A differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In continuation of our earlier work on the effects of amino acid replacements on the thermodynamics of the thermal unfolding of T4 lysozyme [Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, 3788-3792; Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1991) Biochemistry 30, 1887-1891; Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1992) Biochemistry 31, 1643-1647], we report here a study by differential scanning calorimetry of the effects of five replacements at Ile3. Four of these replacements, those with Glu, Phe, Pro, and Thr, caused apparent destabilizations, while the replacement by Leu led to a small apparent stabilization. The largest observed destabilization (Ile3Pro) amounted to -3.0 kcal mol-1 in free energy at pH 2.00 and 38.8 degrees C (the denaturational temperature of the wild-type protein at this pH), and the largest stabilization amounted to +1.2 kcal mol-1 at pH 3.00 and 53.6 degrees C.     

Differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In two recent papers, we reported the effects of several point mutations on the thermodynamics of the thermal unfolding of the lysozyme of phage T4 as determined by differential scanning calorimetry. The mutants studied were R96H [Kitamura, S., & Sturtevant, J.M. (1989) Biochemistry 28, 3788-3792] and T157 replaced by A, E, I, L, N, R, and V [Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J.M. (1991) Biochemistry 30, 1887-1891]. Here we report the results of a similar study of the single mutations A82P, A93P, and G113A and the double mutation C54T:C97A. The three single mutants all show small apparent stabilization at pH 2.5 and 46.2 degrees C (the denaturational temperature of the wild-type protein), amounting to -0.5 +/- 0.4 kcal mol-1 in free energy, whereas the double mutant shows a weak apparent destabilization, +0.8 +/- 0.4 kcal mol-1. As in all our previous studies of mutant proteins, the enthalpy changes produced by these mutations are in general of much larger magnitude than the corresponding free energy changes and frequently of opposite sign.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Importance of each residue within secretin for receptor binding and biological activity

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.     

Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: N-ethylmaleimide-sensitive face of helix II

Cys-scanning mutagenesis of helix II in the lactose permease of Escherichia coli [Frillingos, S., Sun, J. et al. (1997) Biochemistry 36, 269-273] indicates that one face contains positions where Cys replacement or Cys replacement followed by treatment with N-ethylmaleimide (NEM) significantly inactivates the protein. In this study, site-directed sulfhydryl modification is utilized in situ to study this face of helix II. [(14)C]NEM labeling of 13 single-Cys mutants, including the nine NEM-sensitive Cys replacements, in right-side-out membrane vesicles is examined. Permease mutants with a single-Cys residue in place of Gly46, Phe49, Gln60, Ser67, or Leu70 are alkylated by NEM at 25 degrees C in 10 min, and mutants with Cys in place of Thr45 and Ser53 are labeled only in the presence of ligand, while mutants with Cys in place of Ile52, Ser56, Leu57, Leu62, Phe63, or Leu65 do not react. Binding of substrate leads to a marked increase in labeling of Cys residues at positions 45, 49, or 53 in the periplasmic half of helix II and a slight decrease in labeling of Cys residues at positions 60 or 67 in the cytoplasmic half. Labeling studies with methanethiosulfonate ethylsulfonate (MTSES) show that positions 45 and 53 are accessible to solvent in the presence of ligand only, while positions 46, 49, 67, and 70 are accessible to solvent in the absence or presence of ligand. Position 60 is also exposed to solvent, and substrate binding causes a decrease in solvent accessibility. The findings demonstrate that the NEM-sensitive face of helix II participates in ligand-induced conformational changes. Remarkably, this membrane-spanning face is accessible to the aqueous phase from the periplasmic side of the membrane. In the following paper in this issue [Venkatesan, P., Hu, Y., and Kaback, H. R. (2000) Biochemistry 39, 10656-10661], the approach is applied to helix X.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.     

Catalytic convergence of manganese and iron lipoxygenases by replacement of a single amino Acid

Lipoxygenases (LOXs) contain a hydrophobic substrate channel with the conserved Gly/Ala determinant of regio- and stereospecificity and a conserved Leu residue near the catalytic non-heme iron. Our goal was to study the importance of this region (Gly(332), Leu(336), and Phe(337)) of a lipoxygenase with catalytic manganese (13R-MnLOX). Recombinant 13R-MnLOX oxidizes 18:2n-6 and 18:3n-3 to 13R-, 11(S or R)-, and 9S-hydroperoxy metabolites (～80-85, 15-20, and 2-3%, respectively) by suprafacial hydrogen abstraction and oxygenation. Replacement of Phe(337) with Ile changed the stereochemistry of the 13-hydroperoxy metabolites of 18:2n-6 and 18:3n-3 (from ～100% R to 69-74% S) with little effect on regiospecificity. The abstraction of the pro-S hydrogen of 18:2n-6 was retained, suggesting antarafacial hydrogen abstraction and oxygenation. Replacement of Leu(336) with smaller hydrophobic residues (Val, Ala, and Gly) shifted the oxygenation from C-13 toward C-9 with formation of 9S- and 9R-hydroperoxy metabolites of 18:2n-6 and 18:3n-3. Replacement of Gly(332) and Leu(336) with larger hydrophobic residues (G332A and L336F) selectively augmented dehydration of 13R-hydroperoxyoctadeca-9Z,11E,15Z-trienoic acid and increased the oxidation at C-13 of 18:1n-6. We conclude that hydrophobic replacements of Leu(336) can modify the hydroperoxide configurations at C-9 with little effect on the R configuration at C-13 of the 18:2n-6 and 18:3n-3 metabolites. Replacement of Phe(337) with Ile changed the stereospecific oxidation of 18:2n-6 and 18:3n-3 with formation of 13S-hydroperoxides by hydrogen abstraction and oxygenation in analogy with soybean LOX-1.     

The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine.

The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.     

The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.     

Competitive selection in vivo by a cell for one variant over another: implications for RNA virus quasispecies in vivo.

Infidelity of genome applications of RNA viruses leads to the generation of viral quasispecies both in vitro and in vivo. However, the biological significance of such generated variants in vivo is largely unknown and controversial. To study this issue, we continued our evaluation of the tropism of a lymphocytic choriomeningitis virus (LCMV) variant termed clone 13 with its parental virus clonal pool ARM 53b (wild-type parent) for neuronal cells in vivo. Earlier in vivo and in vitro studies noted that the wild-type virus contained a Phe at glycoprotein (GP) residue 260 which correlated with neuron tropism compared with LCMV variants containing a Leu at residue 260 which showed selected tropism for cells of the immune system (C.F. Evans, P. Borrow, J. C. de la Torre, and M. B. A. Oldstone J. Virol. 68:7367-7373, 1994; L. Villarete, T. Somasundaram, and R. Ahmed, J. Virol 68:7490-7496, 1994). Here we (i) evaluated the ability of the viral variants with either a Phe or Leu at GP residue 260 to replicate in vivo in the spleen, liver, or brain, (ii) analyzed the ability of these viruses to compete against each other for cell (neuron)-specific selection following a single viral inoculation of different ratios of both viruses, and (iii) utilized genetic reassortants of both viruses to test their ability to replicate in neurons in vivo. We found that viral variants containing either a Phe or Leu at GP residue 260 were equally capable of replicating in neurons, but when inoculated together, neurons selected for the viral population containing Phe at GP residue 260 over viruses containing a Leu at this position. This was in contrast to selection in the liver and spleen that favored viruses with Leu and not Phe at GP residue 260. Analysis of inoculations with viral reassortants indicated that genes encoded on the short RNA (the GP and nucleoprotein, not the L [polymerase] and Z proteins that are encoded by the large RNA) were associated with neurotropism. Since the nucleoprotein sequences of wild-type Armstrong and clone 13 are identical, it is likely that specific cytoplasmic factors of the neurons play a fundamental role in the selection of virus with Phe at GP residue 260.     

lac permease of Escherichia coli: arginine-302 as a component of the postulated proton relay

The lac permease of Escherichia coli was modified by site-directed mutagenesis such that Arg-302 in putative helix IX was replaced with Leu. In addition, Ser-300 (helix IX) was replaced with Ala, and Lys-319 in putative helix X was replaced with Leu. Permease with Leu at position 302 manifests properties that are similar to those of permease with Arg in place of His-322 [Püttner, I. B., Sarkar, H. K., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4483]. Thus, permease with Leu-302 is markedly defective in active lactose transport, efflux, exchange, and counterflow but catalyzes downhill influx of lactose at high substrate concentrations without H+ translocation. In contrast, permease molecules with Ala at position 300 or Leu at position 319 catalyze lactose/H+ symport in a manner indistinguishable from that of wild-type permease. By molecular modeling, Arg-302 may be positioned in helix IX so that it faces the postulated His-322/Glu-325 ion pair in helix X. In this manner, the guanidino group in Arg-302 may interact with the imidazole of His-322 and thereby play a role in the H+ relay suggested to be involved in lactose/H+ symport [Carrasco, N., Antes, L. M., Poonian, M. S., & Kaback, H. R. (1986) Biochemistry 25, 4486].     

Solution 1H NMR study of the heme cavity and folding topology of the abbreviated chain 118-residue globin from the cyanobacterium Nostoc commune

The globin from the cyanobacterium Nostoc commune, abbreviated GlbN, which appears to serve as a part of a terminal oxidase rather than as a respiratory pigment, displays relatively normal O2 binding properties, despite the highly abbreviated polypeptide chain, (118 residues) relative to more conventional globins [Thorsteinsson, M. V. , Bevan, D. R., Potts, M., Dou, Y., Eich, R. F., Hargrove, M. S., Gibson, Q. H., and Olson, J. S. (1999) Biochemistry 38, 2117-2126]. The nature of the heme cavity and the general folding topology of this cyanoglobin were investigated by solution 1H NMR to establish the extent to which, and the manner in which, this compact globin adheres to the standard globin fold. This represents by far the smallest globin subjected to structural analysis. The paramagnetic cyanomet derivative was selected because its characteristically large magnetic anisotropy imparts significant dipolar shifts which both improve resolution to greatly facilitate assignments and serve as indicators of the folding topology of the globin. Identification of the axial His 70 and highly conserved Phe 35 (CD1) determined the absolute orientation of the heme and proximal His. Sequential assignments of four helical and one loop segments, which exhibit dipolar contacts to the heme and among each other, confirm the presence of well-conserved F, G, and H helices and the FG corner. The majority of the abbreviation of the chain relative to the more conventional length globins is accommodated in the A-D helices, of which the last is completely missing. The distal residue which provides a H-bond to bound ligand is identified as Gln 43, but the expected helical position E7 could not be confirmed. His 46, placed at position E10, is found to adopt alternate orientations into, and out of, the heme cavity depending on protonation state, suggesting the presence of a Bohr effect at low pH. It is shown that the dipolar shifts exhibited by backbone protons for the assigned residues conform well to those observed for other cyanomet globins and further support a conserved Mb fold. Perturbed medium-range dipolar contacts and the pH-independent backbone proton lability of the F helix are interpreted in terms of a holoprotein which is less stable than a conventional length globin.     

The amino acid sequence of hemoglobin II from the symbiont-harboring clamLucina pectinata

The cytoplasmic hemoglobin II from the gill of the clamLucina pectinata consists of 150 amino acid residues, has a calculatedM _m of 17,476, including heme and an acetylated N-terminal residue. It retains the invariant residues Phe 44 at position CD1 and His 65 at the proximal position F8, as well as the highly conserved Trp 15 at position A12 and Pro 38 at position C2. The most likely candidate for the distal residue at position E7, based on the alignment with other globins, is Gln 65. However, optical and EPR spectroscopic studies of the ferri Hb II (Kraus, D. W., Wittenberg, J. B., Lu, J. F., and Peisach, J.,J. Biol. Chem. 265, 16054–16059, 1990) have implicated a tyrosinate oxygen as the distal ligand. Modeling of theLucina Hb II sequence, using the crystal structure of sperm whale aquometmyoglobin, showed that Tyr 30 substituting for the Leu located at position B10 can place its oxygen within 2.8 Å of the water molecule occupying the distal ligand position. This structural alteration is facilitated by the coordinate mutation of the residue at position CD4, from Phe 46 in the sperm whale myoglobin sequence to Leu 47 inLucina Hb II.     

The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe): site-specific Mg2+ binding and coupled conformational transition in DNA analogs.

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence requirements for cytochrome P-450IID1 catalytic activity. A single amino acid change (Ile380 Phe) specifically decreases Vmax of the enzyme for bufuralol but not debrisoquine hydroxylation

A cDNA coding for an allelic variant of rat IID1, designated IID1v, was isolated that produced a P-450 having a 10-fold lower catalytic activity toward the substrate bufuralol when expressed in COS-1 cells (Matsunaga, E., Zanger, U. M., Hardwick, J. P., Gelboin, H. V., Meyer, U. A., and Gonzalez, F. J. (1989) Biochemistry, 28, 7349-7355). IID1 and IID1v cDNA-deduced proteins differed in sequence by 4 amino acid residues. IID1 has Val, Phe, Arg, and Leu while IID1v has Ile, Leu, Gln, and Phe at amino acid positions 123, 124, 173, and 380, respectively. Chimeric cDNAs between IID1 and IID1v were constructed and expressed in hepatoma cells using vaccinia virus. A chimera having the Phe (IID1v) at amino acid 380, with the remaining 3 variant amino acid residues of IID1, was found to have a 17-fold decrease in Vmax and a 2 to 3-fold decrease in Km for (+)-bufuralol 1'-hydroxylation when compared to a converse chimera having Ile (IID1) in a background of IID1v sequence. Although this enzyme lacked significant bufuralol metabolism, it was able to carry out debrisoquine 4-hydroxylation. In contrast, the chimera having Ile (IID1) at position 380 was lacking in debrisoquine 4-hydroxylation. Type I difference spectra analysis revealed that both forms could bind debrisoquine with similar spectral dissociation constants. These data demonstrate that the single amino acid substitution Ile380----Phe differentially decreases the catalytic activity of IID1 toward bufuralol but not debrisoquine.     

Characterization of the signal for rapid internalization of the bovine mannose 6-phosphate/insulin-like growth factor-II receptor.

The signal for rapid internalization of the mannose 6-phosphate/insulin-like growth factor II receptor has been localized to the sequence Tyr-Lys-Tyr-Ser-Lys-Val in positions 24-29 of its 163-residue cytoplasmic tail. Most of the activity of this signal is mediated by the carboxyl 4 amino acids, especially Tyr26 and Val29 (Canfield, W. M., Johnson, K. F., Ye, R. D., Gregory, W. and Kornfeld, S. (1991) J. Biol. Chem. 266, 5682-5688). In this study, we have tested the effect of a series of mutations on the internalization rate of a mutant receptor that contains a 29-amino acid cytoplasmic tail terminating with the 4-amino acid internalization sequence Tyr-Ser-Lys-Val. Replacement of Tyr26 with Phe or Trp gave rise to mutant receptors that were internalized at 10% the wild-type rate, while receptors with Ala, Leu, Ile, Val, or Asn at this position were totally inactive. Val29 could be replaced by other large hydrophobic residues (Phe, Leu, Ile, or Met) with no loss of activity, but the presence of Ala, Gly, Arg, Gln, or Tyr in this position inactivated the signal. Ser27 could be effectively replaced by many different amino acids, but not by Pro or Gly. However, Gly27 could be tolerated if the residues at positions 28 and 29 were also changed. A change in the 2-residue spacing between Tyr26 and Val29 destroyed the signal. These data show that the essential elements of this signal are an aromatic residue, especially a Tyr in the first position, separated from a large hydrophobic residue in the last position by 2 amino acids. The residues in positions 2 and 3 of the signal may have a modulating effect on its activity. The Tyr-Ser-Lys-Val signal could be moved to a more proximal region of the cytoplasmic tail with only a modest loss of activity. In addition, the signal could be effectively replaced by the putative 4-residue signals of seven other receptors and membrane proteins known to undergo rapid endocytosis, including the Tyr-Thr-Arg-Phe sequence of the transferrin receptor, a Type II membrane protein. These results are compatible with the 4-residue signals of this type being interchangeable, even among Type I and Type II membrane proteins.     

Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin.

The binding of calmodulin (CaM) to four synthetic peptide analogues of the skeletal muscle myosin light chain kinase (sk-MLCK) target sequence has been studied using 1H-NMR. The 18-residue peptide WFF is anchored to CaM via the interaction of the Trp 4 side chain with the C-domain and the Phe 17 side chain with the N-domain of the protein. A peptide corresponding to the first 10 residues (WF10) does not provide the second anchoring residue and is not long enough to span both domains of CaM. 1H-NMR spectroscopy indicates that the WF10 peptide interacts specifically with the C-domain of CaM, and the chemical shifts of the bound Trp side chain are very similar in the CaM:WF10 and CaM:WFF complexes. Binding of the C-domain of CaM to the strongly basic region around Trp 4 of this MLCK sequence may be an important step in target recognition. Comparison of 1H-NMR spectra of CaM bound to WFF, a Trp 4-->Phe analogue (FFF), or a Trp 4-->Phe/Phe 17-->Trp analogue (FFW) suggests that all three peptides bind to CaM in the same orientation, i.e., with the peptide side chain in position 4 interacting with the C-domain and the side chain in position 17 interacting with the N-domain. This indicates that a Trp residue in position 4 is not an absolute requirement for binding this target sequence and that interchanging the Trp 4 and Phe 17 residues does not reverse the orientation of the bound peptide, in confirmation of the deduction from previous indirect studies using circular dichroism (Findlay WA, Martin SR, Beckingham K, Bayley PM, 1995, Biochemistry 34:2087-2094). Molecular modeling/energy minimization studies indicate that only minor local changes in the protein structure are required to accommodate binding of the bulkier Trp 17 side chain of the FFW peptide to the N-domain of CaM.     

Active site of mu-conotoxin GIIIA, a peptide blocker of muscle sodium channels.

The amino acid sequence of mu-conotoxin GIIIA (otherwise called geographutoxin I), a peptide having 22 amino acid residues with three disulfide bridges, was modified by replacing each residue with Ala or Lys to elucidate its active center for blocking sodium channels of skeletal muscle. NMR and CD spectra were virtually identical between native and modified toxins, indicating the similarity of their conformation including disulfide bridges. The inhibitory effect of these modified peptides on twitch contractions of the rat diaphragm showed that Arg at the 13th position and the basicity of the molecule are crucial for the biological action. The segment Lys11-Asp12-Arg13 has been reported to be flexible (Lancelin, J.-M., Kohda, D., Tate, S., Yanagawa, Y., Abe, T., Satake, M., and Inagaki, F. (1991) Biochemistry, in press), and this may represent a clue for the subtle fit of Arg13 to the specific site of sodium channels. Since known ligands to sodium channels, such as tetrodotoxin, anthopleulin-A, etc., contain guanidino groups as a putative binding moiety, Arg may be a general residue for peptide toxins to interact with the receptor site on sodium channels.     

Subsite mapping of an acidic amino acid-specific endopeptidase from Streptomyces griseus, GluSGP, and protease V8.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.     

Structural analysis of specificity: alpha-lytic protease complexes with analogues of reaction intermediates

To better understand the structural basis of enzyme specificity, the structures of complexes formed between alpha-lytic protease, an extracellular serine protease of Lysobacter enzymogenes, and five inhibitory peptide boronic acids (R2-boroX, where R2 is methoxysuccinyl-Ala-Ala-Pro- and boroX is the alpha-aminoboronic acid analogue of Ala, Val, Ile, Norleu, or Phe) have been studied at high resolution by X-ray crystallography. The enzyme has primary specificity for Ala in the P1 position of peptide substrates with catalytic efficiency decreasing with increasing side-chain volume. Enzyme affinity for inhibitors with boroVal, boroIle, and boroPhe residues is much higher than expected on the basis of the catalytic efficiencies of homologous substrates. Covalent tetrahedral adducts are formed between the active-site serine and the boronic acid moieties of R2-boroAla, R2-boroVal R2-boroIle, and R2-boroNorleu. Though R2-boroVal is a slowly bound inhibitor and R2-boroAla is rapidly bound [Kettner, C. A., Bone, R., Agard, D. A., & Bachovchin, W. W. (1988) Biochemistry 27, 7682-7688], there appear to be no structural differences that could account for slow binding. The removal from solution of 20% more hydrophobic surface on binding accounts for the improved affinity of alpha-lytic protease for R2-boroVal relative to R2-boroAla. The high affinity of the enzyme for R2-boroIle derives from the selective binding of the L-allo stereoisomer of the boroIle residue, which can avoid bad steric interactions in the binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)