Solution Structure of the 128 kDa Enzyme I Dimer from <i>Escherichia coli</i> and Its 146 kDa Complex with HPr Using Residual Dipolar Couplings and Small- and Wide-Angle X-ray Scattering

Schwieters, Charles D.; Suh, Jeong‐Yong; Grishaev, Alexander; Ghirlando, Rodolfo; Takayama, Yoshihisa; Clore, G. Marius

doi:10.1021/ja105485b

Cited by 118 publications

(183 citation statements)

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“…However, despite a number of landmark studies (4)(5)(6)(7), only a small percentage of structures solved by NMR and deposited in the Protein Data Bank exceed 20 kDa in molecular weight. Larger structures need to be assembled by combining structural information from individual domains, and require additional techniques to elucidate the spatial arrangement, such as shape fitting (5) and/or paramagnetic restraints (8).…”

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confidence: 99%

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

Lange

Rossi

Sgourakis

et al. 2012

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

202

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We have developed an approach for determining NMR structures of proteins over 20 kDa that utilizes sparse distance restraints obtained using transverse relaxation optimized spectroscopy experiments on perdeuterated samples to guide RASREC Rosetta NMR structure calculations. The method was tested on 11 proteins ranging from 15 to 40 kDa, seven of which were previously unsolved. The RASREC Rosetta models were in good agreement with models obtained using traditional NMR methods with larger restraint sets. In five cases X-ray structures were determined or were available, allowing comparison of the accuracy of the Rosetta models and conventional NMR models. In all five cases, the Rosetta models were more similar to the X-ray structures over both the backbone and side-chain conformations than the "best effort" structures determined by conventional methods. The incorporation of sparse distance restraints into RASREC Rosetta allows routine determination of high-quality solution NMR structures for proteins up to 40 kDa, and should be broadly useful in structural biology.nuclear magnetic resonance | sparse data | maltose binding protein | structural genomics | genetic algorithms A dvances in hardware, sample preparation, pulse sequence development, and refinement techniques have expanded the size and complexity of proteins accessible to structure determination by solution-state NMR to include proteins that, until recently, were exclusively the realm of X-ray crystallography (1-3). However, despite a number of landmark studies (4-7), only a small percentage of structures solved by NMR and deposited in the Protein Data Bank exceed 20 kDa in molecular weight. Larger structures need to be assembled by combining structural information from individual domains, and require additional techniques to elucidate the spatial arrangement, such as shape fitting (5) and/or paramagnetic restraints (8).The 20-kDa general limit coincides with the two fundamental problems in solution-state NMR: resonance overlap and progressive increase in the transverse relaxation rate (1∕T 2 ). As the size of a molecule increases, so does the rotational correlation time and, consequently, the efficiency of 1 H-1 H relaxation mechanisms. One way to suppress these effects is to incorporate deuterium into the protein sample, diluting the 1 H-1 H relaxation networks and increasing 13 C and 15 N relaxation times, resulting in sharper line widths and dramatic improvement of the signalto-noise ratios (2, 9, 10). Perdeuteration is generally required for studies of larger proteins (11-14), particularly membrane proteins (15, 16).Unfortunately, deuteration also eliminates the majority of 1 H-1 H NOEs, the main source of long-range distance information in solution-state NMR. Several methods have emerged for reintroducing protons at selected sites to function as distance probes in the structure (11,17). Methyl groups of isoleucine δ1, leucine, and valine side chains are straightforward to label with 13 C and 1 H isotopes in an otherwise deuterated protein sample (12, ...

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confidence: 99%

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

Lange

Rossi

Sgourakis

et al. 2012

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

202

232

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“…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.…”

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confidence: 99%

“…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.…”

mentioning

confidence: 99%

“…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).…”

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confidence: 99%

“…However, in the orientation of EIN α relative to EIN α/β seen in the closed state (i.e., the B conformation of EIN) the Cα-Cα distance between His189 and His15 of HPr bound to EIN α is too large (∼30 Å) to permit subsequent phosphoryl transfer from EIN to HPr (16). In the open state of EI, with EIN in the A conformation, however, the reverse holds: the orientation of EIN α to EIN α/β places His189 in close proximity to His15 of HPr, thereby permitting in-line phosphoryl transfer to HPr (9).…”

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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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Sample Preparation, Data Collection, and Preliminary Data Analysis in Biomolecular Solution X‐Ray Scattering

Grishaev

2012

CP Protein Science

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In addition to the classic methods of structural biology - X-ray crystallography and NMR, solution X-ray scattering (SAXS) is starting to play an important role in experiential structural investigation of biological macromolecules. Ease of SAXS data collection and sophistication of its data analysis tools increasingly used as black boxes can be seen as both a blessing and a curse. On one hand, a sample set aside for solution scattering will always yield experimental data, including cases when macromolecule cannot be crystallized or when it is too large for application of solution NMR. On the other hand, any sample, whether pure or contaminated, whether mono- or polydisperse, will yield scattering data and it is up to the user to ensure the absence of artifacts in them and to choose a proper structural modeling strategy. We will discuss experimental aspects of X-ray solution scattering including sample preparation, data collection, as well as the steps in data processing and preliminary analysis that need to be carried out to ensure the absence of artifacts. Our goal is to summarize everything than can possibly go wrong with SAXS data measurement so that the user can have confidence in the data before they enter structural modeling.

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Solution Structure of the 128 kDa Enzyme I Dimer from Escherichia coli and Its 146 kDa Complex with HPr Using Residual Dipolar Couplings and Small- and Wide-Angle X-ray Scattering

Cited by 118 publications

References 77 publications

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

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

Sample Preparation, Data Collection, and Preliminary Data Analysis in Biomolecular Solution X‐Ray Scattering

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