Two Modes of Ligand Binding in Maltose-binding Protein ofEscherichia coli

Hall, Jason A.; Thorgeirsson, Thorgeir E.; Liu, Jun; Shin, Yeon‐Kyun; Nikaido, Hiroshi

doi:10.1074/jbc.272.28.17610

Cited by 72 publications

(56 citation statements)

References 24 publications

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“…3C). In agreement with the reduced closing movement in the presence of longer ␣-1,4-oligomaltoside chains observed in spectroscopic analyses (15,20), the maximum change in ratio (at saturation) decreased with the length of the maltose chain, soluble starch giving the lowest ratio, whereas the affinity remained at a similar range (Fig. 3C).…”

Section: Resultssupporting

confidence: 71%

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Fehr

Frommer

Lalonde

2002

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

315

279

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Compartmentation of metabolic reactions and thus transport within and between cells can be understood only if we know subcellular distribution based on nondestructive dynamic monitoring. Currently, methods are not available for in vivo metabolite imaging at cellular or subcellular levels. Limited information derives from methods requiring fixation or fractionation of tissue (1, 2). We thus developed a flexible strategy for designing proteinbased nanosensors for a wide spectrum of solutes, allowing analysis of changes in solute concentration in living cells. We made use of bacterial periplasmic binding proteins (PBPs), where we show that, on binding of the substrate, PBPs transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. By using the maltose-binding protein as a prototype, nanosensors were constructed allowing in vitro determination of FRET changes in a concentration-dependent fashion. For physiological applications, mutants with different binding affinities were generated, allowing dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. Control sensors allow the exclusion of the effect from other cellular or environmental parameters on ratio imaging. Thus the myriad of PBPs recognizing a wide spectrum of different substrates is suitable for FRET-based in vivo detection, providing numerous scientific, medical, and environmental applications. Limited information is derived from methods requiring fixation or fractionation of tissue (1, 2). Static analysis of metabolite composition in organs, tissues, and cellular compartments involves cell disruption. Most techniques neither measure metabolite changes in real-time nor account for likely variations in local metabolite concentration at the cellular level. Current methods have low resolution and are prone to artifacts, e.g., contamination by other cell types or subcellular compartments. Thus, little is known about the dynamic changes in concentration of metabolites such as sugars and amino acids͞neurotransmit-ters at the site of transport, i.e., in the synaptic cleft relative to the cytosol of adjacent neurons and glia or at the loading site of the phloem in plants, but also in the distribution of different sugars within cellular compartments. To better understand metabolism and compartmentation, a noninvasive technique would be of significant advantage.To generate a set of multifunctional nanosensors with specificity to a large number of different compounds, suitable binding proteins fulfilling a number of criteria are required. First, the binding proteins must undergo a conformational change on substrate binding. Ideally, they should belong to a family covering a wide spectrum of substrates. Furthermore, high-affinity binding would be advantageous, because it would provide a comparatively fast way to generate mutants with lower affinities suited for an optimal physiological detection range. Finally, for measurements in eukaryotes, ...

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Section: Resultssupporting

confidence: 71%

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Fehr

Frommer

Lalonde

2002

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

315

279

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“…On this basis, the occupancy of the wildtype conformation is estimated at 0.4 and that of the alternative conformation is 0. 6.…”

Section: Energetic Coupling In the Maltosementioning

confidence: 99%

“…In the periplasm, MBP binds maltose, which stabilizes a change from an "open" to a "closed" conformation, enabling it to stimulate the MalFGK 2 ATPase (5,6). Interactions with closed, maltose-bound MBP lead to exposure of the MalFGK 2 maltose-binding site to the periplasmic side where maltose can move from MBP into an occluded translocation pathway (7,8).…”

mentioning

confidence: 99%

Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation without Direct Recognition of Substrate

Gould

Shilton

2010

Journal of Biological Chemistry

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The ATPase activity of the maltose transporter (MalFGK 2 ) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK 2 are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose-and sucrose-bound sMBP stimulate equal levels of MalFGK 2 ATPase activity. Therefore, the ATPase activity of MalFGK 2 is coupled to translocation of maltose solely by interactions between MalFGK 2 and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the ATP-binding cassette (ABC)2 transporters move various substrates across membranes, with substrate movement coupled to the hydrolysis of ATP. Although the ATPase activity of ABC exporters like P-glycoprotein is generally stimulated by substrate binding, the ATPase activity of ABC importers is activated by a peripheral substrate-binding protein and not the free substrate (for recent reviews see Refs.

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“…Analysis of the dipolar broadening function (in Fourier space) indicated that between 60 and 90% of the proteins were doubly labeled, in other words that 10 -40% were monoradical species. (12). It has been demonstrated that accurate inter-nitroxide distance calculations can be obtained from populations containing as little as 40% doubly labeled proteins using the Fourier deconvolution technique when spectra are collected at low temperatures (13) (see below).…”

Section: Methodsmentioning

confidence: 99%

“…Low temperature measurements were employed to minimize the effects due to variability in spin label coupling efficiency as well as to minimize the exchange interactions, dynamic and static magnetic dipolar interactions, and the effects of molecular tumbling that complicate the calculation of distances using spectral broadening (12). Data were collected from 3320 to 3420 G. The center point of the nitroxide spectra was located at 3368 G. All spectra were measured using 10 milliwatts of microwave power with a modulation amplitude of 2 G. Ten scans were signal-averaged for each spectra.…”

Section: Methodsmentioning

confidence: 99%

Arachidonic Acid and Nonsteroidal Anti-inflammatory Drugs Induce Conformational Changes in the Human Prostaglandin Endoperoxide H2 Synthase-2 (Cyclooxygenase-2)

Smith

McCracken

Shin

et al. 2000

Journal of Biological Chemistry

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By using the technique of site-directed spin labeling combined with EPR spectroscopy, we have observed that binding of arachidonic acid and nonsteroidal antiinflammatory drugs induces conformational changes in the human prostaglandin endoperoxide H 2 synthase enzyme (PGHS-2). Line shape broadening resulting from spin-spin coupling of nitroxide pairs introduced into the membrane-binding helices of PGHS-2 was used to calculate the inter-helical distances and changes in these distances that occur in response to binding various ligands. The inter-residue distances determined for the PGHS-2 holoenzyme using EPR were 1-7.9 Å shorter than those of the crystal structure of the PGHS-2 holoenzyme. However, inter-helical distances calculated and determined by EPR for PGHS-2 complexed with arachidonic acid, flurbiprofen, and SC-58125 were in close agreement with those obtained from the cognate crystal structures. These results indicate that the structure of the solubilized PGHS-2 holoenzyme measured in solution differs from the crystal structure of PGHS-2 holoenzyme obtained by x-ray analysis. Furthermore, binding of ligands induces a conformational change in the holo-PGHS-2, converting it to a structure similar to those obtained by x-ray analysis. Proteolysis protection assays had previously provided circumstantial evidence that binding of heme and non-steroidal anti-inflammatory drugs alters the conformation of PGHS, but the present experiments are the first to directly measure such changes. The finding that arachidonate can also induce a conformational change in PGHS-2 was unexpected, and the magnitude of changes suggests this structural flexibility may be integral to the cyclooxygenase catalytic mechanism.Two prostaglandin endoperoxide H 2 synthase (PGHS) 1 isozymes, PGHS-1 and -2 (also known as cyclooxygenase-1 and -2, or COX-1 and COX-2), produce the intermediate PGH 2 from which prostaglandins, prostacyclin, and thromboxanes are derived. These two enzymes are both pharmacological targets of traditional nonsteroidal anti-inflammatory drugs (NSAIDs) (1). Recently, two new drugs have been developed, Celebrex and Vioxx, that selectively inhibit PGHS-2. These "COX-2 inhibitors" have an improved gastrointestinal safety profile, presumably because they do not interfere with prostaglandin production by PGHS-1 (2). Preferential inhibition by COX-2 inhibitors results in part because these drugs are bulky and fit more readily in the larger active site of COX-2 (2, 3). However, the hallmark of COX-2 inhibitors is their differential mechanism of inhibition for PGHS-2. These drugs are time-dependent inhibitors of the PGHS-2, forming tight binding and slowly reversible complexes with this isozyme but are simple competitive reversible inhibitors of the PGHS-1 isozyme.Formation of the NSAID-enzyme complexes has been proposed to result in conformational changes in PGHS (4). This hypothesis is supported by studies that show that time-dependent inhibitors protect from proteolytic cleavage at Arg-277, 2 an amino acid located on a peptide...

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Two Modes of Ligand Binding in Maltose-binding Protein ofEscherichia coli

Cited by 72 publications

References 24 publications

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Visualization of maltose uptake in living yeast cells by fluorescent nanosensors

Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation without Direct Recognition of Substrate

Arachidonic Acid and Nonsteroidal Anti-inflammatory Drugs Induce Conformational Changes in the Human Prostaglandin Endoperoxide H2 Synthase-2 (Cyclooxygenase-2)

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