Amino Acid Substitutions In Domain Research Articles

Disabled insecticidal proteins: A novel tool to understand differences in insect receptor utilization

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or “stacking” of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.

Significant increase in toxicity of the Cry1Ac1 protein of Bacillus thuringiensis by the exchange of two amino acids in its domain III

The LBIT-1200 strain of Bacillus thuringiensis was recently isolated from soil, and showed high biochemical and genetic similarities with the HD-73 standard strain, besides expressing only a Cry1Ac protein: Cry1Ac39. However, LBIT-1200 was 6.4 and 9.5 times more toxic against Manduca sexta and Trichoplusia ni larvae, respectively, than HD-73, which expresses the Cry1Ac1 protein. Sequencing of Cry1Ac1 and Cry1Ac39 proteins showed only four amino acid differences, including two amino acid replacements in domain III, as compared to the rest of Cry1Ac proteins reported to date: N547Y and R602G. These two amino acid substitutions in domain III were introduced in the Cry1Ac1 protein from HD-73 through site directed mutagenesis. Three mutants were obtained: two single and one double mutant, which were expressed in an acrystaliferous B. thuringiensis strain, as well as the native Cry1Ac1 and Cry1Ac39 toxins. Bipyramidal crystals produced by the single mutant strains were larger than the original Cry1Ac1 protein; however, the high toxicity levels previously observed in the strain LBIT-1200 were restored in the three mutants, when tested against M. sexta and T. ni. The substitution N547Y seems to have a higher contribution to LBIT-1200 toxicity.

Mutations in Gene fusA1 as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa.

Resistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1 and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.

Identification and characterization of two CD4 alleles in Microminipigs.

BackgroundWe previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies.ResultsWe identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation.ConclusionsWe identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-016-0856-8) contains supplementary material, which is available to authorized users.

A novel Cry9Aa with increased toxicity for Spodoptera exigua (Hübner)

Cry9Aa, produced by Bacillus thuringiensis is reported to be not active against Spodoptera exigua (beet armyworm). In this study we have cloned a new cry9Aa5 gene encoding a protoxin with increased activity against S. exigua as compared to Cry9Aa1. When aligned to Cry9Aa1, four amino acid substitutions in domain I and one substitution in the C-terminal protein extension of Cry9Aa5 were identified. Toxicity of Cry9Aa5, produced in recombinant Escherichia coli was assessed and compared to the activity of Cry9Aa1, produced under the same conditions.

Mutation analysis of the cross-reactive epitopes of Japanese encephalitis virus envelope glycoprotein

Group and serocomplex cross-reactive epitopes have been identified in the envelope (E) protein of several flaviviruses and have proven critical in vaccine and diagnostic antigen development. Here, we performed site-directed mutagenesis across the E gene of a recombinant expression plasmid that encodes the Japanese encephalitis virus (JEV) premembrane (prM) and E proteins and produces JEV virus-like particles (VLPs). Mutations were introduced at I135 and E138 in domain I; W101, G104, G106 and L107 in domain II; and T305, E306, K312, A315, S329, S331, G332 and D389 in domain III. None of the mutant JEV VLPs demonstrated reduced activity to the five JEV type-specific mAbs tested. Substitutions at W101, especially W101G, reduced reactivity dramatically with all of the flavivirus group cross-reactive mAbs. The group and JEV serocomplex cross-reactive mAbs examined recognized five and six different overlapping epitopes, respectively. Among five group cross-reactive epitopes, amino acids located in domains I, II and III were involved in one, five and three epitopes, respectively. Recognition by six JEV serocomplex cross-reactive mAbs was reduced by amino acid substitutions in domains II and III. These results suggest that amino acid residues located in the fusion loop of E domain II are the most critical for recognition by group cross-reactive mAbs, followed by residues of domains III and I. The amino acid residues of both domains II and III of the E protein were shown to be important in the binding of JEV serocomplex cross-reactive mAbs.

Identical Amino Acid Substitutions in the Repression Domain of Auxin/Indole-3-Acetic Acid Proteins Have Contrasting Effects on Auxin Signaling

Auxin/indole-3-acetic acid (Aux/IAA) proteins function as repressors of auxin response gene expression when auxin concentrations in a cell are low. At elevated auxin concentrations, these repressors are destroyed via the ubiquitin-proteasome pathway, resulting in derepression/activation of auxin response genes. Most Aux/IAA repressors contain four conserved domains, with one of these being an active, portable repression domain (domain I) and a second being an auxin-dependent instability domain (domain II). Here, we have analyzed the effects of amino acid substitutions in the repression domain of selected Aux/IAA proteins. We show that stabilized versions of Aux/IAA proteins with amino acid substitutions in domain I display contrasting phenotypes when expressed in transformed Arabidopsis (Arabidopsis thaliana) plants. An alanine-for-leucine substitution in the LxLxL (where L is leucine and x is another amino acid) repression domain of IAA3, IAA6, or IAA19 confers enhanced auxin response gene expression and "high-auxin" phenotypes when expressed from the 35S or IAA19 promoter (as tested with IAA19) in transformed Arabidopsis plants. In marked contrast, a single alanine-for-leucine substitution in domain I of IAA12 or IAA17 confers repression of auxin response genes and "low-auxin" phenotypes. These results point to intrinsic differences in the repression domain(s) of IAA proteins and suggest that some IAA proteins have stronger or more complex repression domains than others.

A temperature sensitive VSV identifies L protein residues that affect transcription but not replication

To investigate the polymerase components selectively involved in transcription versus replication of vesicular stomatitis virus (VSV), we sequenced the polymerase gene of a conditionally RNA defective, temperature sensitive VSV: ts(G)114, which has a phenotype upon shift from permissive to non-permissive temperature of shut-down of mRNA transcription and unaffected genome replication. Sequence analysis of the ts(G)114 L gene identified three altered amino acid residues in the L protein. These three changes were specifically engineered individually and in combinations into a functional cDNA clone encoding the VSV genome and tested for association with the temperature sensitive and RNA defective phenotypes in the background of recovered engineered viruses. The data presented in this study show a specific amino acid substitution in domain II of the VSV L protein that significantly affects total RNA synthesis, but when in combination with two additional amino acid substitutions identified in the ts(G)114 L protein, leads to a specific reduction in mRNA transcription, but not replication.

Analysis of the cellular immune response induced by Bacillus thuringiensis Cry1A toxins in mice: Effect of the hydrophobic motif from diphtheria toxin

Insecticidal Cry1A toxins from Bacillus thuringiensis elicit strong humoral immune response in mice. Previously, we showed that an eight hydrophobic motif amino acid substitution in Domain I did not affect the antibody inducing capacity of the Cry1A toxins, on the contrary, it was enhanced after intranasal immunization. In addition, Cry1A mutants (carrying a substitution of a motif from fragment B of diphtheria toxin into the structurally similar hydrophobic α-helix 7 motif of Cry1A toxins) were able to modulate the ratio of IgG subclasses, IgG1/IgG2a. However, the capacity of these toxins to induce cellular immune response has not been studied. Thus, in this work, we investigated the cytokine profile induced after in vitro stimulation with the toxins, in spleen cell cultures from unprimed mice, and intranasally primed mice, with either wild-type Cry1Aa or with mutant toxin Cry1Aa8. Spleen cells from unprimed mice stimulated with Cry1Aa produced very low levels of Th1 (IFN-γ, IL-12p70) and Th2 type cytokines (IL-10, IL-4), whereas immunization with Cry1Aa8 toxin led to higher production of these cytokines. Restimulation of spleen cells from primed mice with the Cry1Aa induced the production of significant levels of IL-12p70 whereas with Cry1Aa8, IFN-γ production was stimulated. Interestingly, we found that the capacity of Cry1A toxins to induce cytokine production by lymphocytes was inhibited by N-acetylgalactosamine. Altogether these data demonstrate the immunogenic properties of Cry1A toxins and show that amino acid substitution in Domain I principally affects its ability to induce Th1 cytokines in lymphocytes.

Recovery of Argininosuccinate Lyase Activity in Duck δ1 Crystallin

Delta-crystallin, the major soluble protein component in the avian eye lens, is homologous to argininosuccinate lyase (ASL). Two delta-crystallin isoforms exist in ducks, delta1- and delta2-crystallin, which are 94% identical in amino acid sequence. While duck delta2-crystallin (ddeltac2) has maintained ASL activity, evolution has rendered duck delta1-crystallin (ddeltac1) enzymatically inactive. Previous attempts to regenerate ASL activity in ddeltac1 by mutating the residues in the 20s (residues 22-31) and 70s (residues 74-89) loops to those found in ddeltac2 resulted in a double loop mutant (DLM) which was enzymatically inactive (Tsai, M. et al. (2004) Biochemistry 43, 11672-82). This result suggested that one or more of the remaining five amino acid substitutions in domain 1 of the DLM contributes to the loss of ASL activity in ddeltac1. In the current study, residues Met-9, Val-14, Ala-41, Ile-43, and Glu-115 were targeted for mutagenesis, either alone or in combination, to the residues found in ddeltac2. ASL activity was recovered in the DLM by changing Met-9 to Trp, and this activity is further potentiated in the DLM-M9W mutant when Glu-115 is changed to Asp. The roles of Trp-9 and Asp-115 were further investigated by site-directed mutagenesis in wild-type ddeltac2. Changing the identity of either Trp-9 or Asp-115 in ddeltac2 resulted in a dramatic drop in enzymatic activity. The loss of activity in Trp-9 mutants indicates a preference for an aromatic residue at this position. Truncation mutants of ddeltac2 in which the first 8, 9, or 14 N-terminal residues were removed displayed either decreased or no ASL activity, suggesting residues 1-14 are crucial for enzymatic activity in ddeltac2. Our kinetic studies combined with available structural data suggest that the N-terminal arm in ASL/delta2-crystallin is involved in stabilizing regions of the protein involved in substrate binding and catalysis, and in completely sequestering the substrate from the solvent.

A duck delta1 crystallin double loop mutant provides insight into residues important for argininosuccinate lyase activity.

Delta-crystallin is directly related to argininosuccinate lyase (ASL), and catalyzes the reversible hydrolysis of argininosuccinate to arginine and fumarate. Two delta-crystallin isoforms exist in duck lenses, delta1 and delta2, which are 94% identical in amino acid sequence. Although the sequences of duck delta2-crystallin (ddeltac2) and duck delta1-crystallin (ddeltac1) are 69 and 71% identical to that of human ASL, respectively, only ddeltac2 has maintained ASL activity. Domain exchange experiments and comparisons of various delta-crystallin structures have suggested that the amino acid substitutions in the 20's (residues 22-31) and 70's (residues 74-89) loops of ddeltac1 are responsible for the loss of enzyme activity in this isoform. To test this hypothesis, a double loop mutant (DLM) of ddeltac1 was constructed in which all the residues that differ between the two isoforms in the 20's and 70's loops were mutated to those of ddeltac2. Contrary to expectations, kinetic analysis of the DLM found that it was enzymatically inactive. Furthermore, binding of argininosuccinate by the DLM, as well as the ddeltac1, could not be detected by isothermal titration calorimetry (ITC). To examine the conformation of the 20's and 70's loops in the DLM, and to understand why the DLM is unable to bind the substrate, its structure was determined to 2.5 A resolution. Comparison of this structure with both wild-type ddeltac1 and ddeltac2 structures reveals that the conformations of the 20's and 70's loops in the DLM mutant are very similar to those of ddeltac2. This suggests that the five amino acid substitutions in domain 1 which lie outside of the two loop regions and which are different in the DLM, and ddeltac2, must be important enzymatically. The structure of the DLM in complex with sulfate was also determined to 2.2 A resolution. This structure demonstrates that the conformational changes of the 280's loop and domain 3, previously observed in ddeltac1, also occur in the DLM upon sulfate binding, reinforcing the hypothesis that these events may occur in the active ddeltac2 protein during catalysis.

Neuroadapted Yellow Fever Virus 17D: Determinants in the Envelope Protein Govern Neuroinvasiveness for SCID Mice

A molecular clone of mouse-neuroadapted yellow fever 17D virus (SPYF-MN) was used to identify critical determinants of viral neuroinvasiveness in a SCID mouse model. Virus derived from this clone differs from nonneuroinvasive YF5.2iv virus at 29 nucleotide positions, encoding 13 predicted amino acid substitutions and 2 substitutions in the 3' untranslated region (UTR). The virulence determinants of SPYF-MN for SCID mice were identified by constructing and characterizing intratypic viruses in which the E protein of SPYF-MN was expressed in the YF5.2iv background (SPYF-E) or the E protein of YF5.2iv was expressed in the SPYF-MN background (YF5.2-E). SPYF-E caused lethal encephalitis in young adult SCID mice after intraperitoneal inoculation, with average survival times and tissue virus burdens resembling those of mice inoculated with the parental SPYF-MN virus. To define which domains of the E protein are involved in neuroinvasiveness, two viruses were tested in which the amino acid substitutions in domains I-II and III were segregated. This revealed that substitutions in domain III (residues 305, 326, and 380) were critical for the neuroinvasive phenotype, based on average survival times and tissue burdens of infectious virus. Comparison of growth properties of the various intratypic viruses in cell culture indicated that no inherent defects in replication efficiency were likely to account for the biological differences observed in these experiments. These findings demonstrate that the E protein is a critical factor for yellow fever virus neuropathogenesis in the SCID mouse model and that the neuroinvasive properties depend principally on functions contributed by domain III of this protein. To assess whether critical determinants for neuroinvasion of normal ICR mice by SPYF virus were also in the E protein, sequences of viruses recovered from brains of ICR mice succumbing to encephalitis with the parental SPYF virus were derived. No differences were found in the E protein; however, two substitutions were present in the 3' UTR compared to that of SPYF-MN, one of which is predicted to alter RNA secondary structure in this region. These findings suggest that the 3' UTR may also affect neuroinvasiveness of SPYF virus in the mouse model.

Domain exchange experiments in duck delta-crystallins: functional and evolutionary implications.

Delta-crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two delta crystallins, denoted delta1 and delta2. Duck delta2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to delta2/ASL in the amino acid sequence, delta1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras--221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, delta1 or delta2, respectively--were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to delta1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in delta1. The thermodynamic characterization of human ASL (hASL) and duck delta1 and delta2 indicate that delta crystallins are slightly less stable than hASL, with the delta1 being the least stable. The deltaGs of unfolding are 57.25, 63.13, and 70.71 kcal mol(-1) for delta1, delta2, and hASL, respectively. This result was unexpected, and we speculate that delta crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein-protein and protein-solvent interactions.

Glucose Transporter Isoforms GLUT1 and GLUT3 Transport Dehydroascorbic Acid

Dehydroascorbic acid (DHA) is rapidly taken up by cells and reduced to ascorbic acid (AA). Using the Xenopus laevis oocyte expression system we examined transport of DHA and AA via glucose transporter isoforms GLUT1-5 and SGLT1. The apparent Km of DHA transport via GLUT1 and GLUT3 was 1.1 +/- 0.2 and 1.7 +/- 0.3 mM, respectively. High performance liquid chromatography analysis confirmed 100% reduction of DHA to AA within oocytes. GLUT4 transport of DHA was only 2-4-fold above control and transport kinetics could not be calculated. GLUT2, GLUT5, and SGLT1 did not transport DHA and none of the isoforms transported AA. Radiolabeled sugar transport confirmed transporter function and identity of all cDNA clones was confirmed by restriction fragment mapping. GLUT1 and GLUT3 cDNA were further verified by polymerase chain reaction. DHA transport activity in both GLUT1 and GLUT3 was inhibited by 2-deoxyglucose, D-glucose, and 3-O-methylglucose among other hexoses while fructose and L-glucose showed no inhibition. Inhibition by the endofacial inhibitor, cytochalasin B, was non-competitive and inhibition by the exofacial inhibitor, 4,6-O-ethylidene-alpha-glucose, was competitive. Expressed mutant constructs of GLUT1 and GLUT3 did not transport DHA. DHA and 2-deoxyglucose uptake by Chinese hamster ovary cells overexpressing either GLUT1 or GLUT3 was increased 2-8-fold over control cells. These studies suggest GLUT1 and GLUT3 isoforms are the specific glucose transporter isoforms which mediate DHA transport and subsequent accumulation of AA.

DNA Sequence Variation in Domain a of the Acetolactate Synthase Genes of Herbicide-Resistant and -Susceptible Weed Biotypes

The DNA sequence of a 196 base pair (bp) region of the acetolactate synthase (ALS) genes of three weed species, kochia, prickly lettuce, and Russian thistle, was determined. This region encompasses the coding sequence for Domain A, a region of the amino acid sequence previously demonstrated to play a pivotal role in conferring resistance to herbicides that inhibit ALS. The Domain A DNA sequence from a chlorsulfuron-resistant (R) prickly lettuce biotype from Idaho differed from that of a chlorsulfuron-susceptible (S) biotype by a single point mutation, which substituted a histidine for a proline. The Domain A DNA sequence from an R kochia biotype from Kansas also differed from that of an S biotype by a single point mutation in the same proline codon. This point mutation, however, conferred substitution of threonine for proline. Two different ALS-homologous sequences were isolated from an R biotype of Russian thistle. Neither sequence encoded amino acid substitutions in Domain A that differed from the consensus S sequence. The DNA sequence variation among the R and S kochia biotypes was used to characterize six Ada County, Idaho, kochia collections for correlation between phenotypic chlorsulfuron susceptibility and restriction digest patterns (RFLPs) of polymerase chain reaction amplification products. Most collections showed excellent correspondence between the RFLP patterns and the phenotypic response to chlorsulfuron application. However, one entirely R collection had the RFLP pattern of the S biotype, suggesting that resistance was not due to mutation in the proline codon.

Three amino acid substitutions in domain I of calmodulin prevent the activation of chicken smooth muscle myosin light chain kinase.

TaM-BMI is a genetically engineered chimeric protein consisting of the first 55 amino acids of cardiac troponin C (but with the normally inactive first Ca2+ binding domain reactivated by site- directed mutagenesis) ligated to the last three domains of chicken calmodulin (George, S.E., VanBerkum, M.F., Ono, T., Cook, R., Hanley, R.M., Putkey, J.A., and Means, A. R. (1990) J. Biol. Chem. 265, 9228-9235). This protein binds chicken smooth muscle myosin light chain kinase (smMLCK) but fails to activate the enzyme, thus functioning as a potent competitive inhibitor (Ki = 66 nM). We have created 29 mutants of calmodulin designed to identify the minimal number of alterations which must be introduced in the first domain to convert the protein to a competitive inhibitor of smMLCK. Alterations of three amino acids predicted to lie on the external surface of calmodulin (E14A, T34K, S38M) recapitulated the phenotype of TaM-BMI and exhibited a Ki of 38 nM. Both the triple mutant and TaM-BMI activated phosphodiesterase and bound a synthetic peptide analog of the calmodulin binding region of smMLCK with an affinity similar to that of native calmodulin (Kact and Kd values of approximately 2 and 3 nM respectively). When a synthetic peptide analog of the myosin light chain phosphorylation site was used as substrate rather than the 20-kDa light chains, TaM-BMI and the triple mutant were partial agonists: the Km for peptide substrate was increased 100- and 60-fold, and catalytic activity was 45 and 60%, respectively, relative to calmodulin. These data suggest TaM-BMI and E14A/T34K/S38M may interact with the calmodulin binding domain of smMLCK in a manner similar to calmodulin. However, alterations in electrostatic and hydrophobic interactions created by the three amino acid substitutions prevent the conformational change in the enzyme usually produced by calmodulin binding. Lack of such changes results in loss of catalytic activity and light chain binding. Additionally, our results show that altering only 3 amino acids residues converts calmodulin to an enzyme-selective antagonist, thus demonstrating the ability to separate calmodulin binding to smMLCK from calmodulin-induced activation of the enzyme.

Changes in stability and allosteric properties of aspartate transcarbamoylase resulting from amino acid substitutions in the zinc-binding domain of the regulatory chains.

Changes in subunit interaction energies linked to the allosteric transition of the regulatory enzyme aspartate transcarbamoylase (ATCase; EC 2.1.3.2) from Escherichia coli are localized in part at interfaces between the six catalytic (c) and six regulatory (r) polypeptide chains. Site-directed mutagenesis has been used to construct enzymes with amino acid substitutions in a limited region of the zinc-binding domain of the r chains. Substitution of Ser or His for r114 Cys, one of four cysteines binding the structural zinc ion in the regulatory chain, leads to incorrectly folded chains as shown by the inability to detect stable assembled holoenzyme in cell extracts. Replacement of r111 Asn by Ala at the interface between an r chain and a c chain in the apposing catalytic trimer causes a complete loss of the homotropic and heterotropic effects characteristic of wild-type ATCase. Moreover, sedimentation velocity experiments demonstrated that this mutant enzyme exists in the R ("relaxed") conformation in the absence of active site ligands due to preferential destabilization of the T ("taut") conformation relative to the R state. In contrast, replacement of r113 Asn by Ala at the interface between adjacent r and c chains leads to an increase in the cooperativity of the enzyme. When r139 Lys is replaced by Met, Vmax is reduced by 50% compared to wild-type ATCase, whereas it is increased about 2-fold when r142 Glu is replaced by Asp. Amino acid substitutions in this domain significantly affect subunit interaction energy as measured by rate of subunit exchange when holoenzymes are incubated with isolated catalytic subunits, thus permitting measurements of the effect of the bisubstrate analog N-(phosphonacetyl)-L-asparatate in weakening intersubunit interactions. Subunit exchange increased about 9-fold for the r142 Glu----Asp mutant and almost 20-fold for the r142 Glu----Ala mutant in the presence of the ligand.