Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I

Venditti, Vincenzo; Tugarinov, Vitali; Schwieters, Charles D.; Grishaev, Alexander; Clore, G. Marius

doi:10.1038/ncomms6960

Cited by 36 publications

(70 citation statements)

References 43 publications

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“…Although the closed state of EI WT , in the form of a phosphoryl transfer intermediate, was fortuitously selected by crystallization (14), both the closed and partially closed states of the wild-type protein are very sparsely populated in solution and their presence cannot be ascertained by SAXS or RDCs (16,19 autophosphorylation by PEP and the predominant open state in solution needed to effect subsequent phosphoryl transfer to the downstream partner protein HPr. From a purely experimental perspective, the existence of a dynamic equilibrium between two distinct states of the EI A -PEP complex could not be ascertained from SAXS or RDC measurements alone because these data, when treated independent of one another, can each be accounted for reasonably well by a single-structure representation.…”

Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

“…In the wild-type EI (EI WT )-PEP complex, the latter is undetectable in solution by small angle X-ray scattering (SAXS) (19) despite the fact that the closed conformation could be selectively crystallized from a solution of EI, PEP, and Mg 2+ in which the autophosphorylation reaction was quenched by the inhibitor oxalate (14). These observations can be attributed to steric and electrostatic repulsion between phosphorylated His189 and bound PEP (19) (14)]. This improved agreement is simply a reflection of the fact that the structure of the EIN-HPr complex was determined using RDCs, albeit in a charged alignment medium of phage fd (9) (hence, the excellent agreement of SVD analysis was carried out using the calcTensor helper of Xplor-NIH (29).…”

Section: Significancementioning

confidence: 99%

“…We recently showed that the open/closed interconversion of PEP-bound EI is modulated by the volume of the active site side chain at position 189, with smaller side chains favoring the closed conformation (19). In the wild-type EI (EI WT )-PEP complex, the latter is undetectable in solution by small angle X-ray scattering (SAXS) (19) despite the fact that the closed conformation could be selectively crystallized from a solution of EI, PEP, and Mg 2+ in which the autophosphorylation reaction was quenched by the inhibitor oxalate (14).…”

Section: Significancementioning

confidence: 99%

“…The target value of χ 2 for the SAXS data is 1.0. *SAXS curves were back-calculated from the coordinates of the EI structures using the calcSAXS-bufsub helper function (19) of Xplor-NIH (29). † RDCs arising from steric alignment were back-calculated from the molecular shapes generated from the coordinates of the EI structures using the calcSARDC helper function of Xplor-NIH (29).…”

Section: Significancementioning

confidence: 99%

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Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering

Venditti

Schwieters

Grishaev

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Enzyme I (EI) is the first component in the bacterial phosphotransferase system, a signal transduction pathway in which phosphoryl transfer through a series of bimolecular protein-protein interactions is coupled to sugar transport across the membrane. EI is a multidomain, 128-kDa homodimer that has been shown to exist in two conformational states related to one another by two large (50-90°) rigid body domain reorientations. The open conformation of apo EI allows phosphoryl transfer from His189 located in the N-terminal domain α/β (EIN α/β ) subdomain to the downstream protein partner bound to the EIN α subdomain. The closed conformation, observed in a trapped phosphoryl transfer intermediate, brings the EIN α/β subdomain into close proximity to the C-terminal dimerization domain (EIC), thereby permitting in-line phosphoryl transfer from phosphoenolpyruvate (PEP) bound to EIC to His189. Here, we investigate the solution conformation of a complex of an active site mutant of EI (H189A) with PEP. Simulated annealing refinement driven simultaneously by solution small angle X-ray scattering and NMR residual dipolar coupling data demonstrates unambiguously that the EI(H189A)-PEP complex exists in a dynamic equilibrium between two approximately equally populated conformational states, one corresponding to the closed structure and the other to a partially closed species. EI is a 128-kDa homodimer, with each subunit comprising two domains (4-6) (Fig. 1A). The N-terminal domain (EIN) is itself subdivided into two subdomains: EIN α includes the binding site for the His phosphocarrier protein (HPr), the downstream partner in the phosphorylation cascade, and EIN α/β contains the site of phosphorylation at His189 (7-9). The C-terminal dimerization domain (EIC) possesses the PEP binding site (10-13). EIN α and EIN α/β are connected to one another by two extended loops (7-9), whereas EIN α/β is connected to EIC via a long swivel helix (14, 15) (Fig. 1A). The structures of free EI from Escherichia coli and Staphylococcus aureus in solution (16,17) and crystal states (15) display open conformations (Fig. 1A, Left), whereas the structure of a trapped phosphoryl transfer intermediate of phosphorylated E. coli EI has a closed conformation (14) (Fig. 1A, Right). The open-to-closed state transition involves two large rigid body conformational transitions accompanied by an ∼50-70°reorientation of EIN α/β relative to EIC and an ∼90°reorientation of EIN α relative to EIN α/β (16). We refer to the EIN α /EIN α/β orientation found in the open and closed structures as the A and B conformations of EIN, respectively. Only the A conformation has been observed in solution and crystal structures of isolated EIN, free (7,8), complexed to HPr (9), or phosphorylated (18). Modeling suggests that either both domain reorientations occur concurrently or reorientation of EIN α/β relative to EIC precedes reorientation of EIN α to avoid a steric clash between EIN α and EIC, resulting in the formation of an intermediate (16).In the closed structure, the pos...

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Section: Discussionmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering

Venditti

Schwieters

Grishaev

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

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Dimerization facilitates the conformational transitions for bacterial phosphotransferase enzyme I autophosphorylation in an allosteric manner

et al. 2017

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The bacterial phosphotransferase system is central to sugar uptake and phosphorylation. Enzyme I ( EI ), the first enzyme of the system, autophosphorylates as a dimer using phosphoenolpyruvate ( PEP ), but it is not clearly understood how dimerization activates the enzyme activity. Here, we show that EI dimerization is important for proper conformational transitions and the domain association required for the autophosphorylation. EI (G356S) with reduced dimerization affinity and lower autophosphorylation activity revealed that significantly hindered conformational transitions are required for the phosphoryl transfer reaction. The G356S mutation does not change the binding affinity for PEP , but perturbs the domain association accompanying large interdomain motions that bring the active site His189 close to PEP . The interface for the domain association is separate from the dimerization interface, demonstrating that dimerization can prime the conformational change in an allosteric manner.

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Coupling between overall rotational diffusion and domain motions in proteins and its effect on dielectric spectra

Ryabov¹

2015

Proteins

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In this work, we formulate a closed-form solution of the model of a semirigid molecule for the case of fluctuating and reorienting molecular electric dipole moment. We illustrate with numeric calculations the impact of protein domain motions on dielectric spectra using the example of the 128 kDa protein dimer of Enzyme I. We demonstrate that the most drastic effect occurs for situations when the characteristic time of protein domain dynamics is comparable to the time of overall molecular rotational diffusion. We suggest that protein domain motions could be a possible explanation for the high-frequency contribution that accompanies the major relaxation dispersion peak in the dielectric spectra of protein aqueous solutions. We propose that the presented computational methodology could be used for the simultaneous analysis of dielectric spectroscopy and nuclear magnetic resonance data. Proteins 2015; 83:1571-1581. © 2015 Wiley Periodicals, Inc.

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Large interdomain rearrangement triggered by suppression of micro- to millisecond dynamics in bacterial Enzyme I

Cited by 36 publications

References 43 publications

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering

Dynamic equilibrium between closed and partially closed states of the bacterial Enzyme I unveiled by solution NMR and X-ray scattering

Dimerization facilitates the conformational transitions for bacterial phosphotransferase enzyme I autophosphorylation in an allosteric manner

Coupling between overall rotational diffusion and domain motions in proteins and its effect on dielectric spectra

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