Specific RecA amino acid changes affect RecA–UmuD'C interaction

The UmuD'C mutagenesis complex accumulates slowly and parsimoniously after a 12 J m⁻² UV flash to attain after 45 min a low cell concentration between 15 and 60 complexes. Meanwhile, RecA monomers go up to 72 000 monomers. By contrast, when the UmuD'C complex is constitutively produced at a high concentration, it inhibits recombinational repair and then markedly reduces bacterial survival from DNA damage. We have isolated novel recA mutations that enable RecA to resist UmuD'C recombination inhibition. The mutations, named recA [UmuR], are located on the RecA three-dimensional structure at three sites: (i) the RecA monomer tail domain (four amino acid changes); (ii) the RecA monomer head domain (one amino acid change, which appears to interface with the amino acids in the tail domain); and (iii) in the core of a RecA monomer (one amino acid change). RecA [UmuR] proteins make recombination more efficient in the presence of UmuD'C while SOS mutagenesis is inhibited. The UmuR amino acid changes are located at a head-tail joint between RecA monomers and some are free to possibly interact with UmuD'C at the tip of a RecA polymer. These two RecA structures may constitute possible sites to which the UmuD'C complex might bind, hampering homologous recombination and favouring SOS mutagenesis.     

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity.

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.     

Fabry disease: twenty novel alpha-galactosidase A mutations and genotype-phenotype correlations in classical and variant phenotypes

BACKGROUND: Fabry disease (OMIM 301500) is an X-linked inborn error of glycosphingolipid metabolism resulting from mutations in the alpha-galactosidase A (alpha-Gal A) gene. The disease is phenotypically heterogeneous with classic and variant phenotypes. To assess the molecular heterogeneity, define genotype/phenotype correlations, and for precise carrier identification, the nature of the molecular lesions in the alpha-Gal A gene was determined in 40 unrelated families with Fabry disease. MATERIALS AND METHODS: Genomic DNA was isolated from affected males or obligate carrier females and the entire alpha-Gal A coding region and flanking sequences were amplified by PCR and analyzed by automated sequencing. Haplotype analyses were performed with polymorphisms within and flanking the alpha-Gal A gene. RESULTS: Twenty new mutations were identified (G43R, R49G, M72I, G138E, W236X, L243F, W245X, S247C, D266E, W287C, S297C, N355K, E358G, P409S, g1237del15, g10274insG, g10679insG, g10702delA, g11018insA, g11185-delT), each in a single family. In the remaining 20 Fabry families, 18 previously reported mutations were detected (R49P, D92N, C94Y, R112C [two families], F113S, W162X, G183D, R220X, R227X, R227Q, Q250X, R301X, R301Q, G328R, R342Q, E358K, P409A, g10208delAA [two families]). Haplotype analyses indicated that the families with the R112C or g10208delAA mutations were not related. The proband with the D266E lesion had a severe classic phenotype, having developed renal failure at 15 years. In contrast, the patient with the S247C mutation had a variant phenotype, lacking the classic manifestations and having mild renal involvement at 64 years. CONCLUSIONS: These results further define the heterogeneity of alpha-Gal A mutations causing Fabry disease, permit precise heterozygote detection and prenatal diagnosis in these families, and provide additional genotype/phenotype correlations in this lysosomal storage disease.     

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.     

Mutational Analysis of Bacterial NAD^＋-dependent DNA Ligase： Role of Motif IV in Ligation Catalysis

The bacterial DNA ligase as a multiple domain protein is involved in DNA replication, repair and recombination. Its catalysis of ligation can be divided into three steps. To delineate the roles of amino acid residues in motif IV in ligation catalysis, site-directed mutants were constructed in a bacterial NAD^＋-dependent DNA ligase from Thermus sp. TAK16D. It was shown that four conserved residues （D286, G287, V289 and K291） in motif IV had significant roles on the overall ligation. Under single turnover conditions, the observed apparent rates of D286E, G287A, V289I and K291R mutants were clearly reduced compared with that of WT ligase on both match and mismatch nicked substrates. The effects of D286E mutation on overall ligation may not only be ascribed to the third step. The G287A mutation has a major effect on the second step. The effects of V289I and K291R mutation on overall ligation are not on the third step, perhaps other aspects, such as conformation change of ligase protein in ligation catalysis, are involved. Moreover, the amino acid substitutions of above four residues were more sensitive on mismatch nicked substrate, indicating an enhanced ligation fidelity.     

Enhanced monomer–monomer interactions can suppress the recombination deficiency of the recA142 allele

The RecA142 protein, in which valine is substituted for isoleucine-225, is defective for genetic recombination in vivo and for DNA strand exchange activity in vitro under conventional growth and reaction conditions respectively. However, we show that mildly acidic conditions restore both the in vitro DNA strand exchange activity and the in vivo function of RecA142 protein, suggesting that recombination function can be restored by a slight change in protein structure elicited by protonation. Indeed, we identified an intragenic suppressor of the recombination deficiency of the recA142 allele. This suppressor mutation is a substitution of leucine for glutamine at position 124. Based on the three-dimensional structure, the Q-124L substitution is predicted to make a new monomer-monomer contact with residue phenylalanine-21 of the adjacent RecA monomer. The Q-124L mutation is not allele specific, because it also suppresses the recombination deficiency of a recA deletion (Delta9), lacking nine amino acids at the amino-terminus, presumably by reinforcing the monomer-monomer interactions that are attenuated by the Delta9 deletion. Expression of RecA(Q-124L) protein is toxic to Escherichia coli, presumably because of enhanced affinity for DNA. We speculate as to how enhanced monomer-monomer interactions and acidic pH conditions can restore the recombination activity of some defective recA alleles.     

Cloning and DNA sequence of a mycoplasmal recA gene.

Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.     

Effect of Cleavage Mutants on Syncytium Formation Directed by the Wild-Type Fusion Protein of Newcastle Disease Virus

The effects of Newcastle disease virus (NDV) fusion (F) glycoprotein cleavage mutants on the cleavage and syncytium-forming activity of the wild-type F protein were examined. F protein cleavage mutants were made by altering amino acids in the furin recognition region (amino acids 112 to 116) in the F protein of a virulent strain of NDV. Four mutants were made: Q114P replaced the glutamine residue with proline; K115G replaced lysine with glycine; double mutant K115G, R113G replaced both a lysine and an arginine with glycine residues; and a triple mutant, R112G, K115G, F117L, replaced three amino acids to mimic the sequence found in avirulent strains of NDV. All mutants except Q114P were cleavage negative and fusion negative. However, addition of exogenous trypsin cleaved all mutant F proteins and activated fusion. As expected for an oligomeric protein, the fusion-negative mutants had a dominant negative phenotype: cotransfection of wild-type and mutant F protein cDNAs resulted in an inhibition of syncytium formation. The presence of the mutant F protein did not inhibit cleavage of the wild-type protein. Furthermore, evidence is presented that suggests that the mutant protein and the wild-type protein formed heterooligomers. By measuring the syncytium-forming activity of the wild-type protein at various ratios of expression of mutant and wild-type protein, results were obtained that are most consistent with the notion that the size of the functionally active NDV F protein in these assays is a single oligomer, likely a trimer. That a larger oligomer, containing a mix of both wild-type and mutant F proteins, has partial activity cannot, however, be ruled out.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

Novel single nucleotide polymorphisms of organic cation transporter 1 (SLC22A1) affecting transport functions

Organic cation transporter OCT1 (SLC22A1) plays an essential role in absorption, distribution, and excretion of various xenobiotics including therapeutically important drugs. In the present study, we analyzed the functional properties of the single nucleotide polymorphisms (SNPs) in SLC22A1 gene found in Japanese control individuals. Four mutations resulting in the amino acid changes (F160L, P283L, R287G, and P341L) were functionally characterized in Xenopus oocyte expression system. Two new SNPs, identified in Japanese population, P283L and R287G exhibited no uptake of both [14C]TEA and [3H]MPP+, although their protein expressions were detected in the plasma membrane of the oocytes injected with their cRNAs. Uptake of [14C]TEA by P341L was reduced to 65.1% compared to wild type, whereas F160L showed no significant change in its transport activity. This study suggests that the newly found OCT1 variants will contribute to inter-individual variations leading to the differences in cationic drug disposition and perhaps certain disease processes.     

A recA null mutation may be generated in Streptomyces coelicolor

The recombinase RecA plays a crucial role in homologous recombination and the SOS response in bacteria. Although recA mutants usually are defective in homologous recombination and grow poorly, they nevertheless can be isolated in almost all bacteria. Previously, considerable difficulties were experienced by several laboratories in generating recA null mutations in Streptomyces, and the only recA null mutants isolated (from Streptomyces lividans) appeared to be accompanied by a suppressing mutation. Using gene replacement mediated by Escherichia coli-Streptomyces conjugation, we generated recA null mutations in a series of Streptomyces coelicolor A3(2) strains. These recA mutants were very sensitive to mitomycin C but only moderately sensitive to UV irradiation, and the UV survival curves showed wide shoulders, reflecting the presence of a recA-independent repair pathway. The mutants segregated minute colonies with low viability during growth and produced more anucleate spores than the wild type. Some crosses between pairs of recA null mutants generated no detectable recombinants, showing for the first time that conjugal recombination in S. coelicolor is recA mediated, but other mutants retained the ability to undergo recombination. The nature of this novel recombination activity is unknown.     

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X-ray crystallography.

The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 A resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.     

Functional determinants of ras interference 1 mutants required for their inhbitory activity on endocytosis

In this study, we initiated experiments to address the structure-function relationship of Rin1. A total of ten substitute mutations were created, and their effects on Rin1 function were examined. Of the ten mutants, four of them (P541A, E574A, Y577F, T580A) were defective in Rab5 binding, while two other Rin1 mutants (D537A, Y561F) partially interacted with Rab5. Mutations in several other residues (Y506F, Y523F, T572A, Y578F) resulted in partial loss of Rab5 function. Biochemical studies showed that six of them (D537A, P541A, Y561F, E574A, Y577F, T580A) were unable to activate Rab5 in an in vitro assay.In addition, Rin1: D537A and Rin1: Y561F mutants showed dominant inhibition of Rab5 function. Consistent with the biochemical studies, we observed that these two Rin1 mutants have lost their ability to stimulate the endocytosis of EGF, form enlarged Rab5-positive endosomes, or support in vitro endosome fusion. Based on these data, our results showed that mutations in the Vps9 domain of Rin1 lead to a loss-of-function phenotype, indicating a specific structure-function relationship between Rab5 and Rin1.     

Pertussis toxin-catalyzed ADP-ribosylation of G(o) alpha with mutations at the carboxyl terminus.

The guanine nucleotide-binding protein G(o alpha) has been implicated in the regulation of Ca2+ channels in neural tissues. Covalent modification of G(o alpha) by pertussis toxin-catalyzed ADP-ribosylation of a cysteine (position 351) four amino acids from the carboxyl terminus decouples G(o alpha) from receptor. To define the structural requirements for ADP-ribosylation, preparations of recombinant G(o alpha) with mutations within the five amino acids at the carboxyl terminus were evaluated for their ability to serve as pertussis toxin substrates. As expected, the mutant in which cysteine 351 was replaced by glycine (C351G) was not a toxin substrate. Other inactive mutants were G352D and L353 delta/Y354 delta. Mutations that had no significant effect on toxin-catalyzed ADP-ribosylation included G350D, G350R, Y354 delta, and L353V/Y354 delta. Less active mutants were L353G/Y354 delta, L353A/Y354 delta, and L353G. ADP-ribosylation of the active mutants, like that of wild-type G(o alpha), was enhanced by the beta gamma subunits of bovine transducin. It appears that three of the four terminal amino acids critically influence pertussis toxin-catalyzed ADP-ribosylation of G(o alpha).     

Design and characterization of stabilized derivatives of human CD4D12 and CD4D1

CD4 is present on the surface of T-lymphocytes and is the primary cellular receptor for HIV-1. CD4 consists of a cytoplasmic tail, one transmembrane region, and four extracellular domains, D1-D4. A construct consisting of the first two domains of CD4 (CD4D12) is folded and binds gp120 with similar affinity as soluble 4-domain CD4 (sCD4). However, the first domain alone (CD4D1) was previously shown to be largely unfolded and had 3-fold weaker affinity for gp120 when compared to sCD4 [Sharma, D.; et al. (2005) Biochemistry 44, 16192-16202]. We now report the design and characterization of three single-site mutants of CD4D12 (G6A, L51I, and V86L) and one multisite mutant of CD4D1 (G6A/L51I/L5K/F98T). G6A, L51I, and V86L are cavity-filling mutations while L5K and F98T are surface mutations which were introduced to minimize the aggregation of CD4D1 upon removal of the second domain. Two mutations, G6A and V86L in CD4D12 increased the stability and yield of the protein relative to the wild-type protein. The mutant CD4D1 (CD4D1a) with the 4 mutations was folded and more stable compared to the original CD4D1, but both bound gp120 with comparable affinity. In in vitro neutralization assays, both CD4D1a and G6A-CD4D12 were able to neutralize diverse HIV-1 viruses with similar IC(50)s as 4-domain CD4. These stabilized derivatives of human CD4 can be useful starting points for the design of other more complex viral entry inhibitors.     

Disruption of an ATP-dependent isomerization of the recA protein by mutation of histidine 163.

We have used site-directed mutagenesis to replace histidine 163 of the recA polypeptide with an alanine residue. The new [Ala-163]recA protein catalyzes single-stranded (ss) DNA-dependent ATP hydrolysis with a turnover number that is similar to that of the wild-type recA protein. Despite being proficient in ssDNA-dependent ATP hydrolysis, the [Ala-163]recA protein is unable to promote the ATP-dependent three-strand exchange reaction under standard reaction conditions, pH 7.5. The [Ala-163]recA protein does exhibit three-strand exchange activity at pH 6.0-7.0, however, and the induction of strand exchange activity at low pH correlates directly with the activation of an ATP-dependent isomerization of the mutant protein. Thus, the [Ala-163]recA protein is functionally similar to our previously described mutant [Asn-160]recA protein (Bryant, F.R. (1988) J. Biol. Chem. 263, 8716-8723; Muench, K.A., and Bryant, F. R. (1990) J. Biol. Chem. 265, 11560-11566). Trypsin proteolysis studies indicate that the [Ala-163]recA and [Asn-160]recA proteins, like the wild-type recA protein, are organized into carboxyl-terminal and amino-terminal domains of nearly equal size. According to this structural model, the [Ala-163]recA and [Asn-160]recA mutations may lie in a linker region joining these two domains. We speculate that the [Ala-163]recA and [Asn-160]recA mutations interfere with an ATP-dependent conformational change of the recA protein that perhaps involves a change in the relative orientation of the carboxyl-terminal and amino-terminal domains.     

Exploring the cause of drug resistance by the detrimental missense mutations in KIT receptor: computational approach

In this work, we computationally identified the most detrimental missense mutations of KIT receptor causing gastrointestinal stromal tumors and analyzed the drug resistance of these missense mutations. Out of 31 missense mutations, 19 variants were commonly found less stable, deleterious and damaging by I-Mutant 2.0, SIFT and PolyPhen programs, respectively. Subsequently, we performed modeling of these 19 variants to understand their change in conformations with respect to native KIT receptor by computing their RMSD. Further, the native and 19 mutants were docked with the drug ‘Imatinib’ to explain the drug resistance of these detrimental missense mutations. Among the 19 mutants, we found by docking studies that 12 mutants, namely, F584C, F584L, V654A, L656P, T670I, R804W, D816F, D816V, D816Y, N822K, Y823D and E839K had less binding affinity with Imatinib than the native type. Finally, we analyzed that the loss of binding affinity of these 12 mutants, was due to altered flexibility in their binding amino acids with Imatinib as compared with native type by normal mode analysis. In our work, we found the novel data that the majority of the drug-binding amino acids in those 12 mutants had encountered loss of flexibility, which could be the theoretical basis for the cause of drug insensitivity.     

新城疫病毒F蛋白细胞融合活性位点中保守氨基酸基因突变分析

为了确定新城疫病毒融合蛋白(F)分子上活性位点中保守氨基酸在F蛋白的细胞融合作用,弄清F细胞融合的分子机理,采用基因定点突变法,创造一个酶切位点,用酶切反应初步筛选突变株,然后用DNA序列分析进一步确定,并于真核细胞内进行表达,Giemsa染色定性和指示基因法定量检测细胞融合功能,荧光强度分析(FACS)检测表达效率情况。结果表明,NDV F第117位苯丙氨酸(F)突变成亮氨酸(L)时对细胞融合作用没有显著影响。R112和K115同为保守序列,分别突变为G时,细胞融合活性只有原来的44%,下降了56%。细胞表面表达效率没有明显的改变。N147突变为K时,细胞融合活性明显下降,只有原来的15%,而细胞表面表达效率没有明显的改变。L154为保守序列,突变为K时,细胞融合活性消失,说明L154是一个非常关键的氨基酸,对维持F蛋白的细胞融合活性非常重要。细胞表面表达效率也有所下降(为原来的94%)。D462属于高度保守氨基酸,当突变为N时,细胞融合活性消失,但经细胞表面表达效率分析证明,此突变蛋白未表达于细胞表面,证明在细胞浆转运至细胞表面的过程中发生了问题。当突变为R和E时,细胞融合活性未发生改变,但细胞表面表达效率有所下降,分别为野毒株的63%和44%。说明NDV F分子上与HN相互作用的特异性区域中的某些保守氨基酸在细胞融合中发挥着重要作用,对F蛋白的折叠、加工、转运等,发挥着不同作用,从而影响F蛋白的细胞融合作用和/或在细胞表面的表达量。