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. 2012 Apr;4(4):361-72.
doi: 10.1039/c2mt20037f. Epub 2012 Mar 8.

Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport

Sambuddha Banerjee  1 , Claire J Parker Siburt, Shreni Mistry, Jennifer M Noto, Patrick DeArmond, Michael C Fitzgerald, Lisa A Lambert, Cynthia N Cornelissen, Alvin L Crumbliss
Affiliations

Evidence of Fe3+ interaction with the plug domain of the outer membrane transferrin receptor protein of Neisseria gonorrhoeae: implications for Fe transport

Sambuddha Banerjee et al. Metallomics. 2012 Apr.

Abstract

Neisseria gonorrhoeae is an obligate pathogen that hijacks iron from the human iron transport protein, holo-transferrin (Fe(2)-Tf), by expressing TonB-dependent outer membrane receptor proteins, TbpA and TbpB. Homologous to other TonB-dependent outer membrane transporters, TbpA is thought to consist of a β-barrel with an N-terminal plug domain. Previous reports by our laboratories show that the sequence EIEYE in the plug domain is highly conserved among various bacterial species that express TbpA and plays a crucial role in iron utilization for gonococci. We hypothesize that this highly conserved EIEYE sequence in the TbpA plug, rich in hard oxygen donor groups, binds with Fe(3+) through the transport process across the outer membrane through the β-barrel. Sequestration of Fe(3+) by the TbpA-plug supports the paradigm that the ferric iron must always remain chelated and controlled throughout the transport process. In order to test this hypothesis here we describe the ability of both the recombinant wild-type plug, and three small peptides that encompass the sequence EIEYE of the plug, to bind Fe(3+). This is the first report of the expression/isolation of the recombinant wild-type TbpA plug. Although CD and SUPREX spectroscopies suggest that a non-native structure is observed for the recombinant plug, fluorescence quenching titrations indicate that the wild-type recombinant TbpA plug binds Fe (3+) with a conditional log K(d) = 7 at pH 7.5, with no evidence of binding at pH 6.3. A recombinant TbpA plug with mutated sequence (NEIEYEN → NEIAAAN) shows no evidence of Fe(3+) binding under our experimental set up. Interestingly, in silico modeling with the wild-type plug also predicts a flexible loop structure for the EIEYE sequence under native conditions which once again supports the Fe(3+) binding hypothesis. These in vitro observations are consistent with the hypothesis that the EIEYE sequence in the wild-type TbpA plug binds Fe(3+) during the outer membrane transport process in vivo.

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Figures

Fig. 1
Fig. 1
Cartoon representation of the iron acquisition process of N. gonorrhoeae. Holo-transferrin is recognized and bound by TbpA/TbpB at the external surface of the outer membrane. Iron is released from transferrin and the apo-transferrin is released from the outer membrane receptor using TonB derived energy followed by transport of iron into the periplasm. In the periplasm apo-FbpA binds with TbpA and acquires the iron. FbpA then transports the iron across the periplasm and delivers it to the inner membrane receptors FbpB and FbpC which ultimately transport iron into the cytoplasm (the TbpA plug is not shown in the cartoon for clarity).
Fig. 2
Fig. 2
Cartoon representation of the wild-type recombinant TbpA plug, the mutated recombinant TbpA plug and the short peptides (S1, S2 and S3). The proposed iron binding sequence (EIEYE) is preserved in the wild-type recombinant plug and in the model peptides. However in the mutated recombinant plug this sequence is altered by alanine substitution of the three key potential iron binding residues making the sequence EIAAA. The three other tyrosine residues, Tyr58, 75 and 98, are also shown in the figure for both wild type and mutated recombinant plug proteins.
Fig. 3
Fig. 3
Characterization of purified wild-type and mutated recombinant TbpA plug proteins. Plug proteins were separated by SDS-PAGE and subsequently visualized with Coomassie blue. The lanes contain the following samples: Lane 1, 20 μg lysozyme; Lane 2, molecular weight markers; Lane 3, 20 μg wild-type plug protein; Lane 4, 20 μg EYE mutant plug protein.
Fig. 4
Fig. 4
CD spectra of A) wild-type recombinant and B) mutated recombinant TbpA plug in 50 mM NaClO4 buffer at pH 7.5, [wild-type recombinant TbpA plug] = 10 μM, [mutated recombinant TbpA plug] = 20 μM and [Fe(ClO4)3] = 0–10 μM. The solid line spectra shown in A) and B) are for wild-type or mutated recombinant TbpA plug proteins in the absence of Fe3+, and the dashed and dotted lines are after addition of Fe3+. Both spectra show strong negative bands centered at 200 nm indicating a mostly unfolded structure. Fe3+ addition did not induce any significant structural change for either, which is reflected by very small change in CD absorption signals.
Fig. 5
Fig. 5
SUPREX behavior of wild-type recombinant TbpA plug at pD 7.4 (dark squares) and pD 6.5 (open circles). Change in observed protein mass due to H/D exchange as a function of denaturant concentration is plotted to obtain the SUPREX curve. Conditions: exchange time 5 min, trypsin inhibitor internal standard, 50 mM MES buffer/200 mM KCl pD 6.5 or 20 mM phosphate buffer/pD 7.4. Error bars indicate the variation between the 10 mass spectra collected for each denaturant concentration. When error bars are not visible the mass variation is smaller than the symbol designating the datum point.
Fig. 6
Fig. 6
Repesentative plots of % quenching of the 310 nm fluorescence emission band (Q%) vs total Fe3+ added ([Fe]T) according to Equation (7) for A) wild-type recombinant TbpA plug and B) S1 peptide in 100 mM Tris buffer at pH 7.5, [wild type recombinant plug] = 20 μM; [S1] = 20 μM. The dots represent actual data points and the smooth lines are the best fit for the data according to Equation (7) where Qmax = 40% and Kd = 1 × 10−7 M for A) and Qmax = 100% and Kd = 1 × 10−4 M for B). The insets show the titration spectra of A) recombinant wild-type TbpA plug (20 μM) and B) S1 peptide (20 μM), in the absence (highest intensity band) and in presence of increasing aliquots of Fe(ClO4)3. Conditions: 100 mM Tris at pH 7.5, excitation at 285 nm, using a 1 cm path length cuvette at room temperature the 310 nm band is for surface exposed tyrosine(s); fluorescence data collected in a quartz cuvette of 1 cm path length at room temperature; the final concentration for the wild-type plug titration is [Protein]:[Fe3+] = 20:1 and that of for S1 is [S1]:[Fe3+] = 1:50.
Fig. 7
Fig. 7
Predicted structure of Neisseria gonorrhoeae TbpA plug domain. (A) Wild-type; (B) Mutant (EIEYE → EIAAA). The EIEYE sequence is postulated to be important for iron transport functions. Tyrosine side chains are shown as wire-frames. The possible importance of the “Top Hat” region (red circle) for interaction with transferrin is discussed in the text. The model was created with I-TASSER based on amino acids 25–187 from NCBI sequence YP_208545.
Fig. 8
Fig. 8
Conservation of the NEIEYEN sequence and ‘Top Hat’ regions among gonococci. Dark shading represents conserved sequence positions; the orange block highlights the sequence conserved in Neisseria sequences. Stars indicated the positions of Tyr98 and Ala110.
Fig. 9
Fig. 9
Comparison of topology of wild type and mutant plug domain structures. Pink arrows represent beta strands; red tubes represent alpha helices. While the beta strand backbones of the TbpA model are nearly identical to the ShuA protein structure used as a template (data not shown), a change of just three amino acids in the TbpA sequence had dramatic effects on the overall predicted structure. Yellow spots indicate position of tyrosines. Images created with PDBsum.
Fig. 10
Fig. 10
A) Cartoon representation of the proposed stepwise movement of Fe3+ from transferrin across the outer membrane and insertion into periplasmic iron transport protein FbpA: (i) Diffusion of Fe loaded Tf (Fe3+ tightly bound with conditional Kd ~10−20 M) to the outer surface of TbpA/TbpB receptor; (ii) Fe2Tf docking at TbpA (Fe2Tf Kd ≫ 10−20 M) where Fe3+ is released; (iii) TbpA plug “catches” released Fe3+ by binding at the EIEYE sequence and moves through the β-barrel of TbpA; (iv) Fe3+ bound to periplasm exposed plug (conditional Kd = 10−7 M) is handed off to apo-FbpA bound to inner lib of TbpA; (v) FeFbpA (conditional Kd ~10−18 M) diffuses through the periplasm ultimately releasing the iron at the inner membrane receptor TbpB/TbpC. Kd values shown are conditional not true thermodynamic constants. Relative values, however, illustrate the feasibility of unidirectional Fe3+ exchange across the outer membrane and into the periplasm.
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