In vivo analyses of interactions between SecE and SecY, core components of the Escherichia coli protein translocation machinery

We have carried out structure-function studies on the cytoplasmic membrane protein, SecE, a component of the Escherichia coli secretion machinery. SecE, along with SecY, form a complex in the cytoplasmic membrane essential for protein translocation. By directed mutagenesis, we altered highly conserved residues of the second cytoplasmic domain (CD2) and of the COOH-terminal periplasmic region (PD2) of SecE. These mutants, as well as previously constructed mutations in the third membrane-spanning segment of SecE (MSS3), were tested for their ability to complement a secE null mutation, for their effects on protein export in vivo, and for their ability to form a stable complex with SecY. Most single mutations at the conserved positions in CD2 caused secretion defects, but had little effect on growth at 37 degrees C. Double mutations in CD2, or the introduction or removal of proline residues, affected growth and protein translocation more severely. Co-immunoprecipitations of SecE and SecY revealed that all mutant proteins, except those altered in PD2, destabilized the SecE-SecY complex. These results suggest that several regions contribute to the formation of a stable SecE-SecY complex but the elimination of a single contact point does not necessarily affect the functionality of the complex.     

The role of beta-sheet interactions in domain stability, folding, and target recognition reactions of calmodulin

Single-residue mutations have been made of the hydrophobic Ile or Val residue in position 8 of each of the four calcium-binding loop sequences (sites I-IV) of Drosophila calmodulin. These highly conserved residues are part of the hydrophobic core of either calmodulin domain and are involved in the structural link of two calcium-binding sites via a short antiparallel beta-sheet. In the apo-form, the replacement of Ile (or Val) by Gly causes a significant destabilization, shown by the unfolding of the secondary structure of the domain carrying the mutation. In the presence of calcium, the deficiency in alpha-helical structure at 20 degrees C is restored for the mutants at site I, II, or III but not at site IV, which requires the further binding of a high-affinity target peptide to re-establish the native conformation. The extent of the destabilization is seen in the depression of the melting temperature of individual domains, which can be as large as 80 degrees C in the case of Ca4-CaM(V136G). However, because of low values of the unfolding enthalpy for calmodulin domains, only relatively low values of <2 kcal/mol are implied for DeltaDeltaG, the free energy of destabilization due to mutation. Consistent with this, the secondary structure of any unfolded mutant domain is highly sensitive to solvent composition and is largely refolded in the presence of 12.5% (v/v) aqueous trifluoroethanol. Compared to wild-type calmodulin, the affinities of the mutants for calcium and target peptides from sk-MLCK at 20 degrees C are significantly reduced but the effects are relatively small. These results indicate that the conformation of calmodulin can be dramatically altered by mutation of a single highly conserved residue but that changes in solvent or the binding of a target sequence can readily compensate for this, restoring the wild-type properties. The results also suggest that the integrity of both the apo- and holo-forms of calmodulin is important for the maintenance of its biological function and confirm the importance of conserving the structural function of the residues involved in the beta-sheet interactions.     

Formation of the myosin.ADP.gallium fluoride complex and its solution structure by small-angle synchrotron X-ray scattering

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190-7194, 1990). To identify elements of the UmuD' protein necessary for its role in translesion synthesis, we began with umuD', a modified form of the umuD gene that directly encodes the UmuD' protein, and obtained missense umuD' mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD'2 homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded beta-sheet and appears to interfere with UmuD'2 homodimer formation indirectly by affecting the structure of the UmuD' monomer. When expressed from a multicopy plasmid, the L40R umuD' mutant gene exhibited a dominant negative effect on a chromosomal umuD+ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD' function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal beta-strand and results in a mutant UmuD' protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD+ strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD'2 homodimer, which are mobile in solution.     

Probing the folate-binding site of human thymidylate synthase by site-directed mutagenesis. Generation of mutants that confer resistance to raltitrexed, Thymitaq, and BW1843U89

Human thymidylate synthase (TS) contains three highly conserved residues Ile-108, Leu-221, and Phe-225 that have been suggested to be important for cofactor and antifolate binding. To elucidate the role of these residues and generate drug-resistant human TS mutants, 14 variants with multiple substitutions of these three hydrophobic residues were created by site-directed mutagenesis and transfected into mouse TS-negative cells for complementation assays and cytotoxicity studies, and the mutant proteins expressed and characterized. The I108A mutant confers resistance to raltitrexed and Thymitaq with respective IC50 values 54- and 80-fold greater than wild-type but less resistance to BW1843U89 (6-fold). The F225W mutant displays resistance to BW1843U89 (17-fold increase in IC50 values), but no resistance to raltitrexed and Thymitaq. It also confers 8-fold resistance to fluorodeoxyuridine. Both the kinetic characterization of the altered enzymes and formation of antifolate-resistant colonies in mouse bone marrow cells that express mutant TS are in accord with the IC50 values for cytotoxicity noted above. The human TS mutants (I108A and F225W), by virtue of their desirable properties, including good catalytic function and resistance to antifolate TS inhibitors, confirm the importance of amino acid residues Ile-108 and Phe-225 in the binding of folate and its analogues. These novel mutants may be useful for gene transfer experiments to protect hematopoietic progenitor cells from the toxic effects of these drugs.     

Probable mechanisms underlying interallelic complementation and temperature-sensitivity of mutations at the shibire locus of Drosophila melanogaster

The shibire locus of Drosophila melanogaster encodes dynamin, a GTPase required for the fission of endocytic vesicles from plasma membrane. Biochemical studies indicate that mammalian dynamin is part of a complex containing multiple dynamin subunits and other polypeptides. To gain insight into sequences of dynamin critical for its function, we have characterized in detail a collection of conditional and lethal shi alleles. We describe a probable null allele of shi and show that its properties are distinct from those of two classes of lethal alleles (termed I and II) that show intergroup, interallelic complementation. Sequenced class I alleles, which display dominant properties, carry missense mutations in conserved residues in the GTPase domain of dynamin. In contrast, the sequenced class II alleles, which appear completely recessive, carry missense mutations in conserved residues of a previously uncharacterized "middle domain" that lies adjacent to the GTPase region. These data suggest that critical interactions mediated by this middle domain are severely affected by the class II lethal mutations; thus, the mutant sequences should be very useful for confirming the in vivo relevance of interactions observed in vitro. Viable heteroallelic combinations of shi lethals show rapid and reversible temperature-sensitive paralytic phenotypes hitherto only described for the ts alleles of shi. When taken together with the molecular analysis of shi mutations, these observations suggest that the GTPase domain of dynamin carries an intrinsically temperature-sensitive activity: hypomorphic mutations that reduce this activity at low temperatures result in conditional temperature-sensitive phenotype. These observations explain why screens for conditional paralytic mutants in Drosophila inevitably recover ts alleles of shi at high frequencies.     

Helicase motifs V and VI of the Escherichia coli UvrB protein of the UvrABC endonuclease are essential for the formation of the preincision complex

The UvrB protein is a subunit of the UvrABC endonuclease which is involved in the repair of a large variety of DNA lesions. We have 91 isolated random uvrB mutants which are impaired in the repair of UV-damage in vivo. These mutants were classified on the basis of the ability to form normal levels of protein and the position of the mutations in the gene. The amino acid substitutions in the N-terminal part or in the C-terminal part of the UvrB protein are exclusively found in the conserved boxes of the so-called "helicase motifs" present in these parts of the protein, indicating that these motifs are essential for UvrB function. The proteins of four C-terminal mutants were purified: two mutants in motif V (E514K and G509S), one mutant in motif VI (R544H) and a double mutant in both motifs (E514K + R541H). In vitro experiments with these mutant proteins show that the helicase motifs V and VI are involved in the induction of ATP hydrolysis in the presence of (damaged) DNA and in the strand-displacement activity of the UvrA2B complex as is observed in a helicase assay. Furthermore, our results suggest that this strand-displacement activity is correlated to a local unwinding, which seems to be used to form the UvrB-DNA preincision complex.     

Roles of the Escherichia coli small heat shock proteins IbpA and IbpB in thermal stress management: comparison with ClpA, ClpB, and HtpG In vivo

We have constructed an Escherichia coli strain lacking the small heat shock proteins IbpA and IbpB and compared its growth and viability at high temperatures to those of isogenic cells containing null mutations in the clpA, clpB, or htpG gene. All mutants exhibited growth defects at 46 degrees C, but not at lower temperatures. However, the clpA, htpG, and ibp null mutations did not reduce cell viability at 50 degrees C. When cultures were allowed to recover from transient exposure to 50 degrees C, all mutations except Deltaibp led to suboptimal growth as the recovery temperature was raised. Deletion of the heat shock genes clpB and htpG resulted in growth defects at 42 degrees C when combined with the dnaK756 or groES30 alleles, while the Deltaibp mutation had a detrimental effect only on the growth of dnaK756 mutants. Neither the overexpression of these heat shock proteins nor that of ClpA could restore the growth of dnaK756 or groES30 cells at high temperatures. Whereas increased levels of host protein aggregation were observed in dnaK756 and groES30 mutants at 46 degreesC compared to wild-type cells, none of the null mutations had a similar effect. These results show that the highly conserved E. coli small heat shock proteins are dispensable and that their deletion results in only modest effects on growth and viability at high temperatures. Our data also suggest that ClpB, HtpG, and IbpA and -B cooperate with the major E. coli chaperone systems in vivo.     

Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA

RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.     

Isolation of a non-classical mutant of the DNA recognition subunit of the type I restriction endonuclease R.EcoR124I

We have used deletion mutagenesis and PCR-based misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I. It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease. While a large percentage of these mutations gave a classical Res- Mod- phenotype, one mutant was isolated with a nonclassical Res- Mod+ phenotype. The loss of restriction activity, but retention of the ability to modify indicates that this mutation cannot affect DNA binding and must alter the assembly of the endonuclease in such a way as to prevent DNA cleavage but allow methylation. This mutant resulted from a single amino acid change Trp212-->Arg. The location of the single amino acid change is at the border of the central conserved region and the second target recognition domain (TRD2) and suggests that this region is extremely important for the assembly of the methylase with the HsdR subunit into a functional restriction endonuclease.     

Analysis of the pathogenic human mitochondrial mutation ND1/3460, and mutations of strictly conserved residues in its vicinity, using the bacterium Paracoccus denitrificans

The human mitochondrial ND1/3460 mutation changes Ala52 to Thr in the ND1 subunit of Complex I, and causes Leber's hereditary optic neuropathy (LHON) [Huoponen et al. (1991) Am. J. Hum. Genet. 48, 1147]. We have used a bacterial counterpart of Complex I, NDH-1 from Paracoccus denitrificans, for studying the effect of mutations in the ND1 subunit on the enzymatic activity. The LHON mutation as well as several other mutations in strictly conserved amino acids in its vicinity were introduced into the NQO8 subunit of NDH-1, a bacterial homologue of ND1. The enzymatic activity of the mutants in the presence of hexammineruthenium (rotenone-insensitive) and ubiquinone-1 (rotenone-sensitive) were assayed. In addition, the kinetics of the interaction of selected mutant enzymes with ubiquinone-1, ubiquinone-2, and decylubiquinone was studied. The results suggest that the mutated residues play an important role in ubiquinone reduction by Complex I.     

Site-directed mutagenesis of the yeast PRP20/SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1, 10-phenanthroline)2SO4 at 0 degrees, 10 degrees, or 20 degreesC, strong a-c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a-c dimer formation was observed in nine other double mutants after treatment at 20 degreesC in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.     

The enzymological basis for resistance of herpesvirus DNA polymerase mutants to acyclovir: relationship to the structure of alpha-like DNA polymerases

Acyclovir (ACV), like many antiviral drugs, is a nucleoside analog. In vitro, ACV triphosphate inhibits herpesvirus DNA polymerase by means of binding, incorporation into primer/template, and dead-end complex formation in the presence of the next deoxynucleoside triphosphate. However, it is not known whether this mechanism operates in vivo. To address this and other questions, we analyzed eight mutant polymerases encoded by drug-resistant viruses, each altered in a region conserved among alpha-like DNA polymerases. We measured Km and kcat values for dGTP and ACV triphosphate incorporation and Ki values of ACV triphosphate for dGTP incorporation for each mutant. Certain mutants showed increased Km values for ACV triphosphate incorporation, suggesting a defect in inhibitor binding. Other mutants showed reduced kcat values for ACV triphosphate incorporation, suggesting a defect in incorporation of inhibitor into DNA, while the rest of the mutants exhibited both altered km and kcat values. In most cases, the fold increase in Ki of ACV triphosphate for dGTP incorporation relative to wild-type polymerase was similar to fold resistance conferred by the mutation in vivo; however, one mutation conferred a much greater increase in resistance than in Ki. The effects of mutations on enzyme kinetics could be explained by using a model of an alpha-like DNA polymerase active site bound to primer/template and inhibitor. The results have implications for mechanisms of action and resistance of antiviral nucleoside analogs in vivo, in particular for the importance of incorporation into DNA and for the functional roles of conserved regions of polymerases.     

Therapy with monoclonal antibodies. II. The contribution of Fc gamma receptor binding and the influence of C(H)1 and C(H)3 domains on in vivo effector function

An in vivo model is used to define Fc motifs engaged by mAbs to deplete target cells. Human IgG1 and human IgG4 were very potent, and mutations within a motif critical for Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine 234 to valine, and leucine 235 to alanine) completely prevented depletion. Mouse IgG2b was also potent, and mutations to prevent complement activation did not impair depletion with this isotype, as previously shown for human IgG1. In contrast, a mutation that impaired binding to mouse Fc gammaRII (glutamate 318 to alanine) eliminated effector function of mouse IgG2b and also reduced the potency of human IgG4. To reveal potential contributions of domains other than C(H)2, domain switch mutants were created between human IgG1 and rat IgG2a. Two hybrid mAbs were generated with potencies exceeding anything previously seen in this model. While their mechanism of depletion was not defined, their activity appeared dependent upon interdomain interactions in the Fc region.     

Glycogen storage disease type II: identification of four novel missense mutations (D645N, G648S, R672W, R672Q) and two insertions/deletions in the acid alpha-glucosidase locus of patients of differing phenotype

Glycogen storage disease type II (GSDII), an autosomal recessive myopathic disorder, results from deficiency of lysosomal acid alpha-glucosidase. We searched for mutations in an evolutionarily conserved region in 54 patients of differing phenotype. Four novel mutations (D645N, G448S, R672W, and R672Q) and a previously described mutation (C647W) were identified in five patients and their deleterious effect on enzyme expression demonstrated in vitro. Two novel frame-shifting insertions/deletions (delta nt766-785/insC and +insG@nt2243) were identified in two patients with exon 14 mutations. The remaining three patients were either homozygous for their mutations (D645N/D645 and C647W/C647W) or carried a previously described leaky splice site mutation (IVS1-13T-->G). For all patients "in vivo" enzyme activity was consistent with clinical phenotype. Agreement of genotype with phenotype and in vitro versus in vivo enzyme was seen in three patients (two infantile patients carrying C647W/C647W and D645N/+insG@nt2243 and an adult patient heteroallelic for G648S/IVS1-13T-->G). Relative discordance was found in a juvenile patient homozygous for the non-expressing R672Q and an adult patient heterozygous for the minimally expressing R672W and delta nt766-785/+insC. Possible explanations include differences in in vitro assays vs in vivo enzyme activity, tissue specific expression with diminished enzyme expression/stability in fibroblasts vs muscle, somatic mosaicism, and modifying genes.     

Essential functions and actin-binding surfaces of yeast cofilin revealed by systematic mutagenesis

Cofilin stimulates actin filament turnover in vivo. The phenotypes of twenty yeast cofilin mutants generated by systematic mutagenesis were determined. Ten grew as well as the wild type and showed no cytoskeleton defects, seven were recessive-lethal and three were conditional-lethal and caused severe actin organization defects. Biochemical characterization of interactions between nine mutant yeast cofilins and yeast actin provided evidence that F-actin binding and depolymerization are essential cofilin functions. Locating the mutated residues on the yeast cofilin molecular structure allowed several important conclusions to be drawn. First, residues required for actin monomer binding are proximal to each other. Secondly, additional residues are required for interactions with actin filaments; these residues might bind an adjacent subunit in the actin filament. Thirdly, despite striking structural similarity, cofilin interacts with actin in a different manner from gelsolin segment-1. Fourthly, a previously unrecognized cofilin function or interaction is suggested by identification of spatially proximal residues important for cofilin function in vivo, but not for actin interactions in vitro. Finally, mutation of the cofilin N-terminus suggests that its sequence is conserved because of its critical role in actin interactions, not because it is sometimes a target for protein kinases.