The plug domain of a neisserial TonB‐dependent transporter retains structural integrity in the absence of its transmembrane β‐barrel

Transferrin binding protein A (TbpA) is a TonB‐dependent outer membrane protein expressed by pathogenic bacteria for iron acquisition from human transferrin. The N‐terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of Escherichia coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB‐dependent transporters. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. These experiments suggest that the plug domain may fold independently of the β‐barrel, but extracellular loops of the β‐barrel are required for ligand binding.

[1]  M. Mcintosh,et al.  Mutations in the Escherichia coli receptor FepA reveal residues involved in ligand binding and transport , 2001, Molecular microbiology.

[2]  F. Archibald,et al.  Removal of iron from human transferrin by Neisseria meningitidis , 1979 .

[3]  B. Ahmer,et al.  Energy transduction between membranes. TonB, a cytoplasmic membrane protein, can be chemically cross-linked in vivo to the outer membrane receptor FepA. , 1993, The Journal of biological chemistry.

[4]  P. Sparling,et al.  Effects of serum carrier proteins on the growth of pathogenic neisseriae with heme-bound iron , 1987, Infection and immunity.

[5]  J. Konisky,et al.  Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli , 1990, Journal of bacteriology.

[6]  David P. Chimento,et al.  Substrate-induced transmembrane signaling in the cobalamin transporter BtuB , 2003, Nature Structural Biology.

[7]  James E. Anderson,et al.  Identification of Discrete Domains within Gonococcal Transferrin-Binding Protein A That Are Necessary for Ligand Binding and Iron Uptake Functions , 2000, Infection and Immunity.

[8]  R. Kadner,et al.  Suppression of the btuB451 mutation by mutations in the tonB gene suggests a direct interaction between TonB and TonB-dependent receptor proteins in the outer membrane of Escherichia coli. , 1988, Gene.

[9]  P. Sparling,et al.  Binding and surface exposure characteristics of the gonococcal transferrin receptor are dependent on both transferrin-binding proteins , 1996, Journal of bacteriology.

[10]  R. Kadner.  Vitamin B12 transport in Escherichia coli: energy coupling between membranes , 1990, Molecular microbiology.

[11]  Luc Moulinier,et al.  Transmembrane Signaling across the Ligand-Gated FhuA Receptor Crystal Structures of Free and Ferrichrome-Bound States Reveal Allosteric Changes , 1998, Cell.

[12]  C. Elkins,et al.  Phase Variation of Hemoglobin Utilization inNeisseria gonorrhoeae , 1998, Infection and Immunity.

[13]  P. Mickelsen,et al.  Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from transferrin and iron compounds , 1981, Infection and immunity.

[14]  A. Schryvers,et al.  Identification and characterization of the transferrin receptor from Neisseria meningitidis , 1988, Molecular microbiology.

[15]  J. T. Yang,et al.  Circular dichroic analysis of protein conformation: inclusion of the beta-turns. , 1978, Analytical biochemistry.

[16]  Z. Cao,et al.  Exchangeability of N Termini in the Ligand-gated Porins ofEscherichia coli * , 2001, The Journal of Biological Chemistry.

[17]  K. Postle,et al.  FepA with Globular Domain Deletions Lacks Activity , 2002, Journal of bacteriology.

[18]  J F Brandts,et al.  Rapid measurement of binding constants and heats of binding using a new titration calorimeter. , 1989, Analytical biochemistry.

[19]  V. Braun,et al.  The β‐barrel domain of FhuAΔ5‐160 is sufficient for TonB‐dependent FhuA activities of Escherichia coli , 1999, Molecular microbiology.

[20]  P. Sparling,et al.  Energy‐dependent changes in the gonococcal transferrin receptor , 1997, Molecular microbiology.

[21]  B. Roe,et al.  Molecular characterization of hpuAB, the haemoglobin–haptoglobin‐utilization operon of Neisseria meningitidis , 1997, Molecular microbiology.

[22]  C. N. Cornelissen,et al.  Specific Ligand Binding Attributable to Individual Epitopes of Gonococcal Transferrin Binding Protein A , 2002, Infection and Immunity.

[23]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[24]  P. Mickelsen,et al.  Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from lactoferrin , 1982, Infection and immunity.

[25]  S. Gray-Owen,et al.  Bacterial transferrin and lactoferrin receptors. , 1996, Trends in microbiology.

[26]  P. Mickelsen,et al.  Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae , 1988, Infection and immunity.

[27]  Andrew D. Ferguson,et al.  Structural Basis of Gating by the Outer Membrane Transporter FecA , 2002, Science.

[28]  W. Yue,et al.  Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. , 2003, Journal of molecular biology.

[29]  C. Cornelissen,et al.  Antigenic and Sequence Diversity in Gonococcal Transferrin-Binding Protein A , 2000, Infection and Immunity.

[30]  B. C. Lee,et al.  Comparative analysis of the transferrin and lactoferrin binding proteins in the family Neisseriaceae. , 1989, Canadian journal of microbiology.

[31]  Sequence analysis of the structural tbpA gene: protein topology and variable regions within neisserial receptors for transferrin iron acquisition. , 1997, Microbial pathogenesis.

[32]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[33]  R. P. Williams,et al.  Effects of iron and culture filtrates on killing of Neisseria gonorrhoeae by normal human serum , 1978, Infection and immunity.

[34]  M. Gipson,et al.  Phase variation of HpuAB and HmbR, two distinct haemoglobin receptors of Neisseria meningitidis DNM2 , 1999, Molecular microbiology.

[35]  R. W. Evans,et al.  Transferrin-binding protein B isolated from Neisseria meningitidis discriminates between apo and diferric human transferrin. , 1998, The Biochemical journal.

[36]  J. Brahms,et al.  Determination of protein secondary structure in solution by vacuum ultraviolet circular dichroism. , 1980, Journal of molecular biology.

[37]  S. E. West,et al.  Response of Neisseria gonorrhoeae to iron limitation: alterations in expression of membrane proteins without apparent siderophore production , 1985, Infection and immunity.

[38]  B. Wallace,et al.  Differential absorption flattening optical effects are significant in the circular dichroism spectra of large membrane fragments. , 1987, Biochemistry.

[39]  Christopher D. Kenney,et al.  Demonstration and Characterization of a Specific Interaction between Gonococcal Transferrin Binding Protein A and TonB , 2002, Journal of bacteriology.

[40]  K.,et al.  Gonococcal transferrin-binding protein 1 is required for transferrin utilization and is homologous to TonB-dependent outer membrane receptors , 1992, Journal of bacteriology.

[41]  K. Diederichs,et al.  Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide. , 1998, Science.

[42]  C. Simonson,et al.  Expression of a high-affinity mechanism for acquisition of transferrin iron by Neisseria meningitidis , 1982, Infection and immunity.

[43]  J. Neilands,et al.  Siderophores: Structure and Function of Microbial Iron Transport Compounds (*) , 1995, The Journal of Biological Chemistry.

[44]  W. C. Johnson,et al.  Sensitivity of circular dichroism to protein tertiary structure class , 1983, Nature.

[45]  B. Wallace,et al.  Folding of the mitochondrial proton adenosinetriphosphatase proteolipid channel in phospholipid vesicles. , 1982, Biochemistry.

[46]  I. Stojiljković,et al.  Iron acquisition systems in the pathogenic Neisseria , 1999, Molecular microbiology.

[47]  A. Schryvers,et al.  Identification and characterization of the human lactoferrin-binding protein from Neisseria meningitidis , 1988, Infection and immunity.

[48]  P. I. Higgs,et al.  Interactions in the TonB-Dependent Energy Transduction Complex: ExbB and ExbD Form Homomultimers , 1998, Journal of bacteriology.

[49]  V. Braun,et al.  In Vivo Reconstitution of the FhuA Transport Protein of Escherichia coli K-12 , 2003, Journal of bacteriology.

[50]  Stephen J. Perkins,et al.  Protein volumes and hydration effects , 1986 .

[51]  R. Kadner,et al.  Differential substrate-induced signaling through the TonB-dependent transporter BtuB , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[52]  D. Dyer,et al.  Identification of an iron-regulated outer membrane protein of Neisseria meningitidis involved in the utilization of hemoglobin complexed to haptoglobin , 1995, Journal of bacteriology.

[53]  S. Buchanan,et al.  Expression and purification of functional recombinant meningococcal transferrin-binding protein A. , 2002, The Biochemical journal.

[54]  S. Gray-Owen,et al.  The interaction of primate transferrins with receptors on bacteria pathogenic to humans. , 1993, Microbial pathogenesis.

[55]  J. Deisenhofer,et al.  Crystal structure of the outer membrane active transporter FepA from Escherichia coli , 1999, Nature Structural Biology.

[56]  Volkmar Braum,et al.  Transport across the outer membrane ofEscherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane , 1989, Molecular and General Genetics MGG.

[57]  J. Deisenhofer,et al.  The plug domain of FepA, a TonB-dependent transport protein from Escherichia coli, binds its siderophore in the absence of the transmembrane barrel domain , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[58]  J. E. Anderson,et al.  Gonococcal transferrin-binding protein 2 facilitates but is not essential for transferrin utilization , 1994, Journal of bacteriology.

[59]  B. C. Lee,et al.  Specificity of the lactoferrin and transferrin receptors in Neisseria gonorrhoeae , 1988, Molecular microbiology.

[60]  Stability studies of FhuA, a two-domain outer membrane protein from Escherichia coli. , 2001, Biochemistry.

[61]  J. Neilands.  Microbial iron compounds. , 1981, Annual review of biochemistry.

[62]  V. Braun,et al.  Mutant Analysis of the Escherichia coli FhuA Protein Reveals Sites of FhuA Activity , 2003, Journal of bacteriology.

[63]  J. Coulton,et al.  TonB‐dependent iron acquisition: mechanisms of siderophore‐mediated active transport † , 1998, Molecular microbiology.

[64]  Andrew Gorringe,et al.  Insight into the Structure and Function of the Transferrin Receptor from Neisseria meningitidis Using Microcalorimetric Techniques* , 2003, The Journal of Biological Chemistry.

[65]  E. Jacobs,et al.  Cloning and characterization of Neisseria meningitidis genes encoding the transferrin-binding proteins Tbp1 and Tbp2. , 1993, Gene.