Valence Screening of Water in Protein Crystals Reveals Potential Na+Binding Sites

Nayal, Murad; Di, Enrico

doi:10.1006/jmbi.1996.0081

Cited by 198 publications

(182 citation statements)

References 24 publications

Supporting

Mentioning

167

Contrasting

Order By: Relevance

“…Restraining Na + in the D454A Na1 site during simulations to prevent dissociation results in some accommodation of the Na1 site, but the ν Na+ of 0.82 is still too small to account for significant binding. 24 In contrast to instability of the bound cation in the Na1 site, a Na + ion was stable in the Na3 site of EAAC1 D454A, consistent with previous results showing that Na + can activate the leak anion conductance in the empty mutant transporter. However, it is unclear how Na + would be able to access this binding site, because water is mainly excluded from the core of the transport domain by the D454A mutation ( Figure 7C).…”

Section: ■ Resultssupporting

confidence: 91%

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

Mwaura

Tao

James

et al. 2012

ACS Chem. Neurosci.

View full text Add to dashboard Cite

Substrate transport by glutamate transporters is coupled to the co-transport of 3 Na + ions and counter-transport of 1 K + ion. The highly conserved Asp454, which may be negatively charged, is of interest as its side chain may coordinate cations and/or contribute to charge compensation. Mutation to the nonionizable Asn resulted in a transporter that no longer catalyzed forward transport. However, Na + /glutamate exchange was still functional, as demonstrated by the presence of transient currents following rapid substrate application and voltage jumps. While the kinetics of Na + / glutamate exchange were slowed, the apparent valence (z) of the charge moved in EAAC1 D454N (0.71) was similar to that of EAAC1 WT (0.64). Valences calculated using the Poisson−Boltzmann equation were close to the experimental values for EAAC1 D454N (0.55), and with D454 protonated (0.45). In addition, pK a calculations performed for the bacterial homologue GltPh revealed a highly perturbed pK a (7.6 to >14) for D405 residue (analogous to D454), consistent with this site being protonated at physiological pH. In contrast to the D454N mutation, substitution to alanine resulted in a transporter that still bound glutamate, but could not translocate it. The results are consistent with molecular dynamics simulations, showing that the alanine but not the asparagine mutation resulted in defective Na + coordination. Our results raise the possibility that the protonated state of D454 supports transporter function.

show abstract

Section: ■ Resultssupporting

confidence: 91%

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

Mwaura

Tao

James

et al. 2012

ACS Chem. Neurosci.

View full text Add to dashboard Cite

show abstract

“…In both the coordination number is ∼5 with an average Na + to ligand distance of 2.35 Å. In the highestresolution structure of cotransporters obtained to date, LeuT at 1.6 Å (6), two Na + sites have been proposed on the basis of an indirect valence test (30). Na1 has six bonds between Na + and the protein, including two carbonyl oxygens (A22 and T254), the hydroxyl oxygen from T254, side-chain amide oxygens from N27 3 ± 4 −54 ± 4 0.9 ± 0.1 0.6 ± 0.1 Y290S 18 ± 1 −40 ± 6 0.9 ± 0.1 0.7 ± 0.1 Y290F −49 ± 13 < −120 0.9 ± 0.1 n.m. W291C 12 ± 2 −53 ± 2 0.9 ± 0.1 0.6 ± 0.1 W291F −49 ± 9 < −120 1.0 ± 0.1 n.m. Q457C −55 ± 4 < −120 1.0 ± 0.1 n.m Near Na2 site D204E* −50 ± 2 −67 ± 3 ∼1 ∼1 S392A/C −56 ± 3 −100 to −140 0.9 ± 0.1 n.m. Other F101C −3 ± 3 < −120…”

Section: Resultsmentioning

confidence: 99%

Functional identification and characterization of sodium binding sites in Na symporters

Loo

Jiang

Gorraitz

et al. 2013

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

View full text Add to dashboard Cite

Sodium cotransporters from several different gene families belong to the leucine transporter (LeuT) structural family. Although the identification of Na + in binding sites is beyond the resolution of the structures, two Na + binding sites (Na1 and Na2) have been proposed in LeuT. Na2 is conserved in the LeuT family but Na1 is not. A biophysical method has been used to measure sodium dissociation constants (K d ) of wild-type and mutant human sodium glucose cotransport (hSGLT1) proteins to identify the Na + binding sites in hSGLT1. The Na1 site is formed by residues in the sugar binding pocket, and their mutation influences sodium binding to Na1 but not to Na2. For the canonical Na2 site formed by two -OH side chains, S392 and S393, and three backbone carbonyls, mutation of S392 to cysteine increased the sodium K d by sixfold. This was accompanied by a dramatic reduction in the apparent sugar and phlorizin affinities. We suggest that mutation of S392 in the Na2 site produces a structural rearrangement of the sugar binding pocket to disrupt both the binding of the second Na + and the binding of sugar. In contrast, the S393 mutations produce no significant changes in sodium, sugar, and phlorizin affinities. We conclude that the Na2 site is conserved in hSGLT1, the side chain of S392 and the backbone carbonyl of S393 are important in the first Na + binding, and that Na + binding to Na2 promotes binding to Na1 and also sugar binding.I on coupled symporters, or cotransporters, such as hSGLT1 use electrochemical potential gradients to drive solutes into cells. A common finding for these transporters is that external Na + binds before the substrate. Na + binding induces a conformational change of the protein, resulting in the substrate vestibule becoming open to the external membrane surface. After substrate binding, the two ligands are transported across the membrane and are released into the cytoplasm. The atomic structures of several sodium dependent transporters have been solved [leucine transporter (LeuT), vibrio parahaemolyticus sodium glucose (vSGLT), sodium hydantoin (Mhp1), and sodium betaine (BetP)] (1-6). They share a common structural fold with a five-helix inverted repeat, the "LeuT fold". The substrate binding sites are located in the middle of the protein, isolated from the external and membrane surfaces by hydrophobic gates, and putative Na + sites have been identified. Testing these binding sites is a problem due to the fact that phenomenological kinetic constants for ligand transport (K 0.5 , half-saturation values) are interdependent on each other (7). Here we have developed a method to estimate the intrinsic Na + dissociation constants (K d ) for human sodium glucose cotransport (hSGLT1) that may be broadly applicable to other symporters. We then use this to investigate the importance of hSGLT1 residues predicted to be at or near the two Na + binding sites, Na1 and Na2.The method is based on the fact that voltage-dependent membrane proteins exhibit transient charge movements (capacitive transients) i...

show abstract

“…In both data sets, R-work and R-free converged to approximately 15% and 19%, respectively, and geometrical validation was carried out by several programs such as PROCHECK, 42 STAN, 43 and MOLPROBITY. 44 Analysis of the Ramachandran plot showed that 99.3% of the protein residues are in most favored or additionally allowed regions, while only a small fraction of residues, 0.7%, are in generously allowed or disallowed regions of the plot.…”

Section: Methodsmentioning

confidence: 99%

“…A magnesium ion from the crystallization buffer, not identified previously in the native structure, was added to the two molecular models, as suggested by the STAN program. 43 This atom has a typical octahedral coordination to solvent molecules. The major differences between EDO and GOL structures, relative to the native structure, is the absence of the 2-propanol molecule, which was replaced by these molecules respectively, in similar positions at the active site (Figure 3).The absence of electron density for the 2-propanol proves that the soaking strategy to remove it from the crystallization solution was successful.…”

Section: Epr Spectroscopy Of Dgaor Inhibited With Ethylenementioning

confidence: 99%

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

et al. 2009

View full text Add to dashboard Cite

Aldehyde oxidoreductase from Desulfovibrio gigas (DgAOR) is a member of the xanthine oxidase (XO) family of mononuclear Mo-enzymes that catalyzes the oxidation of aldehydes to carboxylic acids. The molybdenum site in the enzymes of the XO family shows a distorted square pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. We report here steady-state kinetic studies of DgAOR with the inhibitors cyanide, ethylene glycol, glycerol, and arsenite, together with crystallographic and EPR studies of the enzyme after reaction with the two alcohols. In contrast to what has been observed in other members of the XO family, cyanide, ethylene glycol, and glycerol are reversible inhibitors of DgAOR. Kinetic data with both cyanide and samples prepared from single crystals confirm that DgAOR does not need a sulfido ligand for catalysis and confirm the absence of this ligand in the coordination sphere of the molybdenum atom in the active enzyme. Addition of ethylene glycol and glycerol to dithionite-reduced DgAOR yields rhombic Mo(V) EPR signals, suggesting that the nearly square pyramidal coordination of the active enzyme is distorted upon alcohol inhibition. This is in agreement with the X-ray structure of the ethylene glycol and glycerolinhibited enzyme, where the catalytically labile OH/OH 2 ligand is lost and both alcohols coordinate the Mo site in a η 2 fashion. The two adducts present a direct interaction between the molybdenum and one of the carbon atoms of the alcohol moiety, which constitutes the first structural evidence for such a bond in a biological system.

show abstract

Valence Screening of Water in Protein Crystals Reveals Potential Na+Binding Sites

Cited by 198 publications

References 24 publications

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

Functional identification and characterization of sodium binding sites in Na symporters

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

Contact Info

Product

Resources

About

Valence Screening of Water in Protein Crystals Reveals Potential Na+Binding Sites

Cited by 198 publications

References 24 publications

Protonation State of a Conserved Acidic Amino Acid Involved in Na+ Binding to the Glutamate Transporter EAAC1

Protonation State of a Conserved Acidic Amino Acid Involved in Na+ Binding to the Glutamate Transporter EAAC1

Functional identification and characterization of sodium binding sites in Na symporters

Kinetic, Structural, and EPR Studies Reveal That Aldehyde Oxidoreductase from Desulfovibrio gigas Does Not Need a Sulfido Ligand for Catalysis and Give Evidence for a Direct Mo−C Interaction in a Biological System

Contact Info

Product

Resources

About

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1

Protonation State of a Conserved Acidic Amino Acid Involved in Na⁺ Binding to the Glutamate Transporter EAAC1