are the for the resolution of and of of of of of Protein of in are the for the resolution of and of of in a new of the protein were of and to resolution In the structure revealed that the was of two molecules in the and that was However, the protein were of to obtain an of the protein that was used to the native The were using the R. J. 1999; 32: Scopus Google Scholar). of the and were using the E. G. 1997; PubMed Scopus Google Scholar). The resulting were using the D.E. J. Biol. 1999; PubMed Scopus Google and the was against a using the P. J. M. R.J. T. 1998; PubMed Scopus Google Scholar). The high resolution native structure was by a procedure using the from the of 1994; PubMed Scopus Google Scholar). The of the were used as the Because the structure of was in an compared with the two the N- and C-terminal domains of the protein to be to the of of the using the D.E. J. Biol. 1999; PubMed Scopus Google Scholar) use of with R.J. A. PubMed Scopus Google and with E.J. 1997; PubMed Scopus Google Scholar) was to and the using a function and for was the of were used to the and molecules. The final is for all of the from The free using of the is of P. haemolytica FbpA have deposited in the Protein Data the of all of the residues into of The was utilized in to proteins in the based on of Res. 1997; 25: PubMed Scopus Google Scholar). Crystallization and PhFbpA protein was isolated from the periplasm of E. coli and purified by Following protein were to to of a were used to obtain an However, the of the protein because of the that of the molecules of the two in the was This the for a new A form was found for the native protein using the high which us to an of chemical to iron-loaded of PhFbpA have as to a form for The PhFbpA structure was by using the structure as a and are in I. all of the protein the is and The two residues of the and 37 were not in the and were for of residues and was also and were as and The structure of PhFbpA is of high as by the and the which that all of the residues are in in the 1994; PubMed Scopus Google Scholar). of all of the FbpA molecules deposited to an PhFbpA has a structure to the of influenzae periplasmic FbpA with of 40 A using the Res. 1997; 25: PubMed Scopus Google Scholar) for proteins with to PhFbpA in the that PhFbpA the structural with A.J. Mietzner T.A. McRee D.E. Biol. 1997; 4: PubMed Scopus Google Scholar) and sequence and a significant of to other periplasmic ligand-binding proteins as as the of transferrin J. A.B. Protein Sci. PubMed Scopus Google lactoferrin PubMed Scopus Google and M. J. Mol. Biol. PubMed Scopus Google Scholar). of of PhFbpA and can be with a of to other FbpA the the N- and C-terminal domains PhFbpA, to and other periplasmic binding proteins as as is composed of two structural domains the N- and A ligand-binding is at the the two is composed of a by The two domains are by two which form the the two of the N- and C-terminal domains this involving the two at the of the ligand-binding and the protein from an to closed The PhFbpA structure in the form not adopts an conformation, which from the native PhFbpA structure by a body of the N- and the by Iron and on our structural analysis and sequence of PhFbpA with II and we that PhFbpA a Fe3+ ion in an utilizing protein to form the of the with two anions on of the the and The residues the of the are and the of all of the putative iron-binding residues within of and through the for iron Although the residues in the binding are by the of the which on a from the is The of from the binding and is from the putative However, a in the with a of the it on the binding the group in to in iron are and the for the in the are the for the that in the of the is also is probable that and the it iron A structural of the iron-loaded structure with the structure of PhFbpA the different utilized by the two proteins to iron Although and in PhFbpA to have and in the structure, the two proteins the that and the the iron. The in the structure appears to have by in PhFbpA and in the structure is by an in formate at the of formate the the iron in sequence in a new of Fe3+ in and proposed Fe3+ in PhFbpA The of the iron-binding and the of iron The two and PhFbpA were that the binding are in the same with to the protein The binding on protein the of the In a the binding present a different constellation of iron-coordinating to the Fe3+ The the two proteins is the of a and in PhFbpA and HiFbpA, in iron at the of the pocket. In PhFbpA, the in HiFbpA, anions and and the The anions in to with from of residues. of the of formate with from and the two residues the other from the formate with the of binds at the of an residues it through with and and with from and The two anions are by appears to stabilize a closed the N- and C-terminal domains by in a and of the PhFbpA structure that it adopts the periplasmic ligand-binding protein that has in all of the characterized to from ABC transport systems sequence homology and a of all of proteins in which the two domains are by a of at the of the ligand-binding F.A. R. Sci. PubMed Google Scholar, F.A. Mol. Microbiol. 1996; PubMed Scopus Google Scholar). Despite the structural to other FbpAs to be the periplasmic transport proteins that are to a free iron and transport it across the periplasmic Although metal ion is present in the PhFbpA structure, an of binding structure, and sequence have us to assign putative iron and residues with a high of A J. 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A. 2003; PubMed Scopus Google the in the absorbance in PhFbpA can be to the that PhFbpA residues to coordinate iron the two utilized in all of the other characterized FbpAs as as in Tf and In to in the of iron-coordinating protein the high resolution have for the of the formate anions at two binding on opposite sides of the binding pocket. that the residues utilized by PhFbpA to coordinate anions are across all of the bacterial proteins, functional The formate anions in utilizing and also of a as it binds at the of an a that is also for the in A.J. Mietzner T.A. McRee D.E. Biol. 1997; 4: PubMed Scopus Google Scholar). also appears to a in a closed of the binding by a and of the and and and of the C-terminal Both anions are which may have in the iron and removal process in PhFbpA. Because formate was present at high in the and formate anions are of it is not surprising that we to PhFbpA. is probable that the to coordinate iron which has in our of a closed a to the we can that may serve as the of in PhFbpA the high of structural and the and the formate anions. Because the Fe3+ ion in PhFbpA is from the host transferrin or it is not to that be by anions when present in that the iron-loaded of transferrin and lactoferrin contain as the synergistic S.A. J. H. M. A.B. 1998; PubMed Scopus Google Scholar, A.B. PubMed Scopus Google Scholar, D.R. J.M. S. M. M. Biol. PubMed Scopus (95) Google Scholar, H. M. J. Mol. Biol. 1995; PubMed Scopus Google Scholar, S.A. M. J. Mol. Biol. 1997; PubMed Scopus Google Scholar). The binding for the ions be to we for formate be the binding as we for the the from the to coordinate iron from opposite We that the synergistic anions in PhFbpA, of and Fe3+ ion anions residues for iron allowing for of to and from the binding a of iron binding and removal is binding may first to the binding site for iron binding or of the anions from the binding site may iron The which is from the binding site in this structure, as a and iron is to that on the of our structural we as a in PhFbpA because it is and in a to anions such as citrate may be have to a significant of of the binding because of In we have the first structure of a new class of bacterial iron-binding proteins. We a for iron that utilizes a and two anions. We to an iron-loaded structure to our We have also bacterial that coordinate iron utilizing a constellation of and biological to PhFbpA in Because iron is an essential the of proteins in this class may in the of with therapeutic is by the of of of the S. of under at We also the at for

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