Mutant cholinesterases possessing enhanced capacity for reactivation of their phosphonylated conjugates

Selective mutants of mouse acetylcholinesterase (AChE; EC 3.1.1.7) phosphonylated with chiral S(P)- and R(P)-cycloheptyl, -3,3-dimethylbutyl, and -isopropyl methylphosphonyl thiocholines were subjected to reactivation by the oximes HI-6 and 2-PAM and their reactivation kinetics compared with wild-type AChE and butyrylcholinesterase (EC 3.1.1.8). Mutations in the choline binding site (Y337A, Y337A/F338A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) were employed to enlarge active center gorge dimensions. HI-6 was more efficient than 2-PAM (up to 29000 times) as a reactivator of S(P)-phosphonates (k(r) ranged from 50 to 13000 min(-1) M(-1)), while R(P) conjugates were reactivated by both oximes at similar, but far slower, rates (k(r) < 10 min(-1) M(-1)). The Y337A substitution accelerated all reactivation rates over the wild-type AChE and enabled reactivation even of R(P)-cycloheptyl and R(P)-3,3-dimethylbutyl conjugates that when formed in wild-type AChE are resistant to reactivation. When combined with the F295L or F297I mutations in the acyl pocket, the Y337A mutation showed substantial enhancements of reactivation rates of the S(P) conjugates. The greatest enhancement of 120-fold was achieved with HI-6 for the F295L/Y337A phosphonylated with the most bulky alkoxy moiety, S(P)-cycloheptyl methylphosphonate. This significant enhancement is likely a direct consequence of simultaneously increasing the dimensions of both the choline binding site and the acyl pocket. The increase in dimensions allows for optimizing the angle of oxime attack in the spatially impacted gorge as suggested from molecular modeling. Rates of reactivation reach values sufficient for consideration of mixtures of a mutant enzyme and an oxime as a scavenging strategy in protection and treatment of organophosphate exposure.     

Molecular analysis of cyp51 from fluconazole-resistant Candida albicans strains

The target enzyme for fluconazole is sterol 14α-demethylase, a cytochrome P450 encoded by cyp51. One mechanism of fluconazole resistance likely to occur in Candida albicans is through an altered target site. To test this hypothesis DNA sequencing of the cyp51 coding sequence from 19 fluconazole-resistant and 19 fluconazole-sensitive C. albicans was undertaken. A number of point mutations were identified in the resistant isolates which were not present in the sensitive ones: F105L (five), E266D (five), K287R (one), G448G (one), G450E (one), G464S (three) and V488I (one). These alterations are discussed in the light of a molecular model of the enzyme regarding potential roles in resistance. It was also demonstrated that sequence-specific primers can be employed to identify polymorphisms which may be associated with resistance; diagnostic tests for resistant strains will prove of value in combating this serious clinical problem.     

Monoclonal antibodies recognizing surface residues of the beta subunit of Escherichia coli F(1) ATPase: functional importance of the epitope residues

Two monoclonal antibodies, beta 208 and beta 210, against the beta subunit of the F(1) ATPase from Escherichia coli reacted with an intact beta subunit and also a peptide corresponding to a portion of beta between residues 1 and 145. Mutations at Ala-1, Val-15, Glu-16, Phe-17, Leu-29, Gly-65, or Leu-66, and His-110 or Arg-111 for beta 210 and beta 208, respectively, caused decreased antibody binding to beta, suggesting that these residues form the epitopes and are thought to lie close together on the surface of the beta subunit. The topological locations of the corresponding residues in the atomic structure of the bovine beta subunit agree well with these expectations, except for Ala-1 and Leu-29. beta 210 binds to two beta strands including the epitope residues that are 50 residues apart, indicating that this antibody recognizes the tertiary structure of the N-terminal end region. Mutations in the epitope residues of beta 210 do not affect the F(1) ATPase activity, suggesting that surfaces of the two beta strands in the amino-terminal end region are not functionally essential. To analyze the functional importance around His-110 recognized by beta 208 we introduced site specific mutations at residues His-110 and Ile-109. Ile-109 to Ala or Arg, and His-110 to Ala or Asp caused defective assembly of F(1). However, the His-110 to Arg mutation had no effect on molecular assembly, suggesting that Ile-109 and His-110, especially the positive charge of His-110 are essential for the assembly of F(1). The His-110 to Arg mutation caused a large decrease in F(1)-ATPase activity, suggesting that a subtle change in the topological arrangement of the positive charge of His-110 located on the surface of beta plays an important role in the catalytic mechanism of the F(1)-ATPase.     

Interacting residues in an activated state of a G protein-coupled receptor

Ste2p, the G protein-coupled receptor (GPCR) for the tridecapeptide pheromone alpha-factor of Saccharomyces cerevisiae, was used as a model GPCR to investigate the role of specific residues in the resting and activated states of the receptor. Using a series of biological and biochemical analyses of wild-type and site-directed mutant receptors, we identified Asn(205) as a potential interacting partner with the Tyr(266) residue. An N205H/Y266H double mutant showed pH-dependent functional activity, whereas the N205H receptor was non-functional and the Y266H receptor was partially active indicating that the histidine 205 and 266 residues interact in an activated state of the receptor. The introduction of N205K or Y266D mutations into the P258L/S259L constitutively active receptor suppressed the constitutive activity; in contrast, the N205K/Y266D/P258L/S259L quadruple mutant was fully constitutively active, again indicating an interaction between residues at the 205 and 206 positions in the receptor-active state. To further test this interaction, we introduced the N205C/Y266C, F204C/Y266C, and N205C/A265C double mutations into wild-type and P258L/S259L constitutively active receptors. After trypsin digestion, we found that a disulfide-cross-linked product, with the molecular weight expected for a receptor fragment with a cross-link between N205C and Y266C, formed only in the N205C/Y266C constitutively activated receptor. This study represents the first experimental demonstration of an interaction between specific residues in an active state, but not the resting state, of Ste2p. The information gained from this study should contribute to an understanding of the conformational differences between resting and active states in GPCRs.     

Analysis of Trafficking,Stability and Function of Human Connexin 26 Gap Junction Channels with Deafness-Causing Mutations in the Fourth Transmembrane Helix

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.     

Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.     

Clustered charged-to-alanine mutagenesis of poliovirus RNA-dependent RNA polymerase yields multiple temperature-sensitive mutants defective in RNA synthesis.

To generate a collection of conditionally defective poliovirus mutants, clustered charged-to-alanine mutagenesis of the RNA-dependent RNA polymerase 3D was performed. Clusters of charged residues in the polymerase coding region were replaced with alanines by deoxyoligonucleotide-directed mutagenesis of a full-length poliovirus cDNA clone. Following transfection of 27 mutagenized cDNA clones, 10 (37%) gave rise to viruses with temperature-sensitive (ts) phenotypes. Three of the ts mutants displayed severe ts plaque reduction phenotypes, producing at least 10(3)-fold fewer plaques at 39.5 degrees C than at 32.5 degrees C; the other seven mutants displayed ts small-plaque phenotypes. Constant-temperature, single-cycle infections showed defects in virus yield or RNA accumulation at the nonpermissive temperature for eight stable ts mutants. In temperature shift experiments, seven of the ts mutants showed reduced accumulation of viral RNA at the nonpermissive temperature and showed no other ts defects. The mutations responsible for the phenotypes of most of these ts mutants lie in the N-terminal third of the 3D coding region, where no well-characterized mutations responsible for viable mutants had been previously identified. Clustered charged-to-alanine mutagenesis (S. H. Bass, M. G. Mulkerrin, and J. A. Wells, Proc. Natl. Acad. Sci. USA 88:4498-4502, 1991; W. F. Bennett, N. F. Paoni, B. A. Keyt, D. Botstein, J. J. S. Jones, L. Presta, F. M. Wurm, and M. J. Zoller, J. Biol. Chem. 266:5191-5201, 1991; and K. F. Wertman, D. G. Drubin, and D. Botstein, Genetics 132:337-350, 1992) is designed to target residues on the surfaces of folded proteins; thus, extragenic suppression analysis of such mutant viruses may be very useful in identifying components of the viral replication complex.     

Functional consequences of mutations in the conserved 'signature sequence' of the ATP-binding-cassette protein MalK.

The binding-protein-dependent maltose-transport system of enterobacteria, a member of the ATP-binding-cassette (ABC) transporter superfamily, is composed of two integral membrane proteins, MalF and MalG, and two copies of an ATPase subunit, MalK, which hydrolyze ATP, thus energizing the translocation process. Isolated MalK displays spontaneous ATPase activity, whereas in the assembled MalFGK2 complex, reconstituted in liposomes, ATP hydrolysis requires stimulation by the substrate-loaded extracellular maltose-binding protein, MalE. The ATPase domains of ABC transporters, including MalK, share a unique sequence motif ('LSGGQ', 'signature sequence' or 'linker peptide') with as yet unknown function. To elucidate its role in the transport process, we investigated the consequences of mutations affecting two highly conserved residues (G137, Q140) in the MalK-ATPase of Salmonella typhimurium, by biochemical means. Residues corresponding to Q140 in other ABC proteins have not yet been studied. All mutant alleles (G137--> A, V, T; Q140--> L, K, N) fail to restore a functional transport complex in vivo. In addition, the mutations increase the repressing activity of MalK on other maltose-regulated genes when compared with wild-type MalK. Purified variants of G137 have lost the ability to hydrolyze ATP but still display nucleotide-binding activity, albeit with reduced affinity. Binding of MgATP results in similar protection against trypsin, as observed with wild-type, indicating no major change in protein structure. In contrast, the variants of Q140 differ in their properties, depending on the chemical nature of the replacement residue. MalKQ140L fails to hydrolyze ATP and exhibits a strong intrinsic resistance to trypsin in the absence of MgATP, suggesting a drastically altered conformation. In contrast, the purified mutant proteins Q140K and Q140N display ATPase activities and MgATP-induced changes in the tryptic cleavage pattern similar to those of wild-type. However, mutant transport complexes containing the Q140K or Q140N variants, when studied in proteoliposomes, are severely impaired in MalE-maltose-stimulated ATPase activity. These results are discussed with respect to the crystal structure of the homologous HisP protein [Hung, L.-W., Wang, I.X., Nikaido, K., Liu, P.-Q., Ames, G.F.-L. & Kim, S.-H. (1998) Nature (London) 396, 703-707] and are interpreted in favor of a role of the signature sequence in activating the hydrolyzing activity of MalK upon substrate-initiated conformational changes in MalF/MalG.     

The proton pore in the Escherichia coli F0F1-ATPase: a requirement for arginine at position 210 of the a-subunit

Site-directed mutagenesis was used to generate three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli. These mutations directed the substitution of Arg-210 by Gln, or of His-245 by Leu, or of both Lys-167 and Lys-169 by Gln. The mutations were incorporated into plasmids carrying all the structural genes encoding the F0F1-ATPase complex and these plasmids were used to transform strain AN727 (uncB402). Strains carrying either the Arg-210 or His-245 substitutions were unable to grow on succinate as sole carbon source and had uncoupled growth yields. The substitution of Lys-167 and Lys-169 by Gln resulted in a strain with growth characteristics indistinguishable from a normal strain. The properties of the membranes from the Arg-210 or His-245 mutants were essentially identical, both being proton impermeable and both having ATPase activities resistant to the inhibitor DCCD. Furthermore, in both mutants, the F1-ATPase activities were inhibited by about 50% when bound to the membranes. The membrane activities of the mutant with the double lysine change were the same as for a normal strain. The results are discussed in relation to a previously proposed model for the F0 (Cox, G.B., Fimmel, A.L., Gibson, F. and Hatch, L. (1986) Biochim. Biophys. Acta 849, 62-69).     

Insights into oncogenic mutations of plexin-B1 based on the solution structure of the Rho GTPase binding domain

The plexin family of transmembrane receptors are important for axon guidance, angiogenesis, but also in cancer. Recently, plexin-B1 somatic missense mutations were found in both primary tumors and metastases of breast and prostate cancers, with several mutations mapping to the Rho GTPase binding domain (RBD) in the cytoplasmic region of the receptor. Here we present the NMR solution structure of this domain, confirming that the protein has both a ubiquitin-like fold and surface features. Oncogenic mutations T1795A and T1802A are located in a loop region, perturb the average structure locally, and have no effect on Rho GTPase binding affinity. Mutations L1815F and L1815P are located at the Rho GTPase binding site and are associated with a complete loss of binding for Rac1 and Rnd1. Both are found to disturb the conformation of the beta3-beta4 sheet and the orientation of surrounding side chains. Our study suggests that the oncogenic behavior of the mutants can be rationalized with reference to the structure of the RBD of plexin-B1.     

Mutant residues suppressing rho(0)-lethality in Kluyveromyces lactis occur at contact sites between subunits of F(1)-ATPase

Characterisation of 35 Kluyveromyces lactis strains lacking mitochondrial DNA has shown that mutations suppressing rho(0)-lethality are limited to the ATP1, 2 and 3 genes coding for the alpha-, beta- and gamma- subunits of mitochondrial F(1)-ATPase. All atp mutations reduce growth on glucose and three alleles, atp1-2, 1-3 and atp3-1, produce a respiratory deficient phenotype that indicates a drop in efficiency of the F(1)F(0)-ATP synthase complex. ATPase activity is needed for suppression as a double mutant containing an atp allele, together with a mutation abolishing catalytic activity, does not suppress rho(0)-lethality. Positioning of the seven amino acids subject to mutation on the bovine F(1)-ATPase structure shows that two residues are found in a membrane proximal region while five amino acids occur at a region suggested to be a molecular bearing. The intriguing juxtaposition of mutable amino acids to other residues subject to change suggests that mutations affect subunit interactions and alter the properties of F(1) in a manner yet to be determined. An explanation for suppressor activity of atp mutations is discussed in the context of a possible role for F(1)-ATPase in the maintenance of mitochondrial inner membrane potential.     

N-terminal and core-domain random mutations in human topoisomerase II α conferring bisdioxopiperazine resistance

Random mutagenesis of human topoisomerase II α cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer >37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

The region from phenylalanine-17 to phenylalanine-28 of a yeast mitochondrial ATPase inhibitor is essential for its ATPase inhibitory activity.

Mitochondrial ATP synthase (F(1)F(o)-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, we investigated the structure-function relationship of the yeast ATPase inhibitor by amino acid replacement. A total of 22 mutants were isolated and characterized. Five mutants (F17S, R20G, R22G, E25A, and F28S) were entirely inactive, indicating that the residues, Phe17, Arg20, Arg22, Glu25, and Phe28, are essential for the ATPase inhibitory activity of the protein. The activity of 7 mutants (A23G, R30G, R32G, Q36G, L37G, L40S, and L44G) decreased, indicating that the residues, Ala23, Arg30, Arg32, Gln36, Leu37, Leu40, and Leu44, are also involved in the activity. Three mutants, V29G, K34Q, and K41Q, retained normal activity at pH 6.5, but were less active at pH 7.2, indicating that the residues, Val29, Lys34, and Lys41, are required for the protein's action at higher pH. The effects of 6 mutants (D26A, E35V, H39N, H39R, K46Q, and K49Q) were slight or undetectable, and the residues Asp26, Glu35, His39, Lys46, and Lys49 thus appear to be dispensable. The mutant E21A retained normal ATPase inhibitory activity but lacked pH-sensitivity. Competition experiments suggested that the 5 inactivated mutants (F17S, R20G, R22G, E25A, and F28S) could still bind to the inhibitory site on F(1)F(o)-ATPase. These results show that the region from the position 17 to 28 of the yeast inhibitor is the most important for its activity and is required for the inhibition of F(1), rather than binding to the enzyme.     

Further examination of seventeen mutations in Escherichia coli F1-ATPase beta-subunit.

Seventeen mutations in beta-subunit of Escherichia coli F1-ATPase which had previously been characterized in strain AN1272 (Mu-induced mutant) were expressed in strain JP17 (beta-subunit gene deletion). Six showed unchanged behavior, namely: C137Y; G142D; G146S; G207D; Y297F; and Y354F. Five failed to assemble F1F0 correctly, namely: G149I; G154I; G149I,G154I; G223D; and P403S,G415D. Six assembled F1F0 correctly, but with membrane ATPase lower than in AN1272, namely: K155Q; K155E; E181Q; E192Q; D242N; and D242V. AN1272 was shown to unexpectedly produce a small amount of wild-type beta-subunit; F1-ATPase activities reported previously in AN1272 were referable to hybrid enzymes containing both mutant and wild-type beta-subunits. Purified F1 was obtained from K155Q; K155E; E181Q; E192Q; and D242N mutants in JP17. Vmax ATPase values were lower, and unisite catalysis rate and equilibrium constants were perturbed to greater extent, than in AN1272. However, general patterns of perturbation revealed by difference energy diagrams were similar to those seen previously, and the new data correlated well in linear free energy relationships for reaction steps of unisite catalysis. Correlation between multisite and unisite ATPase activity was seen in the new enzymes. Overall, the data give strong support to previously proposed mechanisms of unisite catalysis, steady-state catalysis, and energy coupling in F1-ATPases (Al-Shawi, M. K., Parsonage, D. and Senior, A. E. (1990) J. Biol. Chem. 265, 4402-4410). The K155Q, K155E, D242N, and E181Q mutations caused 5000-fold, 4000-fold, 1800-fold, and 700-fold decrease, respectively, in Vmax ATPase, implying possibly direct roles for these residues in catalysis. Experiments with the D242N mutant suggested a role for residue beta D242 in catalytic site Mg2+ binding.