Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis

A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.     

Analysis of membrane lipids by 500 MHz 1H NMR

A nondestructive method has been developed for rapid analysis of lipid content of membrane extracts based on high field proton NMR spectroscopy. Lipid extraction is done by stepwise sonication of purified membranes into chloroform:methanol:water mixtures, and 1H spectra are recorded at 35 degrees C on final preparations consisting of at least 1 mg dried lipid solubilized in 2:1 CD3OD:CDCl3. Spectral peaks of lipid mixtures are assigned to lipid classes using a data base of standard lipid characteristic resonances derived from purified single membrane lipids and known mixtures of them. Peak intensities of characteristic peaks yield ratios of various lipids such as cholesterol:phospholipid and phosphatidylcholine:phosphatidylethanolamine, degree of unsaturation, average acyl chain length, total glycerol lipid content, and presence or absence of particular lipids, such as glycolipids or lysolipids. This procedure of membrane lipid analysis has been verified using known mixtures of purified standard lipids.     

Identification of residues involved in v-Src substrate recognition by site-directed mutagenesis.

To study the role of the catalytic domain in v-Src substrate specificity, we engineered three site-directed mutants (Leu-472 to Tyr or Trp and Thr-429 to Met). The mutant forms of Src were expressed in Sf9 cells and purified. We analyzed the substrate specificities of wild-type v-Src and the mutants using two series of peptides that varied at residues C-terminal to tyrosine. The peptides contained either the YMTM motif found in insulin receptor substrate-1 (IRS-1) or the YGEF motif identified from peptide library experiments to be the optimal sequence for Src. Mutations at positions Leu-472 or Thr-429 caused changes in substrate specificity at positions P+1 and P+3 (i.e. one or three residues C-terminal to tyrosine). This was particularly evident in the case of the L-472W mutant, which had pronounced alterations in its preferences at the P+1 position. The results suggest that residue Leu-472 plays a role in P+1 substrate recognition by Src. We discuss the results in the light of recent work on the roles of the SH2, SH3 and catalytic domains of Src in substrate specificity.     

Analysis of the binding of 1,3-diacetylchloramphenicol to chloramphenicol acetyltransferase by isotope-edited 1H NMR and site-directed mutagenesis.

The binary complex of diacetylchloramphenicol and chloramphenicol acetyltransferase (CAT) has been studied by a combination of isotope-edited 1H NMR spectroscopy and site-directed mutagenesis. One-dimensional HMQC spectra of the complex between 1,3-[2-13C]diacetylchloramphenicol and the type III natural variant of CAT revealed the two methyl 1H signals arising from each 13C-labeled carbon atom in the acetyl groups of the bound ligand. Slow hydrolysis of the 3-acetyl group by the enzyme precluded further analysis of this binary complex. It was possible to slow down the rate of hydrolysis by use of the catalytically defective S148A mutant of CATIII (Lewendon et al., 1990); in the complex of diacetylchloramphenicol with S148A CATIII, the chemical shifts of the acetyl groups of the bound ligand were the same as in the wild-type complex. The acetyl signals were individually assigned by repeating the experiment using 1-[2-13C],3-[2-12C]diacetylchloramphenicol, where only one signal from the bound ligand was observed. A two-dimensional 1H, 1H NOESY experiment, with 13C(omega 2) half-filter, on the 1,3-[2-13C]diacetylchloramphenicol/S148A CATIII complex showed a number of intermolecular NOEs from each methyl group in the ligand to residues in the chloramphenicol binding site. The 3-acetyl group showed strong NOEs to two aromatic signals which were selected for assignment. The possibility that the NOEs originated from the aromatic protons of diacetylchloramphenicol itself was eliminated by assignment of the signals from enzyme-bound diacetylchloramphenicol and chloramphenicol using perdeuterated CATIII. Examination of the X-ray crystal structure of the chloramphenicol/CATIII binary complex indicated four plausible candidate aromatic residues: Y25, F33, F103, and F158.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of residues involved in G Strand DNA binding by Escherichia coli DNA topoisomerase I

Crystal structures of complexes between type IA DNA topoisomerases and single-stranded DNA suggest that the residues Ser-192, Arg-195, and Gln-197 in a conserved region of Escherichia coli topoisomerase I may be important for direct interactions with phosphates on the G strand of DNA, which is the substrate for DNA cleavage and religation (Changela A., DiGate, R. J., and Mondragón, A. (2001) Nature 411, 1077-1081; Perry, K., and Mondragón, A. (2003) Structure 11, 1349-1358). Site-directed mutagenesis experiments altering these residues to alanines and other amino acids were carried out to probe the relevance of these interactions in the catalytic activities of the enzyme. The results show that the side chains of Arg-195 and Gln-197 are required for DNA cleavage by the enzyme and are likely to be important for positioning of the G strand of DNA at the active site prior to DNA cleavage. Mutation of Ser-192 did not affect DNA binding and cleavage but nevertheless decreased the overall rate of relaxation of supercoiled DNA probably because of its participation in a later step of the reaction pathway.     

Structure-function relationships in human epidermal growth factor studied by site-directed mutagenesis and 1H NMR

In order to elucidate the mechanism of interaction between human epidermal growth factor (EGF) and its receptor, selected variants of EGF, differing by single amino acid substitutions, have been made by site-directed mutagenesis. The receptor affinity of these mutants was determined by a receptor binding competition assay, and the effects of the substitution on the structure of the protein were assessed by 1H nuclear magnetic resonance techniques. Various substitutions of Arg-41 resulted in substantial reduction in receptor affinity of EGF whereas change of Tyr-13 did not affect binding to the receptor. The 1H resonances of all nonexchangeable protons of the Tyr-13----Leu, Arg-41----His, and Leu-47----Glu variants were assigned and compared in order to assess the structural integrity of these mutants, which possess very different spectral and biological properties. In the case of the Leu-47----Glu mutant, only minor localized spectral changes were observed, confirming that the tertiary structure of the protein is preserved upon mutation. In contrast, for both the Arg-41----His and Tyr-13----Leu variants, significant and strikingly similar spectra changes were observed for many residues located far away from the mutated residues. This implies that similar structural alterations have taken place in both proteins, an idea further supported by hydrogen-exchange experiments where the exchange rates of hydrogen-bonded amide protons for both the Tyr-13----Leu and the Arg-41----His mutants were found to be about 4 times faster than in the wild-type protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of cooperative and isolated site binding of T4 gene 32 protein to ssDNA by 1H NMR

Deuteriation of all aromatic protons of gene 32 protein (g32P) from phage T4, followed by selective introduction of specific protons, has allowed the precise identification of the number and magnitude of the chemical shift changes induced in the aromatic protons when g32P binds noncooperatively or cooperatively to nucleotides. Signals from five Tyr residues are shifted by binding of g32P to d(pA)8 or d(pA)40-60; however, the change from noncooperative, d(pA)8, to cooperative, d(pA)40-60, binding causes significant increases in the magnitudes of the shifts for only two of these Tyr signals. These two Tyr residues may interact directly with the nucleotide bases, while the shifts associated with the other three Tyr may be due to conformational changes in g32P upon ssDNA binding. Similar conclusions can be drawn for two of the six Phe residues whose protons undergo shifts upon nucleotide binding. Observation of selected proton signals allows for the first time detection by 1H NMR of changes in the proton signals from two Trp residues upon nucleotide binding. The side chains of two Tyr, one or two Phe, and one Trp are probably directly involved in nucleotide base-protein interactions. As assayed by the signals from the H2 and H8 protons of adenine, the bases of a bound nucleotide are undergoing a fast chemical exchange in the noncooperative mode of binding, but shift to slow exchange upon assuming the cooperative mode of ssDNA interaction. When bound to a polynucleotide, the A domain of g32P (residues 254-301) becomes more mobile, as reflected in sharpening of the 1H NMR signals from the A domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of uracil defect on DNA structure: 1H NMR investigation at 500 MHz.

The local structure of two self complementary oligonucleotides d(GTAC-GTAC) and d(GTACGUAC) which differ only by the presence of uracil, not a normal component of DNA, have been investigated by 1H NMR at 500 MHz. The two octamers exhibit the same thermodynamical constants (t 1/2, delta H), their exchangeable protons broaden and disappear at the same temperature. The T-U substitution did not induce any significant changes on non exchangeable protons resonances from 2-D COSY and 2-D NOESY experiments. So the two octamers exhibit the same global structure. The only variation was detected by 1D NOE measurements: the base orientations around the N glycosidic bonds (chi angles) are different.     

Site-directed mutagenesis identifies residues in uncoupling protein (UCP1) involved in three different functions

Using site-specific mutagenesis, we have constructed several mutants of uncoupling protein (UCP1) from brown adipose tissue to investigate the function of acidic side chains at positions 27, 167, 209, and 210 in H(+) and Cl(-) transport as well as in nucleotide binding. The H(+) transport activity was measured with mitochondria and with reconstituted vesicles. These mutant UCPs (D27N, D27E, E167Q, D209N, D210N, and D209N + D210N) are expressed at near wt levels in yeast. Their H(+) transport activity in mitochondria correlates well with the reconstituted protein except for D27N (intrahelical), which shows strong inhibition of H(+) transport in the reconstituted system and only 50% decrease of uncoupled respiration in mitochondria. In the double adjacent acidic residues (between helix 4 and helix 5), mutation of D210 and of D209 decreases H(+) transport 80% and only 20%, respectively. These mutants retain full Cl(-) transport activity. The results indicate that D210 participates in H(+) uptake at the cytosolic side and D27 in H(+) translocation through the membrane. Differently, E167Q has lost Cl(-) transport activity but retains the ability to transport H(+). The separate inactivation of H(+) and Cl(-) transport argues against the fatty acid anion transport mechanism of H(+) transport by UCP. The mutation of the double adjacent acidic residues (D209, D210) decreases pH dependency for only nucleoside triphosphate (NTP) but not diphosphate (NDP) binding. The results identify D209 and D210 in accordance with the previous model as those residues which control the location of H214 in the binding pocket, and thus contribute to the pH control of NTP but not of NDP binding.     

Characterisation of the Rab binding properties of Rab coupling protein (RCP) by site-directed mutagenesis

Rab coupling protein (RCP) is a member of the Rab11-family of interacting proteins (Rab11-FIPs). Family members are characterised by their ability to interact with Rab11. This property is mediated by a conserved Rab binding domain (RBD) located at their carboxy-termini. Several Rab11-FIPs can also interact with other small GTPases. RCP interacts with Rab4 in addition to Rab11. To dissect out the individual properties of the Rab4 and Rab11 interactions with RCP, conserved amino acids within the RBD of RCP were mutated by site-directed mutagenesis. The effect of these mutations on Rab4 and Rab11 binding, and the intracellular localisation of RCP, was examined. Our results indicate that Rab11, rather than Rab4, mediates the intracellular localisation of RCP, and that the class I Rab11-FIPs compete for binding to Rab11.     

Galactose-1-phosphate uridylyltransferase: identification of histidine-164 and histidine-166 as critical residues by site-directed mutagenesis

Galactose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-glucose and galactose-1-P with UDP-galactose and glucose-1-P by a double-displacement mechanism involving the compulsory formation of a uridylyl enzyme intermediate. The uridylyl group is covalently bonded to the N3 position of a histidine residue in the uridylyl enzyme. The galT gene of Escherichia coli, which codes for the uridylyltransferase and is contained in a plasmid for transformation of E. coli, has been sequenced, and the positions of the 15 histidine residues have been determined from the deduced amino acid sequence of this protein. Fifteen mutant genes, in each of which one of the 15 histidine codons has been changed to an asparagine codon, have been generated and used to transform the E. coli strain JM101. When extracts of the transformants were assayed for uridylyltransferase, 13 exhibited high levels of activity. Two of the extracts containing mutant uridylyltransferase exhibited less than control levels of activity. These mutant proteins, H164N and H166N, were overexpressed, isolated, and tested for their ability to form the compulsory uridylyl enzyme intermediate. Neither the H164N nor the H166N mutant proteins could form the intermediate. Thus, both His-164 and His-166 are critical for activity, and their proximity suggests that both are in the active site. One is the essential nucleophilic catalyst to which the uridylyl group is bonded in the intermediate, and the other serves an equally important, as yet unknown, function. The active-site sequence His(164)-Pro-His(166) is conserved in this enzyme from E. coli, humans, Saccharomyces, and Streptomyces.     

1H NMR studies at 360 MHz of the aromatic amino acid residues in ferrocytochrome c-552 from Euglena gracilis.

The resonances of the aromatic rings in the 1H NMR spectra at 360 MHz of ferrocytochrome c-552 of Euglena gracilis were investigated by double resonance techniques. The spin systems of the two tryptophan and four of the tyrosine residues could be identified. This analysis of the aromatic region of the 1H NMR spectrum provided evidence that His-14 is bound to the heme iron. It gave also some insight into the molecular dynamics of ferrocytochrome c-552 in that it showed that of the six aromatic rings, four tyrosines were rotating rapidly about the Cbeta-Cgamma bond, while one tyrosine and the single phenylalanine were restricted in their rotational mobilities by their environmnent in the protein.     

Identification of critical active-site residues in angiotensin-converting enzyme-2 (ACE2) by site-directed mutagenesis

Angiotensin-converting enzyme-2 (ACE2) may play an important role in cardiorenal disease and it has also been implicated as a cellular receptor for the severe acute respiratory syndrome (SARS) virus. The ACE2 active-site model and its crystal structure, which was solved recently, highlighted key differences between ACE2 and its counterpart angiotensin-converting enzyme (ACE), which are responsible for their differing substrate and inhibitor sensitivities. In this study the role of ACE2 active-site residues was explored by site-directed mutagenesis. Arg273 was found to be critical for substrate binding such that its replacement causes enzyme activity to be abolished. Although both His505 and His345 are involved in catalysis, it is His345 and not His505 that acts as the hydrogen bond donor/acceptor in the formation of the tetrahedral peptide intermediate. The difference in chloride sensitivity between ACE2 and ACE was investigated, and the absence of a second chloride-binding site (CL2) in ACE2 confirmed. Thus ACE2 has only one chloride-binding site (CL1) whereas ACE has two sites. This is the first study to address the differences that exist between ACE2 and ACE at the molecular level. The results can be applied to future studies aimed at unravelling the role of ACE2, relative to ACE, in vivo.     

New late gene, dar, involved in the replication of bacteriophage T4 DNA. II. Overproduction of DNA binding protein (gene 32 protein) and further characterization.

We have previously shown that the arrested DNA synthesis of mutant defective in T4 phage gene 59 can be reversed by a mutation in dar. In this paper, we have examined the effect of the dar mutation on the kinetics of gene 32 protein (DNA binding protein) synthesis, DNA packaging, progeny formation, and several other porcesses. Several lines of evidence are presented showing that the regulation of synthesis of gene 32 protein is abnormal in dar 1-infected cells. In these cells, gene 32 protein, an early protein, is also expressed late in the infectious cycle. Our data also indicate that the packaging og DNA into T4 phage heads is delayed in dar mutant-infected cells, and this in turn results in a 6- to 8-min delay in intracellular progeny formation, although the synthesis of late proteins appears to be normal, as shown by gel electrophoresis. We have also studied the phenotypes of the double mutant dar-amC5 (gene 59). The increased sensitivity to hydroxyurea caused by a mutation in the dar gene can be alleviated by a second mutation in gene 59, but an increased sensitivity to UV irradiation caused by a mutation in gene 59 cannot be alleviated by a second mutation in the dar gene. Therefore, the double mutant still exhibits abnormalities in the repair of UV lesions.     

Spin-labeling proton NMR study on aromatic amino acid residues in the guanine nucleotide binding site of human c-Ha-ras(1-171) protein

A truncated human c-Ha-ras gene product, ras(1-171) protein, was prepared and chemically modified with maleimide spin-label (MSL). By trypsin digestion of the MSL-labeled ras(1-171) protein, MSL-labeled peptide fragments were isolated and sequenced. The cysteine residue in position 118 of the protein, but not the other cysteine residues, Cys-51 or Cys-80, was found to be specifically labeled by MSL. The ESR spectrum of the MSL-labeled ras(1-171) protein indicates that the MSL group attached to Cys-118 is strongly immobilized. Proton NMR spectra at 400-MHz were measured for this MSL-labeled ras(1-171) protein and also for a control sample of a labeled ras(1-171) protein whose MSL was reduced by sodium ascorbate. In the difference spectra for these two proteins, resonances of protons in the vicinity of the MSL group attached to Cys-118 of the ras(1-171) protein were observed. Thus, the MSL group was found to be in the vicinity of the protein-bound GDP. A phenylalanine residue and two histidine residues, which were characterized by 2D HOHAHA and DQF-COSY spectra, were also found to be in the vicinity of MSL. NOE and pH titration analyses indicate that this phenylalanine residue is close to the bound GDP and one of the two histidine residues. By carboxypeptidase digestion, the two histidine residues near MSL were identified as His-27 and His-94.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative homology modeling of human cytochrome P4501A1 (CYP1A1) and confirmation of residues involved in 7-ethoxyresorufin O-deethylation by site-directed mutagenesis and enzyme kinetic analysis

CYP1A1 homology models based on the CYP2C5 and a composite of CYP2C5, CYP2C8, and CYP2C9 X-ray crystal structures were compared to a model generated using the recently published coordinates of CYP1A2. The model using the CYP1A2 coordinates, CYP1A1-(1A2), gave near ideal stereochemical quality and was favored energetically. Docking studies identified the active-site residues potentially involved in binding of the prototypic CYP1A1 substrate 7-ethoxyresorufin. CYP1A1 mutants S122A, F123A, F224A, A317Y, T321G, and I386G were generated to explore the roles of these residues in 7-ethoxyresorufin binding and turnover, and generally confirmed the importance of aromatic interactions over hydrogen bonding in orientating 7-ethoxyresrufin in a catalytically favorable orientation. Although 7-ethoxyresorufin O-deethylation by CYP1A1 and several mutants exhibited substrate inhibition, it is unlikely that inhibition arises from the simultaneous binding of two substrates within the active-site given the geometry of the active site-cavity.     

Identification of putative residues involved in the accessibility of the substrate-binding site of lipoxygenase by site-directed mutagenesis studies

Lipoxygenases (LOXs) are a class of widespread dioxygenases catalyzing the hydroperoxidation of polyunsaturated fatty acids (PUFA). Recently, we isolated a cDNA encoding a LOX, named olive LOX1, from olive fruit of which the deduced amino acid sequence shows more than 50% identity with plant LOXs. In the present study, a model of olive LOX1 based on the crystal structure of soybean LOX-1 as template has been generated and two bulky amino acid residues highly conserved in LOXs (Phe277) and in plant LOXs (Tyr280), located at the putative entrance of catalytic site were identified. These residues may perturb accessibility of the substrate-binding site and therefore were substituted by less space-filling residues. Kinetic studies using linoleic and linolenic acids as substrates were carried out on wild type and mutants. The results show that the removal of steric bulk at the entrance of the catalytic site induces an increase of substrate affinity and of catalytic efficiency, and demonstrate that penetration of substrates into active site of olive LOX1 requires the movement of the side chains of the Phe277 and Tyr280 residues. This study suggests the involvement of these residues in the accessibility of the substrate-binding site in the lipoxygenase family.