Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

Siebold, Christian; Flükiger, Karin; Beutler, Rudolf; Erni, Bernhard

doi:10.1016/s0014-5793(01)02705-3

Cited by 105 publications

(79 citation statements)

References 68 publications

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“…In the absence of external sugar, the PTS is in the resting state with all signal transducers phosphorylated (32). When the bacterium encounters external sugar in the medium, the first event involves the transfer of the phosphoryl group from IIB to the incoming sugar located on IIC, thereby switching on the PTS signaling pathway.…”

Section: Relationship Between Phosphoryl Transfer and Domain Dynamicsmentioning

confidence: 99%

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

Suh

Iwahara

Clore

2007

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

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The Escherichia coli mannitol transporter (II Mtl ) comprises three domains connected by flexible linkers: a transmembrane domain (C) and two cytoplasmic domains (A and B). II Mtl catalyzes three successive phosphoryl-transfer reactions: one intermolecular (from histidine phosphocarrier protein to the A domain) and two intramolecular (from the A to the B domain and from the B domain to the incoming sugar bound to the C domain). A key functional requirement of II Mtl is that the A and B cytoplasmic domains be able to rapidly associate and dissociate while maintaining reasonably high occupancy of an active stereospecific AB complex to ensure effective phosphoryl transfer along the pathway. We have investigated the rate of intramolecular domain-domain association and dissociation in IIBA Mtl by using 1 H relaxation dispersion spectroscopy in the rotating frame. The open, dissociated state (comprising an ensemble of states) and the closed, associated state (comprising the stereospecific complex) are approximately equally populated. The first-order rate constants for intramolecular association and dissociation are 1.7 (؎0.3) ؋ 10 4 and 1.8 (؎0.4) ؋ 10 4 s ؊1 , respectively. These values compare to rate constants of Ϸ500 s ؊1 for A 3 B and B 3 A phosphoryl transfer, derived from qualitative lineshape analysis of 1 H-15 N correlation spectra taken during the course of active catalysis. Thus, on average, Ϸ80 association/ dissociation events are required to effect a single phosphoryltransfer reaction. We conclude that intramolecular phosphoryl transfer between the A and B domains of II Mtl is rate-limited by chemistry and not by the rate of formation or dissociation of a stereospecific complex in which the active sites are optimally apposed.domain motion ͉ NMR ͉ protein dynamics ͉ relaxation dispersion ͉ phosphotransferase system T he bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) is a signaling pathway whereby sequential, reversible phosphoryl transfer via a series of transient bimolecular complexes is coupled to sugar uptake across the membrane (1). The transporter (enzyme II Mtl ) of the mannitol branch of the PTS consists of a single polypeptide chain organized into three domains, IIC Mtl , IIB Mtl , and IIA Mtl (from the N to C terminus), connected by flexible linkers (1). The phosphoryl group is transferred from the histidine phosphocarrier protein (HPr) to IIA Mtl via a bimolecular reaction and subsequently from IIA Mtl to IIB Mtl , and finally onto the incoming sugar bound to the cytoplasmic side of IIC Mtl via unimolecular reactions. A key functional feature of II Mtl is that intramolecular association and dissociation between the A and B domains must be fast to allow for effective phosphoryl transfer along the pathway.Relaxation measurements, including relaxation dispersion, have been used to study dynamics of enzyme function and protein folding on milli-to microsecond time scales (2-10). Recent work has suggested that the dynamics of atomic motions observed by relaxation dispersion a...

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Section: Relationship Between Phosphoryl Transfer and Domain Dynamicsmentioning

confidence: 99%

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

Suh

Iwahara

Clore

2007

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

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“…Unlike enzymes II of the carbohydrate PTS, which have been implicated in various physiological events (14,15), substantially less is known about the functions of EIIA Ntr . In Escherichia coli, the ptsN gene encoding EIIA Ntr was identified as a suppressor of some lethal phenotypes; inactivation of the ptsN gene recovered viability of an era mutant lacking essential GTPase activity (16), whereas overexpression of EIIA Ntr compensated for the loss of σ E activity (17).…”

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confidence: 99%

Salmonella pathogenicity island 2 expression negatively controlled by EIIA ^Ntr –SsrB interaction is required for Salmonella virulence

Choi

Shin

Yoon

et al. 2010

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

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SsrA/SsrB is a primary two-component system that mediates the survival and replication of Salmonella within host cells. When activated, the SsrB response regulator directly promotes the transcription of multiple genes within Salmonella pathogenicity island 2 (SPI-2). As expression of the SsrB protein is promoted by several transcription factors, including SsrB itself, the expression of SPI-2 genes can increase to undesirable levels under activating conditions. Here, we report that Salmonella can avoid the hyperactivation of SPI-2 genes by using ptsN-encoded EIIA Ntr , a component of the nitrogen-metabolic phosphotransferase system. Under SPI-2-inducing conditions, the levels of SsrB-regulated gene transcription increased abnormally in a ptsN deletion mutant, whereas they decreased in a strain overexpressing EIIA Ntr . We found that EIIA Ntr controls SPI-2 genes by acting on the SsrB protein at the posttranscriptional level. EIIA Ntr interacted directly with SsrB, which prevented the SsrB protein from binding to its target promoter. Finally, the Salmonella strain, either lacking the ptsN gene or overexpressing EIIA Ntr , was unable to replicate within macrophages, and the ptsN deletion mutant was attenuated for virulence in mice. These results indicated that normal SPI-2 gene expression maintained by an EIIA Ntr -SsrB interaction is another determinant of Salmonella virulence.virulence gene regulation | nitrogen-metabolic phosphotransferase system (PTS) | protein-protein interaction

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“…However, similarly to the IPL/EPL approach, a mixture of linear and circular forms is obtained, presumably because of hydrolysis of an intermediate [73,75]. On the other hand, artificially split inteins such as PI-PfuI, DnaB, and the RecA intein have been successfully applied for in vivo cyclization, and only circular forms were observed [80][81][82], suggesting that the circular permutation approach is more suitable for cyclization. Compared to the IPL/EPL or the TWIN system, in vivo cyclization does not require any external thiol group for cyclization, similarly to protein ligation with split inteins.…”

Section: In Vivo Cyclizationmentioning

confidence: 99%

“…Nevertheless, intein-mediated backbone cyclization has opened the way to a study of cyclization effects on protein stability (including membrane proteins) in more detail. Experimentally improved thermal and/or chemical stability has been shown to result from backbone cyclization of a range of proteins, including b-lactamase, DHFR, E. coli IIA Glc , a destabilized mutant of SH3 domain and the N-terminal domain of DnaB [74,75,81,82,84]. An additional advantage imparted by backbone cyclization is essentially complete resistance to exopeptidases.…”

Section: Stability Enhancement By Backbone Cyclizationmentioning

confidence: 99%

Modern Methods for the Expression of Proteins in Isotopically Enriched Form

Patzelt

Goto

Iwaï

et al. 2002

BioNMR in Drug Research

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The introduction of stable isotopes into proteins has significantly reduced the time requirements for structure elucidation of biomolecules. Moreover, structural studies of proteins with molecular weights exceeding the 10 kDa limit are usually not possible without uniform isotope labeling because of severe resonance overlap and inefficient coherence transfer along the rather small 3 J 1 H-1 H couplings. Nowadays, efficient expression of recombinant proteins is a prerequisite for many techniques used in structural biology, but the requirement for isotope labeling in particular often precludes NMR (nuclear magnetic resonance) structure determination of proteins isolated from natural sources. Specifically, proteins that have been uniformly labeled with 15 N and/or 13 C are commonly required for NMR spectroscopy, especially for backbone chemical shift assignment procedures, which are greatly facilitated by the use of a series of rather sensitive multi-dimensional triple-resonance NMR experiments (see Chapt. 4) [1], in a process that can also be automated with good success [2]. Moreover, the random replacement of nonexchangeable protons by deuterons reduces 1 H-1 H dipolar interactions and scalar couplings, thereby reducing peak line widths considerably and allowing structure elucidation of proteins exceeding 30 kDa [3,4]. Random fractionally deuterated protein samples also permit the use of longer mixing times in NOESY (nuclear Overhauser effect spectroscopy) experiments, since spin-diffusion pathways are largely eliminated. In addition, transverse-relaxation optimized spectroscopy (TROSY [5], see also Chapt. 10), which has been used for molecules larger than 100 kDa [6], benefits dramatically from deuteration. This stems from the fact that the TROSY component that is narrowed by the DD-CSA compensation is broadened by dipolar interactions with nearby protons.Besides uniform labeling approaches, stable isotopes can also be introduced at specific sites in proteins in order to simplify the assignment process and to isolate spectral information from the region of interest. For example, biosynthetically-directed fractional 13 C labeling offers the possibility of making stereospecific assignments of all isopropyl methyl groups of Val and Leu residues [7]. In another approach often used for solid-state NMR termed residue-specific labeling, isotope labels are introduced at single sites in a protein, as described in another chapter of this volume (Chapt. 11). A related scheme, called amino acid-type labeling, is accomplished by expression in an amino acid-based medium

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Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

Cited by 105 publications

References 68 publications

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

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

Salmonella pathogenicity island 2 expression negatively controlled by EIIA ^Ntr –SsrB interaction is required for Salmonella virulence

Modern Methods for the Expression of Proteins in Isotopically Enriched Form

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Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

Cited by 105 publications

References 68 publications

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

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

Salmonella pathogenicity island 2 expression negatively controlled by EIIA Ntr –SsrB interaction is required for Salmonella virulence

Modern Methods for the Expression of Proteins in Isotopically Enriched Form

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Product

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Salmonella pathogenicity island 2 expression negatively controlled by EIIA ^Ntr –SsrB interaction is required for Salmonella virulence