Two conserved threonines collaborate in the Escherichia coli leucyl-tRNA synthetase amino acid editing mechanism

The aminoacyl-tRNA synthetases covalently link transfer RNAs to their cognate amino acids. Some of the tRNA synthetases have employed an editing mechanism to ensure fidelity in this first step of protein synthesis. The amino acid editing active site for Escherichia coli leucyl-tRNA synthetase resides within the CP1 domain that folds discretely from the main body of the enzyme. A portion of the editing active site is lined with conserved threonines. Previously, we identified one of these threonine residues (Thr(252)) as a critical amino acid specificity factor. On the basis of X-ray crystal structure information, two other nearby threonine residues (Thr(247) and Thr(248)) were hypothesized to interact with the editing substrate near its cleavage site. Single mutations of either of these conserved threonine residues had minimal effects on amino acid editing. However, double mutations that deleted the hydroxyl group from the neighboring threonine residues abolished amino acid editing activity. We propose that these threonine residues, which are also conserved in the homologous isoleucyl-tRNA synthetase and valyl-tRNA synthetase editing active sites, play a central role in amino acid editing. It is possible that they collaborate in stabilizing the transition state.     

The C-terminal domain of Escherichia coli MutY is involved in DNA binding and glycosylase activities

Escherichia coli MutY is an adenine and a weak guanine DNA glycosylase involved in reducing mutagenic effects of 7,8-dihydro-8-oxo-guanine (8-oxoG). The C-terminal domain of MutY is required for 8-oxoG recognition and is critical for mutation avoidance of oxidative damage. To determine which residues of this domain are involved in 8-oxoG recognition, we constructed four MutY mutants based on similarities to MutT, which hydrolyzes specifically 8-oxo-dGTP to 8-oxo-dGMP. F294A-MutY has a slightly reduced binding affinity to A/G mismatch but has a severe defect in A/8-oxoG binding at 20°C. The catalytic activity of F294A-MutY is much weaker than that of the wild-type MutY. The DNA binding activity of R249A-MutY is comparable to that of the wild-type enzyme but the catalytic activity is reduced with both A/G and A/8-oxoG mismatches. The biochemical activities of F261A-MutY are nearly similar to those of the wild-type enzyme. The solubility of P262A-MutY was improved as a fusion protein containing streptococcal protein G (GB1 domain) at its N-terminus. The binding of GB1-P262A-MutY with both A/G and A/8-oxoG mismatches are slightly weaker than those of the wild-type protein. The catalytic activity of GB1-P262A-MutY is weaker than that of the wild-type enzyme at lower enzyme concentrations. Importantly, all four mutants can complement mutY mutants in vivo when expressed at high levels; however, F294A, R249A and P262A, but not F261A, are partially defective in vivo when they are expressed at low levels. These results strongly support that the C-terminal domain of MutY is involved not only in 8-oxoG recognition, but also affects the binding and catalytic activities toward A/G mismatches.     

Influences of amino acid, ATP, pyrophosphate and tRNA on binding of aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600.

Aminoalcohol-AMP esters, structurally related to the assumed intermediates of the amino acid activation reaction, behave as competitive inhibitors both with respect to the amino acid and ATP, when tested in the ATP-(32P) PPi-exchange or the tRNA-charging reaction. However, closer investigation of the binding of norvalinyl adenylate to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 by an equilibrium method shows that only the amino acid is a true competitor, while ATP cannot displace the ester from binding. Pyrophosphate enhances the stability of the ester-enzyme complex whereas tRNA is without detectable influence.     

Involvement of a conserved GFG motif region in substrate binding by RseP,an Escherichia coli S2P protease

RseP, an Escherichia coli S2P family intramembrane cleaving protease, is involved in regulation of the extracytoplasmic stress response and membrane quality control through specific cleavage of substrates. Recent research suggested that the PDZ domains and the MRE β‐loop (m embrane‐r ee ntrant β‐loop) are involved in substrate discrimination; the former would serve to prevent cleavage of substrates with a large periplasmic domain, whereas the latter would directly interact with the substrate's transmembrane segment and induce its conformational change. However, the mechanisms underlying specific substrate recognition and cleavage by RseP are not fully understood. Here, the roles of the N‐terminal part of the first cytoplasmic loop region (C1N) of RseP that contains a highly conserved GFG motif were investigated. A Cys modifiability assay suggested that C1N is partly membrane‐inserted like the MRE β‐loop. Pro, but not Cys, substitutions in the GFG motif region compromised the proteolytic function of RseP, suggesting the importance of a higher order structure of this motif region. Several lines of evidence indicated that the GFG motif region directly interacts with the substrate and also aids the function of the MRE β‐loop that participates in substrate recognition by RseP. These findings provide insights into the substrate recognition mechanisms of S2P proteases.     

The A-loop, a novel conserved aromatic acid subdomain upstream of the Walker A motif in ABC transporters, is critical for ATP binding

ATP-binding cassette (ABC) transporters represent one of the largest families of proteins, and transport a variety of substrates ranging from ions to amphipathic anticancer drugs. The functional unit of an ABC transporter is comprised of two transmembrane domains and two cytoplasmic ABC ATPase domains. The energy of the binding and hydrolysis of ATP is used to transport the substrates across membranes. An ABC domain consists of conserved regions, the Walker A and B motifs, the signature (or C) region and the D, H and Q loops. We recently described the A-loop (Aromatic residue interacting with the Adenine ring of ATP), a highly conserved aromatic residue approximately 25 amino acids upstream of the Walker A motif that is essential for ATP-binding. Here, we review the mutational analysis of this subdomain in human P-glycoprotein as well as homology modeling, structural and data mining studies that provide evidence for a functional role of the A-loop in ATP-binding in most members of the superfamily of ABC transporters.     

Tyr-48, a conserved residue in ribotoxins, is involved in the RNA-degrading activity of alpha-sarcin

Residue Tyr-48 in alpha-sarcin is conserved not only within the ribotoxin family, but also within the larger group of extracellular fungal ribonucleases, best represented by RNase T1. A mutant protein in which this Tyr residue was substituted by Phe has been produced and isolated to homogeneity. It was spectroscopically analyzed by means of circular dichroism, fluorescence emission and NMR. Taken together, these results and those from enzyme characterization have revealed the essential role of the -OH group from the Tyr-48 phenolic ring in the cleavage of polymeric RNA substrates, including the ribosome-embedded 28S rRNA, the natural substrate of ribotoxins. Thus, the mutant protein does not degrade its natural ribosomal RNA substrate. However, it has been shown that this Y48F mutant still retains its ability to cleave a phosphodiester bond in a minimal substrate such as the dinucleoside phosphate ApA. The role of different alpha-sarcin residues within the enzyme reaction catalyzed by this protein is discussed.     

Identification of residues involved in the binding of methionine by Escherichia coli methionyl-tRNA synthetase

Comparison of the amino-acid sequences of several methionyl-tRNA synthetases indicates the occurrence of a few conserved motifs, having a possible functional significance. The role of one of these motifs, centered at position 300 in the E. coli enzyme sequence, was assayed by the use of site-directed mutagenesis. Substitution of the His301 or Trp305 residues by Ala resulted in a large decrease in methionine affinity, whereas the change of Val298 into Ala had only a moderate effect. The catalytic rate of the enzyme was unimpaired by these substitutions. It is concluded that the above conserved amino-acid region is located at or close to the amino-acid binding pocket of methionyl-tRNA synthetase.     

Structure of Escherichia coli tetrahydrodipicolinate N-succinyltransferase reveals the role of a conserved C-terminal helix in cooperative substrate binding

Tetrahydrodipicolinate N-succinyltransferase is an enzyme present in many bacteria that catalyzes the first step of the succinylase pathway for the synthesis of meso-diaminopimelate and the amino acid L-lysine. Inhibition of the synthesis of meso-diaminopimelate, a component of peptidoglycan present in the cell wall of bacteria, is a potential route for the development of novel anti-bacterial agents. Here, we report the crystal structure of the DapD tetrahydrodipicolinate N-succinyltransferase from Escherichia coli at 2.0 A resolution. Comparison of the structure with the homologous enzyme from Mycobacterium bovis reveals the C-terminal helix undergoes a large rearrangement upon substrate binding, which contributes to cooperativity in substrate binding.     

Conformational movements and cooperativity upon amino acid,ATP and tRNA binding in threonyl-tRNA synthetase

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus, with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8A at some C(alpha) positions, of a strand-loop-strand region of the core beta-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.     

The strongly conserved carboxyl-terminus glycine-methionine motif of the Escherichia coli GroEL chaperonin is dispensable

The universally distributed heat-shock proteins (HSPs) are divided into classes based on molecular weight and sequence conservation. The members of at least two of these classes, the HSP60s and the HSP70S, have chaperone activity. Most HSP60s and many HSP70s feature a striking motif at or near the carboxyl terminus which consists of a string of repeated glycine and methionine residues. We have altered the groEL gene (encoding the essential Escherichia coli HSP60 chaperonin) so that the protein produced lacks its 16 final (including nine gly, and five met) residues. This truncated product behaves like the intact protein in several in vitro tests, the only discernible difference between the two proteins being in the rate at which ATP is hydrolysed. GroEL_tr can substitute for GroEL in vivo although cells dependent for survival on the truncated protein survive slightly less well during the stationary phase of growth. Elevated levels of the wild-type protein can suppress a number of temperature-sensitive mutations; the truncated protein lacks this ability.     

Characterization of a conserved alpha-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli

FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short alpha helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli sigma(32) and the bacteriophage lambda CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the alpha helical region is an important structural element of FtsH (HflB).     

A conserved motif in S-layer proteins is involved in peptidoglycan binding in Thermus thermophilus.

There is experimental evidence to suggest that the 100-kDa S-layer protein from Thermus thermophilus HB8 binds to the peptidoglycan cell wall. This property could be related to the presence of a region (SLH) of homology with other S-layer proteins and extracellular enzymes (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). By using specific monoclonal antibodies, we show that similar regions are present in different members of the Deinococcus-Thermus phylogenetic group. To analyze the role that the SLH domain plays in vivo and in vitro in T. thermophilus, we have obtained a mutant form (slpA.X) of the S-layer gene (slpA) in which the SLH domain was deleted. The slpA.X gene was inserted into the chromosome of the thermophile by gene replacement, resulting in a mutant which expressed a major membrane protein with the size expected from the construction (90 kDa). This protein was identified as the product of slpA.X by its differential reaction with monoclonal antibodies. Mutants expressing the SlpA.X protein grow as groups of cells, surrounded by a common external envelope of trigonal symmetry that contains the SlpA.X protein as a main component, thus showing the inability of the SLH-defective protein to attach to the underlying material in vivo. In addition, averaged images of SlpA.X-rich fractions showed a regular arrangement, identical to that built up by the wild-type (SlpA) protein in the absence of peptidoglycan. Finally, we demonstrate by Western blotting (immunoblotting) the direct role of the SLH domain in the binding of the S-layer of T. thermophilus HB8 to the peptidoglycan layer.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

Effect of alanine-293 replacement on the activity, ATP binding, and editing of Escherichia coli leucyl-tRNA synthetase

Leucyl-tRNA synthetase (LeuRS) is a class I aminoacyl-tRNA synthetase that catalyzes leucylation of tRNA(Leu). Several mutants in the CP1 domain of Escherichia coli LeuRS were obtained by introduction of restriction endonuclease sites into its gene, leuS. Of these mutants, only LeuRS-A293F had decreased activity (46%) compared to the native enzyme. To investigate the effect of A293 on enzyme function, A293 was mutated to Y, G, I, R, or D. The mutants were impaired in activity and editing function to varying extents. The decrease in K(m) values for three substrates showed that the binding of ATP to these mutants became much stronger. The inhibition of ATP binding to most of the mutants was also stronger. In particular, LeuRS-A293D had the lowest activity, the strongest ATP binding, and the most impaired editing function. A red shift of the fluorescence emission maximum of LeuRS-A293D indicated a less hydrophobic chromophore environment and a relatively more flexible dynamic conformation. The change in T(m) of LeuRS-A293D was higher than that of all other substitutions. Evidence from sequence alignment and crystal structure of LeuRS from Thermus thermophilus shows that A293 was conserved as R (K) or A and is located at a small helix in the editing domain of the enzyme facing the active site. Hence, any amino acid substitution of A293 may affect the stability of the helix, which may lead to impaired editing function and aminoacylation activity and may be indirectly involved in ATP binding.     

Site-directed mutagenesis of conserved charged amino acid residues in ClpB from Escherichia coli

ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates strongly aggregated proteins. The sequence of ClpB contains two ATP-binding domains, each containing Walker consensus motifs. The N- and C-terminal sequence regions of ClpB do not contain known functional motifs. In this study, we performed site-directed mutagenesis of selected charged residues within the Walker A motifs (Lys212 and Lys611) and the C-terminal region of ClpB (Asp797, Arg815, Arg819, and Glu826). We found that the mutations K212T, K611T, D797A, R815A, R819A, and E826A did not significantly affect the secondary structure of ClpB. The mutation of the N-terminal ATP-binding site (K212T), but not of the C-terminal ATP-binding site (K611T), and two mutations within the C-terminal domain (R815A and R819A) inhibited the self-association of ClpB in the absence of nucleotides. The defects in self-association of these mutants were also observed in the presence of ATP and ADP. The four mutants K212T, K611T, R815A, and R819A showed an inhibition of chaperone activity, which correlated with their low ATPase activity in the presence of casein. Our results indicate that positively charged amino acids that are located along the intersubunit interface (this includes Lys212 in the Walker A motif of the N-terminal ATP-binding domain as well as Arg815 and Arg819 in the C-terminal domain) participate in intersubunit salt bridges and stabilize the ClpB oligomer. Interestingly, we have identified a conserved residue within the C-terminal domain (Arg819) which does not participate directly in nucleotide binding but is essential for the chaperone activity of ClpB.     

The novel transmembrane Escherichia coli proteins involved in the amino acid efflux.

A novel gene of Escherichia coli, rhtB, has been characterized. Amplification of this gene provides resistance to homoserine and homoserine lactone. Another E. coli gene, rhtC, provides resistance to threonine. The homologues of RhtB are widely distributed among various eubacteria and archaea, from one to 12 copies of family members that differ in their primary structure were found in the genomes. Most of them are genes that encode hypothetical transmembrane proteins. Experimental data that indicate participation of the rhtB product in the excretion of homoserine have been obtained.     

The C-terminal region of the Escherichia coli UvrC protein, which is homologous to the C-terminal region of the human ERCC1 protein, is involved in DNA binding and 5'-incision.

The incisions in the DNA at the 3'- and 5'-side of a DNA damage during nucleotide excision repair in Escherichia coli occur in a complex consisting of damaged DNA, UvrB and UvrC. The exact requirements for the two incision events, however, are different. It has previously been shown that the 3'-incision requires the interaction between the C-terminal domain of UvrB and a homologous region in UvrC. This interaction, however, is dispensable for the 5'-incision. Here we show that the C-terminal domain of the UvrC protein is essential for the 5'-incision, whereas this domain can be deleted without affecting the 3'-incision. The C-terminal domain of UvrC is homologous with the C-terminal part of the ERCC1 protein which, in a complex with XPF, is responsible for the 5'-incision reaction in human nucleotide excision repair. Both in the UvrC and the ERCC1 domain a Helix-hairpin-Helix (HhH) motif can be indicated, albeit at different positions. Such a motif also has been found in a large variety of DNA binding proteins and it has been suggested to form a structure involved in non-sequence-specific DNA binding. In contrast to the full length UvrC protein, a truncated UvrC protein (UvrC554) lacking the entire ERCC1 homology including the HhH motif no longer binds to ssDNA. Analysis of protein-DNA complexes using bandshift experiments showed that this putative DNA binding domain of UvrC is required for stabilisation of the UvrBC-DNA complex after the 3'-incision has taken place. We propose that after the initial 3'-incision the HhH motif recognises a specific DNA structure, thereby positioning the catalytic site for the subsequent 5'-incision reaction.