Beyond non-integer Hill coefficients: A novel approach to analyzing binding data, applied to Na+-driven transporters

Ravera, Silvia; Quick, Matthias; Nicola, Juan Pablo; Carrasco, Nancy; Amzel, L. Mario

doi:10.1085/jgp.201511365

Cited by 18 publications

(22 citation statements)

References 35 publications

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“…Far from being surprising, this observation leads to an important mechanistic conclusion: binding of Na + to the Na1 site increases the affinity for Na + of the Na2 site by allowing residues S353 and T354 to coordinate Na + at the Na2 site-either through a defined conformational change or by reducing the magnitude of the fluctuations, thereby increasing the population of the binding-competent conformations. In support of this conclusion, SPA Na + binding data on the S353A/T354A mutant showed that the mutant NIS has two affinities-one in the range of 1-2 mM and the other in the range of 90-120 mM (51). This finding can be interpreted as indicating that, in the double mutant, the cooperative interactions are lost, yielding a highaffinity Na1 site and a disrupted Na2 site (51).…”

Section: Q263n (20%)mentioning

confidence: 71%

“…In support of this conclusion, SPA Na + binding data on the S353A/T354A mutant showed that the mutant NIS has two affinities-one in the range of 1-2 mM and the other in the range of 90-120 mM (51). This finding can be interpreted as indicating that, in the double mutant, the cooperative interactions are lost, yielding a highaffinity Na1 site and a disrupted Na2 site (51).…”

Section: Q263n (20%)mentioning

confidence: 71%

“…Although S353 and T354 of NIS do not directly coordinate Na + at Na2 when only Na2 is occupied (Fig. 2), substitutions at these positions have a dramatic effect on the transport activity of the protein (37,51). Both these residues, however, coordinate Na + at the Na2 site when the Na1 site is occupied.…”

Section: Q263n (20%)mentioning

confidence: 98%

“…In NIS, our analysis of whole-cell transport data carried out using a statistical thermodynamics formalism found that binding to one of the sites significantly increases the affinity for Na + of the other (12). Experiments using 22 Na + in a scintillation proximity assay (SPA) also showed strong cooperativity between the two sites (51). Furthermore, the cooperativity is lost when residues S353 and T354-Na + ligands at the Na2 site-are replaced by alanine (51).…”

Section: Significancementioning

confidence: 99%

“…Experiments using 22 Na + in a scintillation proximity assay (SPA) also showed strong cooperativity between the two sites (51). Furthermore, the cooperativity is lost when residues S353 and T354-Na + ligands at the Na2 site-are replaced by alanine (51). The effect of these mutations suggests that the cooperativity between the sites involves, at least in part, some of the residues that form the Na2 site.…”

Section: Significancementioning

confidence: 99%

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Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Ferrandino

Nicola

Sánchez

et al. 2016

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

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The sodium/iodide symporter (NIS) mediates active I− transport in the thyroid—the first step in thyroid hormone biosynthesis—with a 2 Na+: 1 I− stoichiometry. The two Na+ binding sites (Na1 and Na2) and the I− binding site interact allosterically: when Na+ binds to a Na+ site, the affinity of NIS for the other Na+ and for I− increases significantly. In all Na+-dependent transporters with the same fold as NIS, the side chains of two residues, S353 and T354 (NIS numbering), were identified as the Na+ ligands at Na2. To understand the cooperativity between the substrates, we investigated the coordination at the Na2 site. We determined that four other residues—S66, D191, Q194, and Q263—are also involved in Na+ coordination at this site. Experiments in whole cells demonstrated that these four residues participate in transport by NIS: mutations at these positions result in proteins that, although expressed at the plasma membrane, transport little or no I−. These residues are conserved throughout the entire SLC5 family, to which NIS belongs, suggesting that they serve a similar function in the other transporters. Our findings also suggest that the increase in affinity that each site displays when an ion binds to another site may result from changes in the dynamics of the transporter. These mechanistic insights deepen our understanding not only of NIS but also of other transporters, including many that, like NIS, are of great medical relevance.

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Section: Q263n (20%)mentioning

confidence: 71%

Section: Q263n (20%)mentioning

confidence: 71%

Section: Q263n (20%)mentioning

confidence: 98%

Section: Significancementioning

confidence: 99%

Section: Significancementioning

confidence: 99%

See 3 more Smart Citations

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Ferrandino

Nicola

Sánchez

et al. 2016

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

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Design principles of drug combinations for chemotherapy

Pusuluri

Vogus

et al. 2020

Journal of Controlled Release

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Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS)

Zhekova

Sakuma

Johnson³

et al. 2020

J. Chem. Inf. Model.

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The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na+ ions and I– substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) 125I– uptake of 35 hNIS variants.

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Beyond non-integer Hill coefficients: A novel approach to analyzing binding data, applied to Na+-driven transporters

Abstract: A novel approach to analyzing binding data from proteins with two binding sites for the same substrate provides information beyond that accessible with traditional Hill equation analysis.

Cited by 18 publications

References 35 publications

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Design principles of drug combinations for chemotherapy

Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS)

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Beyond non-integer Hill coefficients: A novel approach to analyzing binding data, applied to Na+-driven transporters

Abstract: A novel approach to analyzing binding data from proteins with two binding sites for the same substrate provides information beyond that accessible with traditional Hill equation analysis.

Cited by 18 publications

References 35 publications

Na + coordination at the Na2 site of the Na + /I − symporter

Na + coordination at the Na2 site of the Na + /I − symporter

Design principles of drug combinations for chemotherapy

Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS)

Contact Info

Product

Resources

About

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter

Na ⁺ coordination at the Na2 site of the Na ⁺ /I ⁻ symporter