Intramolecular domain–domain association/dissociation and phosphoryl transfer in the mannitol transporter of
            <i>Escherichia coli</i>
            are not coupled

Suh, Jeong Yong; Iwahara, Junji; Clore, G. Marius

doi:10.1073/pnas.0609103104

Cited by 21 publications

(24 citation statements)

References 39 publications

(70 reference statements)

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“…We exclude the possibility that contaminating EI and HPr in the preparation of EIC or EIN have caused the phosphorylation reaction in our study. First, EIN was not 15 N-IIA Mtl with EIC using the same buffer composition as was used to phosphorylate EIN, the spectra clearly indicated that IIA Mtl remained in the unphosphorylated state (Supporting Information Fig. 1S).…”

Section: Resultsmentioning

confidence: 99%

“…A covalent linker connecting the domains makes the association a unimolecular reaction which assures fast association kinetics defined by the size of the linker. 15 This can be a useful strategy for PTS in which rapid association and dissociation coupled with phosphotransfer reactions are important for prompt sugar uptake and phosphorylation in bacteria.…”

Section: Resultsmentioning

confidence: 99%

“…The plasmids were transformed into an E. coli strain BL21star(DE3), and then grown in Luria Bertini or minimal media with 13 C 6 -glucose and 15 NH 4 Cl as the sole carbon and nitrogen sources in D 2 O. The protein expression was induced with 1 mM isopropyl-b-D-thiogalactopyranoside at an A 600 of $0.8, and harvested by centrifugation after 4 h of induction.…”

Section: Methodsmentioning

confidence: 99%

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Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

Yun

Suh

2012

Protein Science

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Enzyme I initiates a series of phosphotransfer reactions during sugar uptake in the bacterial phosphotransferase system. Here, we have isolated a stable recombinant C-terminal domain of Enzyme I (EIC) of Escherichia coli and characterized its interaction with the N-terminal domain of Enzyme I (EIN) and also with various ligands. EIC can phosphorylate EIN, but their binding is transient regardless of the presence of phosphoenolpyruvate (PEP). Circular dichroism and NMR indicate that ligand binding to EIC induces changes near aromatic groups but not in the secondary structure of EIC. Binding of PEP to EIC is an endothermic reaction with the equilibrium dissociation constant (K D ) of 0.28 mM, whereas binding of the inhibitor oxalate is an exothermic reaction with K D of 0.66 mM from calorimetry. The binding thermodynamics of EIC and PEP compared to that of Enzyme I (EI) and PEP reveals that domain-domain motion in EI can contribute as large as~23.2 kcal/mol toward PEP binding.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

Yun

Suh

2012

Protein Science

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“…3B). Although binding is weak, in the context of intact IIAB Man , where the A and B domains are connected by a flexible 25-residue linker, one can calculate (11,51), based upon the expected average end-to-end distance of ϳ50 Å for the linker (52), that there would be an ϳ85% probability of the two domains interacting with one another at any given time. Man or IIB Man occurs upon binding (at the level of detection of the NMR data).…”

Section: Resultsmentioning

confidence: 99%

Solution NMR Structures of Productive and Non-productive Complexes between the A and B Domains of the Cytoplasmic Subunit of the Mannose Transporter of the Escherichia coli Phosphotransferase System

Hu¹,

Hu²,

Williams³

et al. 2008

Journal of Biological Chemistry

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The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) 5 is a phosphorylation cascade involved in active sugar transport, signaling, and the regulation of carbon catabolite repression as well as an array of other cellular processes (1-3). The initial phosphorylation steps from phosphoenolpyruvate to His-189 (N⑀2) of enzyme I and subsequently to His-15 (N␦1) of HPr are common to all branches of the PTS. Thereafter, the phosphoryl group is transferred to sugar-specific enzymes II, which fall into four major families, glucose (Glc), mannitol (Mtl), mannose (Man), and chitobiose (Chb). The enzymes II are organized into two cytoplasmic domains A and B, and one or two membrane-bound domains, C and D, which may or may not be covalently linked to one another. The A domains from the four major families bear no sequence or structural similarity to one another, but in all cases the active site residue is a histidine (N⑀2) that accepts a phosphoryl group from His-15 (N␦1) of HPr and donates a phosphoryl group to either a cysteine residue in the case of IIB Glc , IIB Mtl , and IIB Chb or a histidine residue (N␦2) for IIB Man . As in the case of the A domains, the B domains from the four major families bear no sequence similarity to one another, and with the exception of IIB Mtl (4) and IIB Chb (5), which have similar topologies, bear no structural similarity either (2, 3).The various protein-protein complexes of the PTS provide a paradigm to explore the structural basis of protein-protein interactions and the factors that permit the recognition of diverse partners using structurally similar interfaces. Moreover, the complexes of the PTS are generally weak (K D ranging from the micromolar to millimolar range) and to date have proved refractory to crystallization. In a series of reports we have used NMR spectroscopy to solve the structures of complexes of HPr with the N-terminal domain of enzyme I (6), IIA Glc (7), IIA Mtl (8), and IIA Man (9), and complexes of IIA Glc and IIA Mtl with IIB Glc (10) and IIB Mtl (11), respectively. In this report we explore the interaction of the IIA Man dimer (35 kDa) with IIB Man (19 kDa), thereby completing the structure determination of the cytoplasmic complexes of the mannose branch of the E. coli PTS.The mannose transporter of E. coli comprises four domains, expressed as three proteins: IIAB * This work was supported by the intramural program of NIDDK, NationalInstitutes of Health (NIH), and the Intramural AIDS Targeted Antiviral Program of the Office of the Director of the NIH (to G. M. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and experimental NMR restraints (codes 2JZH, 2JZN, 2JZO, and 1VSQ)

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“…40. In the presence of catalytic amounts of EI and excess PEP, both proteins are fully phosphorylated.…”

Section: Table 2 Equilibrium Dissociation Constants (K D ) Binding Fmentioning

confidence: 99%

Impact of Phosphorylation on Structure and Thermodynamics of the Interaction between the N-terminal Domain of Enzyme I and the Histidine Phosphocarrier Protein of the Bacterial Phosphotransferase System

Suh

Cai

Clore

2008

Journal of Biological Chemistry

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The structural and thermodynamic impact of phosphorylation on the interaction of the N-terminal domain of enzyme I (EIN) and the histidine phosphocarrier protein (HPr), the two common components of all branches of the bacterial phosphotransferase system, have been examined using NMR spectroscopy and isothermal titration calorimetry. His-189 is located at the interface of the ␣ and ␣␤ domains of EIN, resulting in rather widespread chemical shift perturbation upon phosphorylation, in contrast to the highly localized perturbations seen for HPr, where His-15 is fully exposed to solvent. Residual dipolar coupling measurements, however, demonstrate unambiguously that no significant changes in backbone conformation of either protein occur upon phosphorylation: for EIN, the relative orientation of the ␣ and ␣␤ domains remains unchanged; for HPr, the backbone / torsion angles of the active site residues are unperturbed within experimental error. His 3 Glu/Asp mutations of the active site histidines designed to mimic the phosphorylated states reveal binding equilibria that favor phosphoryl transfer from EIN to HPr. Although binding of phospho-EIN to phospho-HPr is reduced by a factor of ϳ21 relative to the unphosphorylated complex, residual dipolar coupling measurements reveal that the structures of the unphosphorylated and biphosphorylated complexes are the same. Hence, the phosphorylation states of EIN and HPr shift the binding equilibria predominantly by modulating intermolecular electrostatic interactions without altering either the backbone scaffold or binding interface. This facilitates highly efficient phosphoryl transfer between EIN and HPr, which is estimated to occur at a rate of ϳ850 s ؊1 from exchange spectroscopy.The bacterial phosphotransferase system (PTS) 2 couples sequential phosphoryl transfer via a series of biomolecular protein-protein interactions to active sugar transport across the membrane (1-4). In addition, the phosphorylation state of individual components of the PTS act as switches in the regulation of diverse cellular processes, including transcription, chemotaxis, and glycolysis (4). The first two proteins in the PTS, enzyme I, and the histidine phosphocarrier protein (HPr) are common to all branches, whereas subsequent phosphorylation transfer steps involve sugar-specific permeases, also known as enzymes II. The initial step in the PTS involves autophosphorylation of enzyme I (EI) at the N⑀2 atom of His-189 by phosphoenolpyruvate (PEP) (5, 6). Autophosphorylation requires the presence of the C-terminal domain of EI (EIC), but reversible phosphoryl transfer from EI to the N␦1 atom of His-15 of HPr (7) only requires the N-terminal domain of EI, EIN (8, 9). The phosphoryl group is subsequently transferred from HPr through a sequence of two cytoplasmic enzyme II components, A and B, to the incoming sugar bound to the cytoplasmic side of the membrane-bound sugar permease (1-4).The phosphotransfer reactions within the PTS are reversible (4). In the resting state (in the absence of external sugar),...

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Intramolecular domain–domain association/dissociation and phosphoryl transfer in the mannitol transporter of Escherichia coli are not coupled

Cited by 21 publications

References 39 publications

Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

Solution NMR Structures of Productive and Non-productive Complexes between the A and B Domains of the Cytoplasmic Subunit of the Mannose Transporter of the Escherichia coli Phosphotransferase System

Impact of Phosphorylation on Structure and Thermodynamics of the Interaction between the N-terminal Domain of Enzyme I and the Histidine Phosphocarrier Protein of the Bacterial Phosphotransferase System

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