Kinetics and mechanism of exogenous anion exchange in FeFbpA–NTA: significance of periplasmic anion lability and anion binding activity of ferric binding protein A

Heymann, Jared J.; Gabričević, Mario; Mietzner, Timothy A.; Crumbliss, Alvin L.

doi:10.1007/s00775-009-0589-2

Cited by 9 publications

(6 citation statements)

References 30 publications

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“…In strict contrast to holo-FbpA, phosphate is only weakly bound to apo-FbpA with a K d value of 2.3±0.3 mM at the assumed pH of 6.5 of the periplasm [12]. However, a linear correlation between anion dissociation constants in apo-FbpA and anion exchange rates in holo-FbpA prevailing over three orders of magnitude suggests similar molecular level processes for both events [22].…”

Section: Introductionmentioning

confidence: 95%

“…12 However, a linear relationship between anion dissociation constants in apo-FbpA and anion exchange rates in holo-FbpA prevailing over 3 orders of magnitude suggests similar molecular level processes for both events. 21 In this study, we investigate in detail the interactions between the protein and the anion in the absence of iron using extensive molecular dynamics (MD) simulations. Since the role of phosphate as a synergistic anion is much debated, we inquire the scenario that the rich environmental conditions of the periplasmic space, with its wide distribution of ionic species maintaining a 30 mV Donnan potential 22 and its gel-like properties harboring microenvironments reserved for specific tasks, 23 may be utilized to optimize the binding kinetics of FbpA.…”

Section: ■ Introductionmentioning

confidence: 99%

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Protonation States of Remote Residues Affect Binding–Release Dynamics of the Ligand but Not the Conformation of Apo Ferric Binding Protein

Guven

Atılgan

2014

J. Phys. Chem. B

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We have studied the apo (Fe 3+ free) form of periplasmic ferric binding protein (FbpA) under different conditions and we have monitored the changes in the binding and release dynamics of H 2 PO 4 − that acts as a synergistic anion in the presence of Fe 3+ . Our simulations predict a dissociation constant of 2.2±0.2 mM which is in remarkable agreement with the experimentally measured value of 2.3±0.3 mM under the same ionization strength and pH conditions. We apply perturbations relevant for changes in environmental conditions as (i) different values of ionization strength (IS), and (ii) protonation of a group of residues to mimic a different pH environment. Local perturbations are also studied by protonation or mutation of a site distal to the binding region that is known to mechanically manipulate the hinge-like motions of FbpA.We find that while the average conformation of the protein is intact in all simulations, the H 2 PO 4 − dynamics may be substantially altered by the changing conditions. In particular, the bound fraction which is 20% for the wild type system is increased to 50% with a D52A mutation/protonation and further to over 90% at the protonation conditions mimicking those at pH 5.5. The change in the dynamics is traced to the altered electrostatic distribution on the surface of the protein which in turn affects hydrogen bonding patterns at the active site. The observations are quantified by rigorous free energy calculations. Our results lend clues as to how the environment versus single residue perturbations may be utilized for regulation of binding modes in hFbpA systems in the absence of conformational changes. 3 AUTHOR SUMMARYBacterial ferric binding protein A (FbpA) plays a major role in iron confiscation from its host.Phosphate has been long identified as a synergistic anion for iron in this transport protein, while its mode of action is still debated. We have demonstrated in this work that phosphate binding to apo FbpA is a heavily environment dependent activity. Hydrogen-bond network motifs between the phosphate and its surrounding residues in FbpA become more stable when we lower the pH.With the aid of extensive molecular dynamics simulations conducted for different protonation scenarios, we show that the average structure of the protein is not altered during phosphate binding. Despite the lack of conformational transitions, however, phosphate binding-release dynamics is substantially affected by these changes. We find that the charges are redistributed, giving rise to altered fluctuation patterns of the loops remotely controlling the binding activity.

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Section: Introductionmentioning

confidence: 95%

Section: ■ Introductionmentioning

confidence: 99%

Protonation States of Remote Residues Affect Binding–Release Dynamics of the Ligand but Not the Conformation of Apo Ferric Binding Protein

Guven

Atılgan

2014

J. Phys. Chem. B

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“…It has been shown that FbpA has a high binding affinity for Fe 3þ (K a =10 18 M -1 ) in the presence of phosphate, and that concomitant exogenous anion binding is requisite (16). Many anions complement tight iron binding, including phosphate, arsenate, oxalate, nitrilotriacetate (NTA), sulfate, and citrate, and anion exchange is facile (17)(18)(19)(20)(21). The kinetics of both iron loading and iron release are paramount to the function of FbpA in shuttling iron across the periplasm between two membranebound receptor proteins.…”

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confidence: 99%

Role of Citrate and Phosphate Anions in the Mechanism of Iron(III) Sequestration by Ferric Binding Protein: Kinetic Studies of the Formation of the Holoprotein of Wild-Type FbpA and Its Engineered Mutants

et al. 2010

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Ferric binding protein A (FbpA) plays a central role in the iron acquisition processes of pathogenic Neisseria gonorrhoeae, Neisseria meningitidis, and Haemophilus influenza. FbpA functions as an iron shuttle within the periplasmic space of these Gram-negative human pathogens. Iron is picked up by FbpA at the periplasmic aspect of the outer membrane with concomitant acquisition of a synergistic anion. Here we report the kinetics and mechanisms involved with iron(III) loading into iron-free FbpA using iron(III) citrate as an iron source in the presence of excess citrate or phosphate (physiologically available anions) at pH 6.5. In the presence of excess phosphate, iron(III) citrate loads into apo-FbpA in three kinetically distinguishable steps, while in the presence of excess citrate only two steps are discernible. A stable intermediate containing iron(III) citrate bound FbpA is observed in each case. The observation of an additional kinetic step and moderate increase in apparent rate constants suggests an active role for phosphate in the iron insertion process. To further elucidate a mechanism for iron-loading, we report on the sequestration kinetics of iron(III) citrate in the presence of phosphate with binding site mutant apo-FbpAs, H9E, E57D, E57Q, Q58A, Y195F, and Y196H. Tyrosine mutations drastically alter the kinetics, and reduce iron sequestration ability. H9E, E57D, and E57Q have near native iron sequestration behavior; however iron binding rates are altered enabling assignment of sequential side chain interactions. Additionally, this investigation elaborates on the function of FbpA as a carrier for iron chelates as well as “naked” or free iron as originally proposed.

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“…The native protein binds to one Fe 3+ positively via two Tyr, one His and one Glu residues, and one phosphate as the synergistic anion, with the binding affinity up to 10 18 M À1 , and varies due to the different bound synergetic anions. [17][18][19][20][21] It is also known that the anion plays a vital role in iron binding and release, which has been verified by a series of biochemical and kinetic studies. The synergistic anion has been shown to preorganize the binding site prior to iron insertion for FbpA, 51 and to control the release of iron, because proton-assisted release of the phosphate anion is the preliminary step in iron release.…”

Section: Discussionmentioning

confidence: 88%

“…12 In addition to phosphate, a number of different exogenous anions including sulfate, oxalate, citrate and carbonate can also be utilized to facilitate the binding of Fe 3+ to FbpA. [19][20][21][22] Recently biochemical and X-ray crystallography studies by our group and others revealed that other metal ions including Hf 4+ , Zr 4+ and Ti 4+ as well as Fe 3+ when binding to FbpA, can assemble into the iron binding cleft by means of the oxo-metal clusters that could function with the known two-tyrosine motif (Tyr195 and Tyr196) as ligands, and the proteins were in an open conformation. [23][24][25][26][27][28][29][30] Therefore, it is premised that the binding of these Fe 3+ -like metal ions could either block iron binding to FbpA directly when competing with Fe 3+ , or alter the protein conformation to impede iron transport into the cytoplasmic membrane indirectly.…”

Section: Introductionmentioning

confidence: 99%

Binding of oxo-Cu2 clusters to ferric ion-binding protein A from Neisseria gonorrhoeae: a structural insight

Chen

Wang

et al. 2013

Metallomics

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The ferric ion-binding protein A (FbpA), a member of transferrin superfamily, is a periplasmic iron transporter employed by many Gram-negative pathogens. Our experiments indicated copper(ii) could bind with Neisseria gonorrhoeae FbpA (NgFbpA), and the binding constant reached up to (8.7 ± 0.2) × 10(8) M(-1)via UV-vis titration. The crystal structure of recombinant Cu-NgFbpA at 2.1 Å revealed that the oxo-Cu2 clusters (dinuclear centres) assembled in the iron binding cleft and were bound to the two adjacent tyrosine residues (Y195 and Y196) of the protein, two Cu ions coordinated with two tyrosines, Y195 and Y196, respectively, which was different from the binding model of Fe ion with FbpA, in which Y195 and Y196 coordinated together with one Fe ion. While this was similar to the binding of Zr and Hf ion clusters, Y195 and Y196 coordinated with two metal ions and the μ-oxo-bridges linking the metal ions. Structural superimposition demonstrated that oxo-Cu2-NgFbpA still keeping an open conformation, similar to the apo-form of NgFbpA. The structure presented additional information towards an understanding of the function of FbpA, and provided a detailed binding model for FbpA protein with the possible metal ions in a biological system.

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Kinetics and mechanism of exogenous anion exchange in FeFbpA–NTA: significance of periplasmic anion lability and anion binding activity of ferric binding protein A

Cited by 9 publications

References 30 publications

Protonation States of Remote Residues Affect Binding–Release Dynamics of the Ligand but Not the Conformation of Apo Ferric Binding Protein

Protonation States of Remote Residues Affect Binding–Release Dynamics of the Ligand but Not the Conformation of Apo Ferric Binding Protein

Role of Citrate and Phosphate Anions in the Mechanism of Iron(III) Sequestration by Ferric Binding Protein: Kinetic Studies of the Formation of the Holoprotein of Wild-Type FbpA and Its Engineered Mutants

Binding of oxo-Cu2 clusters to ferric ion-binding protein A from Neisseria gonorrhoeae: a structural insight

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