Energetic localization of saxitoxin in its channel binding site

Saxitoxin (STX) selectively blocks the voltage-gated sodium channel at the outer vestibule lined by P-loops of the four domains. Neosaxitoxin has an additional -OH group at the N1 position of the 1,2,3 guanidinium (N1-OH) that interacts with domains I and IV of the Na(+) channel. Determination of a second toxin interaction with the channel would fix the location of STX. Gonyautoxin 2,3 and Gonyautoxin 1,4 are C-11 sulfated derivatives of saxitoxin and neosaxitoxin, respectively. We used these variants to constrain the STX docking orientation by energetically localizing the C-11 sulfate in the outer vestibule. Interactions between the C-11 sulfate and each of the four domains of the channel were determined by a systematic approach to mutant cycle analysis in which all known carboxyl groups important for site 1 toxin binding were neutralized, allowing energetic triangulation of the toxin sulfate and overcoming some limitations of mutant cycles. Toxin IC(50)s were measured by two-electrode voltage clamp from Xenopus oocytes injected with the channel mRNA. Three unique types of analysis based on the coupling results localized the C-11 sulfate between domains III and IV. Combined with our previous report, the data establish the orientation of STX in the outer vestibule and confirm the clockwise arrangement of the channel domains.     

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.     

Interactions of neosaxitoxin with the sodium channel of the frog skeletal muscle fiber

Neosaxitoxin (neoSTX) differs structurally from saxitoxin (STX) in that the hydrogen on N-1 is replaced by a hydroxyl group. On single frog skeletal muscle fibers in the vaseline-gap voltage clamp, the concentrations for reducing the maximum sodium current by 50% (ED50) at pH's 6.50, 7.25, and 8.25 are, respectively, 4.9, 5.1, and 8.9 nM for STX and 1.6, 2.7, and 17.2 nM for neoSTX. The relative potencies of STX at the different pH's closely parallel the relative abundance of the protonated form of the 7,8,9 guanidinium function, but the relative potencies of neoSTX at the same pH's vary with the relative abundance of the deprotonated N-1 group. In constant-ratio mixtures of the two toxins, the observed ED50's are consistent with the notion that the two toxins compete for the same site. At pH's 6.50 and 7.25, the best agreement between observed and computed values is obtained when the efficacy term (epsilon) for either toxin is 1. At pH 8.25 the best agreement is obtained if the efficacy is 1 for STX but 0.75 for neo-STX. The marked pH dependence of the actions of neoSTX probably reflects the presence of a site in the receptor that interacts with the N-1 -OH, in addition to those interacting with the 7,8,9 guanidinium and the C-12 hydroxyl groups. Considering the three-dimensional structure of the STX and neoSTX molecules, the various site points are probably located in a fold or a crevice of the channel protein, where the extracellular orifice of the sodium channel is located.     

A mu-conotoxin-insensitive Na+ channel mutant: possible localization of a binding site at the outer vestibule.

We describe a mutation in the outer vestibule region of the adult rat skeletal muscle voltage-gated Na+ channel (microliter) that dramatically alters binding of mu-conotoxin GIIIA (mu-CTX). Mutating the glutamate at position 758 to glutamine (E758Q) decreased mu-CTX binding affinity by 48-fold. Because the mutant channel showed both low tetrodotoxin (TTX) and mu-CTX affinities, these results suggested that mu-CTX bound to the outer vestibule and implied that the TTX- and mu-CTX-binding sites partially overlapped in this region. The mutation decreased the association rate of the toxin with little effect on the dissociation rate, suggesting that Glu-758 could be involved in electrostatic guidance of mu-CTX to its binding site. We propose a mechanism for mu-CTX block of the Na+ channel based on the analogy with saxitoxin (STX) and TTX, on the requirement of mu-CTX to have an arginine in position 13 to occlude the channel, and on this experimental result suggesting that mu-CTX binds in the outer vestibule. In this model, the guanidinium group of Arg-13 of the toxin interacts with two carboxyls known to be important for selectivity (Asp-400 and Glu-755), with the association rate of the toxin increased by interaction with Glu-758 of the channel.     

Residues in the epsilon subunit of the nicotinic acetylcholine receptor interact to confer selectivity of waglerin-1 for the alpha-epsilon subunit interface site

Waglerin-1 (Wtx-1) is a 22-amino acid peptide that competitively antagonizes muscle nicotinic acetylcholine receptors (nAChRs). Previous work demonstrated that Wtx-1 binds to mouse nAChRs with higher affinity than receptors from rats or humans, and distinguished residues in alpha and epsilon subunits that govern the species selectivity. These studies also showed that Wtx-1 binds selectively to the alpha-epsilon binding site with significantly higher affinity than to the alpha-delta binding site. Here we identify residues at equivalent positions in the epsilon, gamma, and delta subunits that govern Wtx-1 selectivity for one of the two binding sites on the nAChR pentamer. Using a series of chimeric and point mutant subunits, we show that residues Gly-57, Asp-59, Tyr-111, Tyr-115, and Asp-173 of the epsilon subunit account predominantly for the 3700-fold higher affinity of the alpha-epsilon site relative to that of the alpha-gamma site. Similarly, we find that residues Lys-34, Gly-57, Asp-59, and Asp-173 account predominantly for the high affinity of the alpha-epsilon site relative to that of the alpha-delta site. Analysis of combinations of point mutations reveals that Asp-173 in the epsilon subunit is required together with the remaining determinants in the epsilon subunit to achieve Wtx-1 selectivity. In particular, Lys-34 interacts with Asp-173 to confer high affinity, resulting in a DeltaDeltaG(INT) of -2.3 kcal/mol in the epsilon subunit and a DeltaDeltaG(INT) of -1.3 kcal/mol in the delta subunit. Asp-173 is part of a nonhomologous insertion not found in the acetylcholine binding protein structure. The key role of this insertion in Wtx-1 selectivity indicates that it is proximal to the ligand binding site. We use the binding and interaction energies for Wtx-1 to generate structural models of the alpha-epsilon, alpha-gamma, and alpha-delta binding sites containing the nonhomologous insertion.     

Interactions of the C-11 hydroxyl of tetrodotoxin with the sodium channel outer vestibule

The highly selective sodium channel blocker, tetrodotoxin (TTX) has been instrumental in characterization of voltage-gated sodium channels. TTX occludes the ion-permeation pathway at the outer vestibule of the channel. In addition to a critical guanidinium group, TTX possesses six hydroxyl groups, which appear to be important for toxin block. The nature of their interactions with the outer vestibule remains debatable, however. The C-11 hydroxyl (C-11 OH) has been proposed to interact with the channel through a hydrogen bond to a carboxyl group, possibly from domain IV. On the other hand, previous experiments suggest that TTX interacts most strongly with pore loops of domains I and II. Energetic localization of the C-11 OH was undertaken by thermodynamic mutant cycle analysis assessing the dependence of the effects of mutations of the adult rat skeletal muscle Na(+) channel (rNa(v)1.4) and the presence of C-11 OH on toxin IC(50). Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents were measured by two-electrode voltage clamp. Toxin blocking efficacy was determined by recording the reduction in current upon toxin exposure. Mutant cycle analysis revealed that the maximum interaction of the C-11 OH was with domain IV residue D1532 (DeltaDeltaG: 1.0 kcal/mol). Furthermore, C-11 OH had significantly less interaction with several domain I, II, and III residues. The pattern of interactions suggested that C-11 was closest to domain IV, probably involved in a hydrogen bond with the domain IV carboxyl group. Incorporating this data, a new molecular model of TTX binding is proposed.     

A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel.

Biophysical evidence has placed the binding site for the naturally occurring marine toxins tetrodotoxin (TTX) and saxitoxin (STX) in the external mouth of the Na+ channel ion permeation pathway. We developed a molecular model of the binding pocket for TTX and STX, composed of antiparallel beta-hairpins formed from peptide segments of the four S5-S6 loops of the voltage-gated Na+ channel. For TTX the guanidinium moiety formed salt bridges with three carboxyls, while two toxin hydroxyls (C9-OH and C10-OH) interacted with a fourth carboxyl on repeats I and II. This alignment also resulted in a hydrophobic interaction with an aromatic ring of phenylalanine or tyrosine residues for the brainII and skeletal Na+ channel isoforms, but not with the cysteine found in the cardiac isoform. In comparison to TTX, there was an additional interaction site for STX through its second guanidinium group with a carboxyl on repeat IV. This model satisfactorily reproduced the effects of mutations in the S5-S6 regions and the differences in affinity by various toxin analogs. However, this model differed in important ways from previously published models for the outer vestibule and the selectivity region of the Na+ channel pore. Removal of the toxins from the pocket formed by the four beta-hairpins revealed a structure resembling a funnel that terminated in a narrowed region suitable as a candidate for the selectivity filter of the channel. This region contained two carboxyls (Asp384 and Glu942) that substituted for molecules of water from the hydrated Na+ ion. Simulation of mutations in this region that have produced Ca2+ permeation of the Na+ channel created a site with three carboxyls (Asp384, Glu942, and Glu1714) in proximity.     

Electrostatic couplings in OmpA ion-channel gating suggest a mechanism for pore opening

The molecular forces that drive structural transitions between the open and closed states of channels and transporters are not well understood. The gate of the OmpA channel is formed by the central Glu52-Arg138 salt bridge, which can open to form alternate ion pairs with Lys82 and Glu128. To gain deeper insight into the channel-opening mechanism, we measured interaction energies between the relevant side chains by double-mutant cycle analysis and correlated these with the channel activities of corresponding point mutants. The closed central salt bridge has a strong interaction energy of -5.6 kcal mol(-1), which can be broken by forming the open-state salt bridge Glu52-Lys82 (DeltaDeltaG(Inter) = -3.5 kcal mol(-1)) and a weak interaction between Arg138 and Glu128 (DeltaDeltaG(Inter) = -0.6 kcal mol(-1)). A covalent disulfide bond in place of the central salt bridge completely blocks the channel. Growth assays indicate that this gating mechanism could physiologically contribute to the osmoprotection of Escherichia coli cells from environmental stress.     

Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins

bTyrosine 401 of the skeletal muscle isoform (mu 1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn2+ and Cd2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na+ current for a series of point mutations at residue Tyr401 of the rat mu 1 Na channel expressed in Xenopus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na+ currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with Ki values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7- and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn2+ (Ki = 8-23 microM) required Cys, His, or Asp, three residues commonly found to coordinate directly with Zn2+ in metalloproteins. For the Cys mutant of mu 1 and also for the cardiac isoform Na channel (rh1) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na+ conductance by Zn2+ reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The Asp mutant also displayed enhanced affinity for inhibition of conductance by Ca2+ (Ki = 0.3 mM vs approximately 40 mM in wild type), but block by Ca2+ was incomplete, saturating at approximately 69% inhibition. In contrast, Cd2+ completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the particular residue at position 401 and the species of divalent cation. The His mutant also exhibited enhanced sensitivity to block by H+ with a pKa of approximately 7.5 for the His imidazole group. Our findings provide further evidence that residue 401 of mu 1 is located within the outer vestibule of the Na channel but external to the single-filing region for permeant ions.     

Modeling of the outer vestibule and selectivity filter of the L-type Ca2+ channel

Using the KcsA bacterial K+ channel crystal structure [Doyle, D. A., et al. (1998) Science 280, 69-74] and the model of the outer vestibule of the Na+ channel [Lipkind, G. M., and Fozzard, H. A. (2000) Biochemistry 39, 8161-8170] as structural templates, we propose a structural model of the outer vestibule and selectivity filter of the pore of the Ca2+ channel (alpha1C or Ca(v)1.2). The Ca2+ channel P loops were modeled by alpha-helix-turn-beta-strand motifs, with the glutamate residues of the EEEE motif located in the turns. P loops were docked in the extracellular part of the inverted teepee structure formed by S5 and S6 alpha-helices with backbone coordinates from the M1 and M2 helices of the KcsA crystal structure. This construction results in a conical outer vestibule that tapers to the selectivity filter at the bottom. The modeled selectivity ring forms a wide open pore ( approximately 6 A) in the absence of Ca2+. When Ca2+ is present ( approximately 1 microM), all four glutamate side chains move to the center and form a cage around the dehydrated Ca2+ ion, blocking the pore. In the millimolar concentration range, Ca2+ also interacts with two low-affinity sites located externally and internally, which were modeled by the same carboxylate groups of the selectivity filter. Calculation of the resulting electrostatic potentials show that the single Ca2+ ion is located in an electrostatic trap. Only when three Ca2+ ions are bound simultaneously in the high- and low-affinity sites of the selectivity filter is Ca2+ able to overcome electrostatic attraction, permitting Ca2+ flux.     

Mapping the binding site of a human ether-a-go-go-related gene-specific peptide toxin (ErgTx) to the channel's outer vestibule

The goals of this study are to investigate the mechanism and site of action whereby a human ether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx.Shaker interaction, elevating [K](o) (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx.HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (DeltaDeltaG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic alpha-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx.HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule.     

Differential scanning calorimetric studies of E. coli aspartate transcarbamylase. III. The denaturational thermodynamics of the holoenzyme with single-site mutations in the catalytic chain

Aspartate transcarbamylase (EC 2.1.3.2) from E. coli is a multimeric enzyme consisting of two catalytic subunits and three regulatory subunits whose activity is regulated by subunit interactions. Differential scanning calorimetric (DSC) scans of the wild-type enzyme consist of two peaks, each comprised of at least two components, corresponding to denaturation of the catalytic and regulatory subunits within the intact holoenzyme (Vickers et al., J. Biol. Chem. 253 (1978) 8493; Edge et al., Biochemistry 27 (1988) 8081). We have examined the effects of nine single-site mutations in the catalytic chains. Three of the mutations (Asp-100-Gly, Glu-86-Gln, and Arg-269-Gly) are at sites at the C1: C2 interface between c chains within the catalytic subunit. These mutations disrupt salt linkages present in both the T and R states of the molecule (Honzatko et al., J. Mol. Biol. 160 (1982) 219; Krause et al., J. Mol. Biol. 193 (1987) 527). The remainder (Lys-164-Ile, Tyr-165-Phe, Glu-239-Gln, Glu-239-Ala, Tyr-240-Phe and Asp-271-Ser) are at the C1: C4 interface between catalytic subunits and are involved in interactions which stabilize either the T or R state. DSC scans of all of the mutants except Asp-100-Gly and Arg-269-Gly consisted of two peaks. At intermediate concentrations, Asp-100-Gly and Arg-269-Gly had only a single peak near the Tm of the regulatory subunit transition in the holoenzyme, although their denaturational profiles were more complex at high and low protein concentrations. The catalytic subunits of Glu-86-Gln, Lys-164-Ile and Asp-271-Ser appear to be significantly destabilized relative to wild-type protein while Tyr-165-Phe and Tyr-240-Phe appear to be stabilized. Values of delta delta G degree cr, the difference between the subunit interaction energy of wild-type and mutant proteins, evaluated as suggested by Brandts et al. (Biochemistry 28 (1989) 8588) range from -3.7 kcal mol-1 for Glu-86-Gln to 2.4 kcal mol-1 for Tyr-165-Phe.     

Role of the minor energetic determinants of chicken egg white lysozyme (HEWL) to the stability of the HEWL.antibody scFv-10 complex

Seven of the 13 non-glycine contact amino acids in the hen (chicken) egg white lysozyme (HEWL) epitope for antibody Fab-10 each contribute < or =0.3 kcal/mol to the change in free energy (DeltaDeltaG(D)) from wild type (WT) when replaced by alanine (nullspots), and three others each give (0.7 < DeltaDeltaG(D) < or = 1. 0) kcal/mol (warm spots) (Rajpal et al. Protein Sci 1998;7:1868-1874). The low DeltaDeltaG(D) values introduced by alanine mutations present an opportunity to explore accurately their cumulative effects, as the sum of the combined DeltaDeltaG(D) values is not so large as to destabilize the complex beyond the range of accurate measurement. Substitution of six of the seven null spot residues by alanine leads to a cumulative DeltaDeltaG(D) = 2.25 +/- 0.04 kcal/mol, whereas the sum of the six individual changes is only -0.36 +/- 0.32 kcal/mol. The triple warm spot mutation generates a DeltaDeltaG(D) = 5.11 +/- 0.06 kcal/mol versus DeltaDeltaG(D) = 2.52 +/- 0.22 kcal/mol for the sum of the three individuals. The non-additivity in the individual DeltaDeltaG(D) values for the alanine mutations may indicate that these residues provide a conformationally stabilizing effect on the hot spot residues, each of which exhibits DeltaDeltaG(D) > 4.0 kcal/mol on alanine substitution.     

Novel interactions identified between micro -Conotoxin and the Na+ channel domain I P-loop: implications for toxin-pore binding geometry

micro -Conotoxins ( micro -CTX) are peptides that inhibit Na(+) flux by blocking the Na(+) channel pore. Toxin residue arginine 13 is critical for both high affinity binding and for complete block of the single channel current, prompting the simple conventional view that residue 13 (R13) leads toxin docking by entering the channel along the pore axis. To date, the strongest interactions identified are between micro -CTX and domain II (DII) or DIII pore residues of the rat skeletal muscle (Na(v)1.4) Na(+) channels, but little data is available for the role of the DI P-loop in micro -CTX binding due to the lack of critical determinants identified in this domain. Despite being an essential determinant of isoform-specific tetrodotoxin sensitivity, the DI-Y401C variant had little effect on micro -CTX block. Here we report that the charge-changing substitution Y401K dramatically reduced the micro -CTX affinity ( approximately 300-fold). Using mutant cycle analysis, we demonstrate that K401 couples strongly to R13 (DeltaDeltaG > 3.0 kcal/mol) but not R1, K11, or R14 (<1 kcal/mol). Unlike K401, however, a significant coupling was detected between toxin residue 14 and DI-E403K (DeltaDeltaG = 1.4 kcal/mol for the E403K-Q14D pair). This appears to underlie the ability of DI-E403K channels to discriminate between the GIIIA and GIIIB isoforms of micro -CTX (p < 0.05), whereas Y401K, DII-E758Q, and DIII-D1241K do not. We also identify five additional, novel toxin-channel interactions (>0.75 kcal/mol) in DII (E758-K16, D762-R13, D762-K16, E765-R13, E765-K16). Considered together, these new interactions suggest that the R13 side chain and the bulk of the bound toxin micro -CTX molecule may be significantly tilted with respect to pore axis.     

Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.     

Stabilization of the second oxyanion intermediate by 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase of the menaquinone pathway: spectroscopic evidence of the involvement of a conserved aspartic acid

1,4-Dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) synthase, or MenB, catalyzes an intramolecular Claisen condensation involving two oxyanion intermediates in the biosynthetic pathway of menaquinone, an essential respiration electron transporter in many microorganisms. Here we report the finding that the DHNA-CoA product and its analogues bind and inhibit the synthase from Escherichia coli with significant ultraviolet--visible spectral changes, which are similar to the changes induced by deprotonation of the free inhibitors in a basic solution. Dissection of the structure--affinity relationships of the inhibitors identifies the hydroxyl groups at positions 1 (C1-OH) and 4 (C4-OH) of DHNA-CoA or their equivalents as the dominant and minor sites, respectively, for the enzyme--ligand interaction that polarizes or deprotonates the bound ligands to cause the observed spectral changes. In the meantime, spectroscopic studies with active site mutants indicate that C4-OH of the enzyme-bound DHNA-CoA interacts with conserved polar residues Arg-91, Tyr-97, and Tyr-258 likely through a hydrogen bonding network that also includes Ser-161. In addition, site-directed mutation of the conserved Asp-163 to alanine causes a complete loss of the ligand binding ability of the protein, suggesting that the Asp-163 side chain is most likely hydrogen-bonded to C1-OH of DHNA-CoA to provide the dominant polarizing effect. Moreover, this mutation also completely eliminates the enzyme activity, strongly supporting the possibility that the Asp-163 side chain provides a strong stabilizing hydrogen bond to the tetrahedral oxyanion, which takes a position similar to that of C1-OH of the enzyme-bound DHNA-CoA and is the second high-energy intermediate in the intracellular Claisen condensation reaction. Interestingly, both Arg-91 and Tyr-97 are located in a disordered loop forming part of the active site of all available DHNA-CoA synthase structures. Their involvement in the interaction with the small molecule ligands suggests that the disordered loop is folded in interaction with the substrates or reaction intermediates, supporting an induced-fit catalytic mechanism for the enzyme.     

DeltaG-based prediction and experimental confirmation of SYCRP1-binding sites on the Synechocystis genome

DNA-binding sites for SYCRP1, which is a regulatory protein of the cyanobacterium Synechocystissp. PCC6803, were predicted for the whole genome sequence by estimating changes in the binding free energy () for SYCRP1 for those sites. The values were calculated by summing DeltaDeltaG values derived from systematic single base-pair substitution experiments (symmetrical and cooperative binding model). Of the calculated binding sites, 23 sites with a value <3.9kcal.mol(-1) located upstream or between the ORFs were selected as putative binding sites for SYCRP1. In order to confirm whether SYCRP1 actually binds to these binding sites or not, 11 sites with the lowest values were tested experimentally, and we confirmed that SYCRP1 binds to ten of the 11 sites with a DeltaDeltaG(total) value <3.9kcal.mol(-1). The best correlation coefficient between and the observed DeltaDeltaG(total) for binding of SYCRP1 to those sites was 0.78. These results suggest that the DeltaDeltaG values derived from systematic single base-pair experiments may be used to screen for potential binding sites of a regulatory protein in the genome sequence.     

Thrombomodulin changes the molecular surface of interaction and the rate of complex formation between thrombin and protein C

The interaction of thrombin with protein C triggers a key down-regulatory process of the coagulation cascade. Using a panel of 77 Ala mutants, we have mapped the epitope of thrombin recognizing protein C in the absence or presence of the cofactor thrombomodulin. Residues around the Na(+) site (Thr-172, Lys-224, Tyr-225, and Gly-226), the aryl binding site (Tyr-60a), the primary specificity pocket (Asp-189), and the oxyanion hole (Gly-193) hold most of the favorable contributions to protein C recognition by thrombin, whereas a patch of residues in the 30-loop (Arg-35 and Pro-37) and 60-loop (Phe-60h) regions produces unfavorable contributions to binding. The shape of the epitope changes drastically in the presence of thrombomodulin. The unfavorable contributions to binding disappear and the number of residues promoting the thrombin-protein C interaction is reduced to Tyr-60a and Asp-189. Kinetic studies of protein C activation as a function of temperature reveal that thrombomodulin increases >1,000-fold the rate of diffusion of protein C into the thrombin active site and lowers the activation barrier for this process by 4 kcal/mol. We propose that the mechanism of thrombomodulin action is to kinetically facilitate the productive encounter of thrombin and protein C and to allosterically change the conformation of the activation peptide of protein C for optimal presentation to the thrombin active site.     

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity

Voltage-gated Na⁺ channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca²⁺ permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca²⁺ or Na⁺ ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca²⁺ permeability, suggesting that ion–toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.     

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate

Solution properties of beta recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.