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.     

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.     

Identification of residues within the 727-767 segment of human complement component C3 important for its interaction with factor H and with complement receptor 1 (CR1, CD35)

Mapping approaches employing blocking antibodies and synthetic peptides have implicated the 727-767 segment at the NH2 terminus of C3b alpha'-chain as contributing to the interactions with factor B, factor H, and CR1. Our previous mutagenesis study on the NH2-terminal acidic cluster of this segment identified residues Glu-736 and Glu-737 as contributing to the binding of C3b to factor B and CR1 but not factor H. We have now extended the charged residue mutagenic scan to cover the remainder of the segment (738-767) and have assessed the ability of the C3b-like C3(H2O) form of the mutant molecules to interact with factor H, CR1, and membrane cofactor protein (MCP) using a cofactor-dependent factor I cleavage assay as a surrogate binding assay. We have found that the negatively charged side chains of Glu-744 and Glu-747 are important for the interaction between C3(H2O) and factor H, a result in general agreement with an earlier synthetic peptide study (Fishelson, Z. (1991) Mol. Immunol. 28, 545-552) which implicated residues within the 744-754 segment in H binding. The interactions of the mutants with soluble CR1 (sCR1) revealed two classes of residues. The first are residues required for sCR1 to be an I cofactor for the first two cleavages of alpha-chain. These are all acidic residues and include the Glu-736/Glu-737 pair, Glu-747, and the Glu-754/Asp-755 pairing. The second class affects only the ability of sCR1 to be a cofactor for the third factor I cleavage and include Glu-744 and the Lys-757/Glu-758 pairing. The dominance of acidic residues in the loss-of-function mutants is striking and suggests that H and CR1 contribute basic residues to the interface. Additionally, although there is partial overlap, the contacts required for CR1 binding appear to extend over a wider portion of the 727-767 segment than is the case for factor H. Finally, none of the mutations had any effect on the interaction between soluble MCP and C3(H2O), indicating that despite its functional homology to H and CR1, MCP differs in its mode of binding to C3b/C3(H2O).     

Role for Pro-13 in directing high-affinity binding of anthopleurin B to the voltage-sensitive sodium channel

Anthopleurin A (ApA) and B (ApB) are 49-amino acid polypeptide toxins from the Pacific sea anemone Anthopleura xanthogrammica that interfere with inactivation of voltage-gated sodium channels. ApA, which differs from ApB in seven of the 49 amino acids, displays markedly enhanced isoform selectivity compared with ApB, acting preferentially on cardiac over neuronal sodium channels. Previous studies in this lab have indicated the importance of two unique charged residues in ApB, Arg-12 and Lys-49, in this toxin's ability to discriminate between neuronal and cardiac sodium channels. Likewise, a double mutant (R12S/K49Q) recently characterized in this lab (Khera et al., 1995) displays a greatly reduced affinity for neuronal channels, essentially restoring the discriminatory ability of ApA. When the remaining five residues unique to ApB are individually converted to those of ApA, only ApB (Pro-13) shows a major effect, reducing the affinity of the new mutant toxin (P13V) against both channel isoforms approximately 10-fold. This effect is most likely the result of a conformational rearrangement within the surrounding cationic cluster which includes Arg-12 and -14, as well as Lys-49. However, when placed into the context of the double mutant R12S/K49Q a unique effect is observed: the new triple mutant (R12S/P13V/K49Q) is no longer able to discriminate effectively between channel isoforms. Its affinity for the neuronal sodium channel is significantly enhanced compared to either P13V or to the double mutant R12S/K49Q. These results are consistent both with our proposed model (Khera et al., 1995) and with the recently reported solution structure of ApB, which implicate the cationic cluster in both affinity and channel isoform selectivity. We suggest that the P13V mutation results in a shift in the relative orientation of cationic residues within the large flexible loop between residues 9-18, thus strengthening their interactions with target sequences of the neuronal sodium channel.     

Identification of pairwise interactions in the alpha-neurotoxin-nicotinic acetylcholine receptor complex through double mutant cycles

alpha-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the alpha-neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha-subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha-neurotoxin from N. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alphadelta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the alpha-subunit region between residues 188-200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/pi interactions with Tyr190.     

The substrate-binding site in the lactose permease of Escherichia coli

Site-directed N-ethylmaleimide labeling was studied with Glu-126 and/or Arg-144 mutants in lactose permease containing a single, native Cys residue at position 148 in the substrate-binding site. Replacement of either Glu-126 or Arg-144 with Ala markedly decreases Cys-148 reactivity, whereas interchanging the residues, double-Ala replacement, or replacement of Arg-144 with Lys or His does not alter reactivity, indicating that Glu-126 and Arg-144 are charge-paired. Importantly, although alkylation of Cys-148 is blocked by ligand in wild-type permease, no protection whatsoever is observed with any of the Glu-126 or Arg-144 mutants. Site-directed fluorescence with 2-(4-maleimidoanilino)-naphthalene-6-sulfonic acid (MIANS) in mutant Val-331 --> Cys was also studied. In marked contrast to Val-331 --> Cys permease, ligand does not alter MIANS reactivity in mutant Glu-126 --> Ala/Val-331 --> Cys, Arg-144 --> Ala/Val-331 --> Cys, or Arg-144 --> Lys/Val-331 --> Cys and does not cause either quenching or a shift in the emission maximum of the MIANS-labeled mutants. However, mutation Glu-126 --> Ala or Arg-144 --> Ala and, to a lesser extent, Arg-144 --> Lys cause a red-shift in the emission spectrum and render the fluorophore more accessible to I-. The results demonstrate that Glu-126 and Arg-144 are irreplaceable for substrate binding and suggest a model for the substrate-binding site in the permease. In addition, the findings are consistent with the notion that alterations in the substrate translocation pathway at the interface between helices IV and V are transmitted conformationally to the H+ translocation pathway at the interface between helices IX and X.     

Molecular switch in signal transduction: reaction paths of the conformational changes in ras p21

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the gamma-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD-Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of alpha2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of alpha2 and facilitates the unwinding of a helical turn of alpha2 (residues 66-69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.     

Electrostatic interactions in collagen-like triple-helical peptides

Collagen-like peptides with potential for ion pair formation were studied to investigate the role of electrostatic interactions in the triple-helix conformation. Three peptides--(POG)10, the EK-containing peptide (POG)4EKG(POG)5, and T3-487, a peptide with 18 residues of type III collagen and a C-terminal (GPO)4 tail--all form stable triple helices in aqueous solution, with melting temperatures of 58, 46, and 26 degrees C, respectively, at neutral pH. The thermal stabilities of these peptides correlate with their imino acid content, which is 66%, 60%, and 41%, respectively. Variation of pH over the range of 1-13 led to 8-9 degrees C changes in the Tm of the EK-containing peptide and peptide T3-487, with the greatest stability seen at pH values where both acidic and basic residues are ionized. Equilibrium ultracentrifugation shows these peptides are largely trimeric at low temperature, with no hexamers or larger aggregates, indicating that the pH-dependent stability arises from intramolecular interaction. Computer modeling indicates both intrachain ion pairs and interchain ion pairs can form and stabilize the triple helix. Studies of the pH dependence of the thermal stability of (POG)10 and the N-terminal acetylated form of T3-487 indicate that repulsion of the three charged N-terminal or C-terminal ends has a destabilizing effect. Taking into account these end effects, the energy contribution of two oppositely charged residues in a triple helix which are sterically capable of participating in ion pairs and backbone hydrogen bonding is 0.5-1 kcal/mol ion pair. It is possible that the stabilizing influence of ion pairs arises indirectly, through elimination of like charge repulsion, formation of ion pairs in the single chain form, or solvent effects.     

Role of electrostatic interactions in defining the potency of neurotoxin B-IV from Cerebratulus lacteus

Chemical modification implicates arginine residues of the Cerebratulus lacteus neurotoxin B-IV in biological activity. In the present study, we used site-directed mutagenesis to assess the functional contributions of each of these residues. Panels of mutants at each site have been constructed by polymerase chain reaction and recombinant proteins expressed and purified to homogeneity using an Escherichia coli expression system developed in this laboratory. All substitutions for Arg-17 (Gln, Ala, or Lys) yield proteins having undetectable levels of activity, while charge neutralizing replacement of Arg-25 (R25Q) causes a 400-fold reduction in specific toxicity. However, the R25K mutein is almost as active as natural toxin. Circular dichroism spectroscopy indicates that there are no major conformational changes in any of these muteins. These results therefore demonstrate the requirement for a guanidinium group at position 17, and a positive charge at position 25. NMR analyses (Hansen, P. E., Kem, W. R., Bieber, A. L., and Norton, R. S. (1992) Eur. J. Biochem. 210, 231-240) reveal neurotoxin B-IV to contain two antiparallel alpha-helices, which together include 57% of the sequence. Both Arg-17 and Arg-25 lie on the same face of the N-terminal helix (residues 13-26), as do the carboxyl groups of Glu-13 and Asp-21. However, charge neutralizing mutations of the latter two sites have no effects on biological activity. Arg-34, situated near the N terminus of helix 2 (residues 33-49) is also important for activity, as its replacement by Gln or Ala diminishes activity by 20- and 80-fold, respectively. However, unlike Arg-17 and Arg-25, thermal denaturation experiments suggest that R34Q may be structurally destabilized relative to wild-type B-IV.     

The behavior of the active site salt bridge of bovine neurophysins as monitored by 15N NMR spectroscopy and chemical substitution. Relationship to biochemical properties

The active site of liganded neurophysin contains a salt bridge that involves the side chains of Arg-8 and Glu-47 of the protein and the alpha-amino group of bound hormone or related peptide. The extent to which the Arg-8-Glu-47 salt bridge persists in the absence of peptide, or to which the environment of Arg-8 in the unliganded state differs in monomers and dimers, is relevant to an understanding of allosteric mechanism in this system. In the present study, the behavior of the salt bridge was investigated by 15N NMR and chemical replacement of Arg-8. Bovine neurophysin-I was converted to its des 1-8 derivative, and Arg-8 was replaced by 15N-substituted Arg or by other residues using chemical semisynthesis. The relative abilities of different amino acids to restore peptide affinity to the des 1-8 protein were in good accord with the view of the salt bridge in the liganded state obtained from crystals of bovine neurophysin-II complexes. In the unliganded state, comparison of the 15N and proton NMR signals from Arg-8 with those in smaller arginine systems suggested the absence of significant interactions between the guanidinium of Arg-8 and Glu-47 or between the amino terminal region of Arg-8 and other elements of the protein. No evidence of a difference in Arg-8 environment between unliganded monomers and dimers was found. Marked spectral changes accompanying the binding of oxytocin indicated changes in the environment of both the side chain and amino terminal region of Arg-8. The NMR results were in good agreement with a recently emerging comparison of bovine neurophysin-II derivatives in the liganded and unliganded states, with the notable exception of the extent of salt bridge formation in the unliganded state. The results are shown to be consistent with, and to help explain, significant differences between the two bovine neurophysins in the susceptibility to tryptic cleavage at Arg-8 in the unliganded state and in the pH dependence of peptide binding and additionally constrain potential allosteric mechanisms underlying neurophysin ligand-facilitated dimerization.     

Electrostatic and non-electrostatic contributions to the binding free energies of anthracycline antibiotics to DNA

The knowledge about molecular factors driving simple ligand-DNA interactions is still limited. The aim of the present study was to investigate the electrostatic and non-electrostatic contributions to the binding free energies of anthracycline compounds with DNA. Theoretical calculations based on continuum methods (Poisson-Boltzmann and solvent accessible surface area) were performed to estimate the binding free energies of five selected anthracycline ligands (daunomycin, adriamycin, 9-deoxyadriamycin, hydroxyrubicin, and adriamycinone) to DNA. The free energy calculations also took into account the conformational change that DNA undergoes upon ligand binding. This conformational change appeared to be very important for estimating absolute free energies of binding. Our studies revealed that the absolute values of all computed contributions to the binding free energy were quite large compared to the total free energy of binding. However, the sum of these large positive and negative values produced a small negative value of the free energy around -10 kcal/mol. This value is in good agreement with experimental data. Experimental values for relative binding free energies were also reproduced for charged ligands by our calculations. Together, it was found that the driving force for ligand-DNA complex formation is the non-polar interaction between the ligand and DNA even if the ligand is positively charged.     

A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.     

Mutational analysis of the roles of residues in Escherichia coli elongation factor Ts in the interaction with elongation factor Tu

The crystal structure of the Escherichia coli elongation factor (EF)-Tu.Ts complex indicates that there are extensive contacts between EF-Tu and EF-Ts. To determine the importance of these contacts in the interaction between E. coli EF-Tu and EF-Ts, residues in EF-Ts at the interface of these two proteins were mutated. The binding constants governing the interaction of the resulting EF-Ts variants with E. coli EF-Tu were determined. The effects of these mutations on the ability of EF-Ts to stimulate GDP exchange with EF-Tu.GDP and on its ability to stimulate the activity of EF-Tu in polymerization were tested. The results indicate that Arg-12, Met-19, and Met-20 in the N-terminal domain of EF-Ts and His-147 and Lys-166 and/or His-167 in subdomain C of EF-Ts are crucial in the interaction between EF-Tu and EF-Ts. Lys-23, Val-234, Met-235, and the C-terminal helix h13 are less important. The binding constants of the EF-Ts variants governing their interactions with EF-Tu correlate well with their activities in stimulating GDP exchange with EF-Tu. Mutations prepared in EF-Tu indicate that His-19 and Gln-114 but not Glu-348 in EF-Tu are moderately important for its interaction with EF-Ts.     

From membrane to molecule to the third amino acid from the left with a membrane transport protein

The lac permease of E. coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer. The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane. Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover. Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E. coli is proposed. Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII). In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles. Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa. Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa. In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex. In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325. The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane. Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism. On the other hand, widespread, cooperative conformational changes appear to be involved in turnover. As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover. This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e. helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism. (ABSTRACT TRUNCATED)     

Spatial localization of the K+ channel selectivity filter by mutant cycle-based structure analysis

The structurally well-characterized scorpion toxin Agitoxin2 inhibits ion permeation through Shaker K+ channels by binding to the external pore entryway. Scanning mutagenesis identified a set of inhibitor residues critical for making energetic contacts with the channel. Using thermodynamic mutant cycle analysis, we have mapped channel residues relative to the known inhibitor structure. This study constrains the position of multiple channel residues within the pore-forming loops; in one stretch, we have been able to map five out of seven contiguous residues to the inhibitor interaction surface, including those involved in ion selectivity. One interaction in particular, that of K27M on the inhibitor with Y445F on the channel, is unique in that it depends on the K+ ion concentration. These results reveal a shallow vestibule formed by the pore loops at the K+ channel entryway. The selectivity filter is located at the center of the vestibule close to (approximately 5 A) the extracellular solution.     

Aluminium, neurofibrillary degeneration and Alzheimer''s disease

The calcium-binding protein (parvalbumin), isolated from carp (Cyprinus carpio) muscle, has been specifically fragmented into two polypeptides by tryptic hydrolysis at the single arginine residue at position 75. Fragment A contains residues 1 leads to 75 and fragment B is composed of residues 76 leads to 108. The fragments have been characterized according to size, amino acid composition, carboxyl- and aminoterminal analysis. Both fragments appear to be homogeneous by these criteria. The intact protein is known to bind 2 mol of calcium per mol of parvalbumin, and although each fragment alone contains all of the essential ligands for the coordination of one Ca2+, neither fragment displays calcium binding activity. Attempts to reconstitute the two fragments, under a variety of conditions, into a functional complex which can bind calcium have been unsuccessful. The side chain of Arg-75 is known to occupy an internal position in the crystalline structure of parvalbumin (Kretsinger, R.H. and Nockolds, C.E. (1973) J. Biol. Chem. 248, 3313), where it is stabilized by an intricate network of hydrogen bonding involving the side chain of Glu-81. Although this internal salt bridge is approx. 20 A from either calcium binding site, it has been suggested that this structural feature of the molecule plays an essential role in the reversible binding of Ca2+. That the side chain of Arg-75 likewise occupies an internal position in the solution structure is indicated by its unavailability for reaction with 1,2-cyclohexanedione under conditions of physiological pH and temperature. However, in the presence of EDTA and at pH 8, it is readily modified by cyclohexanedione. This modification is accompanied by a concomitant loss in calcium binding activity. Reversal of the modification by treatment with hydroxylamine is accompanied by restoration of calcium binding activity. The serum of these data support the hypothesis that Arg-75 plays a critical role in the structural organization and calcium binding activity of the molecule, and in addition, suggests that the integrity of the peptide bond between Arg-75 and Ala-76 may be necessary for establishing the proper micro-environment required for formation of the internal salt bridge between Arg-75 and Glu-81.     

Suc1-associated neurotrophic factor target (SNT) protein is a major FGF-stimulated tyrosine phosphorylated 90-kDa protein which binds to the SH2 domain of GRB2

Low pH enhances tumor necrosis factor alpha (TNF)-induced cytolysis of cancer cells and TNF-membrane interactions that include binding, insertion, and ion-channel formation. We have also found that TNF increases Na+ influx in cells. Here, we examined the structural features of the TNF-membrane interaction pathway that lead to channel formation. Fluorometric studies link TNF's acid-enhanced membrane interactions to rapid but reversible acquisition of hydrophobic surface properties. Intramembranous photolabeling shows that (i) protonation of TNF promotes membrane insertion, (ii) the physical state of the target bilayer affects the kinetics and efficiency of TNF insertion, and (iii) binding and insertion of TNF are two distinct events. Acidification relaxes the trimeric structure of soluble TNF so that the cryptic carboxyl termini, centrally located at the base of the trimer cone, become susceptible to carboxypeptidase Y. After membrane insertion, TNF exhibits a trimeric configuration in which the carboxyl termini are no longer exposed; however, the proximal salt-bridged Lys-11 residues as well as regional surface amino acids (Glu-23, Arg-32, and Arg-44) are notably more accessible to proteases. The sequenced cleavage products bear the membrane-restricted photoreactive probe, proof that surface-cleaved TNF has an intramembranous disposition. In summary, the trimer's structural plasticity is a major determinant of its channel-forming ability. Channel formation occurs when cracked or partially splayed trimers bind and penetrate the bilayer. Reannealing leads to a slightly relaxed trimeric structure. The directionality of bilayer penetration conforms with x-ray data showing that receptor binding to the monomer interfaces of TNF poises the tip of the trimeric cone directly above the target cell membrane.     

Identification of active site residues in the "GyrA" half of yeast DNA topoisomerase II

Site-directed mutagenesis was carried out at 10 highly conserved polar residues within the C-terminal half of yeast DNA topoisomerase II, which corresponds to the A subunit of bacterial DNA gyrase, to identify amino acid side chains that augment the active site tyrosine Tyr-782 in the breakage and rejoining of DNA strands. Complementation tests show that alanine substitution at Arg-690, Asp-697, Lys-700, Arg-704, or Arg-781, but not at His-735, His-736, Glu-738, Gln-750, or Asn-828, inactivates the enzyme in vivo. Measurements of DNA relaxation and cleavage by purified mutant enzymes show that these activities are abolished in the R690A mutant and are much reduced in the mutants D697A, K700A, R704A, and R781A. When a Y782F polypeptide with a phenylalanine substituting for the active site tyrosine was expressed in cells that also express the R690A polypeptide, the resulting heterodimeric yeast DNA topoisomerase II was found to nick plasmid DNA. Thus in a dimeric wild-type enzyme, Tyr-782 in one protomer and Arg-690 in the other cooperate in trans in the catalysis of DNA cleavage. For the residues D697A, K700A, R704A, and R781A, their locations in the crystal structures of type II DNA topoisomerase fragments suggest that Arg-781 and Lys-700 might be involved in anchoring the 5' and 3' sides of the broken DNA, respectively, and the roles of Asp-697 and Arg-704 are probably less direct.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Helix-capping interaction in lambda Cro protein: a free energy simulation analysis

The stability mutant Tyr-26-->Asp was studied in the Cro protein from bacteriophage lambda using free energy molecular dynamics simulations. The mutant was calculated to be more stable than the wild type by 3.0 +/- 1.7 kcal/mol/monomer, in reasonable agreement with experiment (1.4 kcal/mol/monomer). Moreover, the aspartic acid in the mutant was found to form a capping interaction with the amino terminus of the third alpha-helix of Cro. The simulations were analyzed to understand better the source of the stability of this helix-capping interaction and to examine the results in light of previous explanations of stabilizing helix caps--namely, a model of local unsatisfied hydrogen bonds at the helix termini and the helix macrodipole model. Analysis of the simulations shows that the stabilizing effect of this charged helical cap is due both to favorable hydrogen bonds with backbone NH groups at the helix terminus and to favorable electrostatic interactions (but not hydrogen bonds) with their carbonyls (effectively the next row of local dipoles in the helix). However, electrostatic interactions are weak or negligible with backbone dipolar groups in the helix further away from the terminus. Moreover, the importance of other local electrostatic interactions with polar side chains near the helix terminus, which are neglected in most treatments of this effect, are shown to be important. Thus, the results support a model that is intermediate between the two previous explanations: both unsatisfied hydrogen bonds at the helix terminus and other, local preoriented dipolar groups stabilize the helix cap. These findings suggest that similar interactions with preoriented dipolar groups may be important for cooperativity in other charge-dipole interactions and may be employed to advantage for molecular design.