Synthetic receptors as models for alkali metal cation-π binding sites in proteins

Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K m and K d for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22 Na + binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.secondary transport | symmetry | membrane protein | alkali metal ion | osmoregulation

Section: Discussionmentioning

confidence: 67%

Section: Simulations Of Betp Suggest the Coordination In The Proposedmentioning

confidence: 98%

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

“…If steric forces controlled complexation, a complex analogous to 1⅐KI should form. Instead, iodide was in contact with ring-bound K ϩ in 4⅐KI as expected in the absence of a cation-interaction (27).…”

Section: Resultsmentioning

confidence: 95%

“…The major focus of our previous efforts (26) has been on the neutral arenes benzene (27), phenol (28), and tryptophan (29). These comprise three of the four aromatic amino acid side chains.…”

mentioning

confidence: 99%

Probing alkali metal–π interactions with the side chain residue of tryptophan

Barbour

Gokel

2002

Self Cite

Feeble forces play a significant role in the organization of proteins. These include hydrogen bonding, hydrophobic interactions, salt bridge formation, and steric interactions. The alkali metal cationinteraction is a force of potentially profound importance but its consideration in biology has been limited by the lack of experimental evidence. Our previous studies of cation-interactions with Na ؉ and K ؉ involved the side arms of tryptophan (indole), tyrosine (phenol), and phenylalanine (benzene) as the arene donors. The receptor system possesses limiting steric constraints. In this report, we show that direct interactions between alkali metals and arenes occur at or within the van der Waals contact distance.

“…T he interactions of cations with aromatic rings play an important role in a range of biological processes, including ion channels, membrane receptors, and enzyme substrate interactions (1)(2)(3)(4)(5)(6)(7)(8)(9). Supramolecular chemical model systems have been instrumental in establishing the basic properties of this important class of noncovalent interactions, but cation-interactions are still poorly understood at a quantitative level, and it is difficult to predict substituent effects.…”

mentioning

confidence: 99%

Substituent effects on cation–π interactions: A quantitative study

Hunter

Low

Rotger

et al. 2002

122

A synthetic supramolecular complex has been adapted to quantify cation-interactions in chloroform by using chemical doublemutant cycles. The interaction of a pyridinium cation with the -face of an aromatic ring is found to be very sensitive to the -electron density. Electron-donating substituents lead to a strong attractive interaction (؊8 kJ͞mol ؊1 ), but electron-withdrawing groups lead to a repulsive interaction (؉2 kJ͞mol ؊1 ).T he interactions of cations with aromatic rings play an important role in a range of biological processes, including ion channels, membrane receptors, and enzyme substrate interactions (1-9). Supramolecular chemical model systems have been instrumental in establishing the basic properties of this important class of noncovalent interactions, but cation-interactions are still poorly understood at a quantitative level, and it is difficult to predict substituent effects. Dougherty and coworkers (10) used the interaction between a synthetic aromatic host and a pyridinium guest to estimate a value of Ϫ10 kJ͞mol Ϫ1 for the interaction of a cation with four -systems in water. This value agrees well with the value of Ϫ11 kJ͞mol Ϫ1 measured by using protein engineering for the interaction of S-methylmethionine with a cavity lined by three -systems (11). Schneider et al. (12) obtained a value of Ϫ3 kJ͞mol Ϫ1 for a single cation-interaction by using a positively charged lipophilic host and an aromatic guest in water.We have developed an approach to the quantitative measurement of noncovalent functional group interactions based on chemical double-mutant cycles. This approach has proved particularly valuable for investigating structure-activity relationships in edge-to-face aromatic interactions, providing new insight into the physical basis for substituent effects on the strengths of these interactions (13,14). Here, we apply this approach to the cation-interaction, or more specifically, to the interaction of a pyridinium cation with the -face of functionalized aromatic rings. The double-mutant cycle is illustrated in Fig. 1. The difference between the stabilities of complexes A and B (⌬G A -⌬G B ) provides an indication of the magnitude of the cation-interaction in complex A, but the value is perturbed by changes in H-bond strength and other secondary interactions associated with the A3B mutation. The secondary effects can be quantified by using complexes C and D where there are no cation-interactions, but the same chemical mutation is made. Thus, the difference ⌬G C -⌬G D provides a direct measure of the changes in H-bond strength and secondary interactions associated with the A3B mutation, and it is possible to dissect out the thermodynamic contribution of the pyridinium-interaction from all of the other interactions present in complex A (⌬⌬G in Eq.