Oligomerization of a molecular chaperone modulates its activity

Molecular chaperones alter the folding properties of cellular proteins via mechanisms that are not well understood. Here, we show that Trigger Factor (TF), an ATP-independent chaperone, exerts strikingly contrasting effects on the folding of non-native proteins as it transitions between a monomeric and a dimeric state. We used NMR spectroscopy to determine the atomic resolution structure of the 100 kDa dimeric TF. The structural data show that some of the substrate-binding sites are buried in the dimeric interface, explaining the lower affinity for protein substrates of the dimeric compared to the monomeric TF. Surprisingly, the dimeric TF associates faster with proteins and it exhibits stronger anti-aggregation and holdase activity than the monomeric TF. The structural data show that the dimer assembles in a way that substrate-binding sites in the two subunits form a large contiguous surface inside a cavity, thus accounting for the observed accelerated association with unfolded proteins. Our results demonstrate how the activity of a chaperone can be modulated to provide distinct functional outcomes in the cell.

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To be, or not to be two sites: that is the question about LeuT substrate binding

The Journal of General Physiology ◽

10.1085/jgp.201110652 ◽

2011 ◽

Vol 138 (4) ◽

pp. 467-471 ◽

Cited By ~ 15

Author(s):

Nicolas Reyes ◽

Sotiria Tavoulari

Keyword(s):

Binding Sites ◽

Journal Club ◽

Substrate Binding ◽

Structural Data ◽

Transport Proteins ◽

Model System ◽

Transport Mechanisms ◽

High Affinity ◽

The Central Nervous System ◽

Substrate Binding Sites

Transport proteins of the neurotransmitter sodium symporter (NSS) family regulate the extracellular concentration of several neurotransmitters in the central nervous system. The only member of this family for which atomic-resolution structural data are available is the prokaryotic homologue LeuT. This protein has been used as a model system to study the molecular mechanism of transport of the NSS family. In this Journal Club, we discuss two strikingly different LeuT transport mechanisms: one involving a single high-affinity substrate binding site and one recently proposed alternative involving two high-affinity substrate binding sites that are allosterically coupled.

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Faculty Opinions recommendation of Identification of the TRiC/CCT substrate binding sites uncovers the function of subunit diversity in eukaryotic chaperonins.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1046453.496422 ◽

2006 ◽

Author(s):

Amnon Horovitz

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Faculty Opinions recommendation of Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718076922.793482351 ◽

2013 ◽

Author(s):

Robert Vandenberg

Keyword(s):

Dopamine Transporter ◽

Binding Sites ◽

Substrate Binding ◽

Multiple Substrate ◽

Substrate Binding Sites

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Covalent modification of substrate-binding sites of Escherichia coli ADP-glucose synthetase. Isolation and structural characterization of 8-azido-ADP-glucose-incorporated peptides.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)36052-0 ◽

1986 ◽

Vol 261 (3) ◽

pp. 1058-1064

Author(s):

Y M Lee ◽

J Preiss

Keyword(s):

Escherichia Coli ◽

Structural Characterization ◽

Binding Sites ◽

Substrate Binding ◽

Covalent Modification ◽

Substrate Binding Sites

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Substrate-binding sites in acetylcholinesterase

Trends in Pharmacological Sciences ◽

10.1016/0165-6147(91)90621-x ◽

1991 ◽

Vol 12 ◽

pp. 422-426 ◽

Cited By ~ 26

Author(s):

Ferdinand Hucko ◽

Jaak Järv ◽

Christoph Weise

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Opposite stereospecificity of two tropinone reductases is conferred by the substrate-binding sites.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32627-3 ◽

1994 ◽

Vol 269 (16) ◽

pp. 11695-11698 ◽

Cited By ~ 1

Author(s):

K. Nakajima ◽

T. Hashimoto ◽

Y. Yamada

Keyword(s):

Binding Sites ◽

Substrate Binding ◽

Substrate Binding Sites

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Human organic anion transporter MRP4 (ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites

AJP Renal Physiology ◽

10.1152/ajprenal.00133.2004 ◽

2005 ◽

Vol 288 (2) ◽

pp. F327-F333 ◽

Cited By ~ 147

Author(s):

Rémon A. M. H. Van Aubel ◽

Pascal H. E. Smeets ◽

Jeroen J. M. W. van den Heuvel ◽

Frans G. M. Russel

Keyword(s):

Binding Sites ◽

Efflux Pump ◽

Substrate Binding ◽

Organic Anion ◽

Apical Cell ◽

Cell Uptake ◽

Hek293 Cells ◽

Hill Coefficient ◽

Anion Transporter ◽

Substrate Binding Sites

The end product of human purine metabolism is urate, which is produced primarily in the liver and excreted by the kidney through a well-defined basolateral blood-to-cell uptake step. However, the apical cell-to-urine efflux mechanism is as yet unidentified. Here, we show that the renal apical organic anion efflux transporter human multidrug resistance protein 4 (MRP4), but not apical MRP2, mediates ATP-dependent urate transport via a positive cooperative mechanism ( Km of 1.5 ± 0.3 mM, Vmax of 47 ± 7 pmol·mg−1·min−1, and Hill coefficient of 1.7 ± 0.2). In HEK293 cells overexpressing MRP4, intracellular urate levels were lower than in control cells. Urate inhibited methotrexate transport (IC50 of 235 ± 8 μM) by MRP4, did not affect cAMP transport, whereas cGMP transport was stimulated. Urate shifted cGMP transport by MRP4 from positive cooperativity ( Km and Vmax value of 180 ± 20 μM and 58 ± 4 pmol·mg−1·min−1, respectively, Hill coefficient of 1.4 ± 0.1) to single binding site kinetics ( Km and Vmax value of 2.2 ± 0.9 mM and 280 ± 50 pmol·mg−1·min−1, respectively). Finally, MRP4 could transport urate simultaneously with cAMP or cGMP. We conclude that human MRP4 is a unidirectional efflux pump for urate with multiple allosteric substrate binding sites. We propose MRP4 as a candidate transporter for urinary urate excretion and suggest that MRP4 may also mediate hepatic export of urate into the circulation, because of its basolateral expression in the liver.

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