Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase

The catalytic residues of levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) from Zymomonas mobilis were analyzed by random mutation and site-directed mutagenesis. We found that substitution of Glu278 with Asp and His reduced the k(cat) for sucrose hydrolysis 30- and 210-fold, respectively, strongly suggesting Glu278 plays a key role in catalyzing this reaction. Given the likelihood that another acidic amino residue was also involved, we constructed variants in which acidic amino acids located within homologous regions among bacterial levansucrases and fructosyltransferases were substituted, and found that substitution of Asp194, located in homologous region III, abolished sucrose hydrolysis. In addition, Glu278 was determined to be situated within the DXXER motif in homologous region IV conserved among bacterial levansucrases and fructosyltransferases, while Asp194 was within the triplet RDP motif conserved among bacterial levansucrases, fructosyltransferases and fructofuranosidases. Finally, comparison of our findings with published data on other site-directed mutated enzymes indicated His296, also located in homologous region IV, is crucial for catalysis of the transfructosylation reaction.     

Site specific mutants of beta-galactosidase show that Tyr-503 is unimportant in Mg2+ binding but that Glu-461 is very important and may be a ligand to Mg2+

The Mg2+ concentrations required for half maximal activity, the dissociation constants, and the free energies of binding for Mg2+ bound to wild type beta-galactosidase and several site specific mutants are reported. The mutants have one of the following substitutions: Glu-461 substituted with Asp, Gln, Gly, His, or Lys; or Tyr-503 substituted with Phe, His or Cys. Substitutions for Tyr-503 had little effect on the affinity of the enzyme for Mg2+, implying that Tyr-503 is not involved in Mg2+ binding. Neutrally charged amino acids substituted for the negatively charged Glu-461 significantly decreased the affinity of the enzyme for Mg2+ and substitution of positively charged amino acids at this position further decreased the affinity. On the other hand, substitution by Asp (negative charge) at position 461 had no effect on the binding. Thus, the negatively charged side chain of Glu-461 is important for divalent cation binding to beta-galactosidase.     

Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase

As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.     

Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.     

Structure-function studies of Na,K-ATPase. Site-directed mutagenesis of the border residues from the H1-H2 extracellular domain of the alpha subunit

It has recently been shown that replacement of the border residues (Gln-111 and Asn-122) of the H1-H2 extracellular domain of the sheep Na,K-ATPase alpha subunit with the charged amino acids Arg and Asp generates a ouabain-resistant enzyme (Price, E. M. and Lingrel, J. B. (1988) Biochemistry 27, 8400-8408). In order to further study structure-function relationships in Na,K-ATPase, six additional mutations have been made at these border positions. Two of these mutants were single amino acid substitutions (Gln-111 to Arg or Asn-122 to Asp). These mutations change one or the other H1-H2 border residue to a charged amino acid. The remaining substitutions were double mutants in which both of the H1-H2 border residues were simultaneously changed to charged amino acids. Changes were made which introduced either positively charged amino acids (Lys at positions 111 and 122), negatively charged amino acids (Glu at positions 111 and 122) or oppositely charged amino acids (Lys at position 111 and Glu at 122; Asp at position 111 and Arg at 122) at the borders of the H1-H2 extracellular domain. HeLa cells transfected with any of these sheep Na,K-ATPase alpha subunit mutants were able to grow in concentrations of ouabain that were toxic to untransfected cells or cells transfected with the wild type sheep alpha subunit. Crude membranes isolated from the transfectants were analyzed for ouabain inhibitable Na,K-ATPase activity. All of the transfectants contained a relatively ouabain-resistant component of enzyme activity, with the ouabain I50 values ranging from 4 x 10(-3) M to 1 x 10(-6) M. The most resistant enzyme was the double mutant that contained Asp at position 111 and Arg at 122, whereas the least resistant were the enzymes containing the single amino acid substitutions. There was no correlation between the type of charged amino acid present at the border position and the degree of ouabain resistance. These data demonstrate the functional importance, in terms of ouabain binding, of the border positions of the H1-H2 extracellular domain of the Na,K-ATPase alpha subunit.     

Probing the critical residues for intramolecular fructosyl transfer reaction of a levan fructotransferase

Levan fructotransferase (LFTase) preferentially catalyzes the transfructosylation reaction in addition to levan hydrolysis, whereas other levan-degrading enzymes hydrolyze levan into a levan-oligosaccharide and fructose. Based on sequence comparisons and enzymatic properties, the fructosyl transfer activity of LFTase is proposed to have evolved from levanase. In order to probe the residues that are critical to the intramolecular fructosyl transfer reaction of the Microbacterium sp. AL-210 LFTase, an error-prone PCR mutagenesis process was carried out, and the mutants that led to a shift in activity from transfructosylation towards hydrolysis of levan were screened by the DNS method. After two rounds of mutagenesis, TLC and HPLC analyses of the reaction products by the selected mutants revealed two major products; one is a di-D-fructose- 2,6':6,2'-dianhydride (DFAIV) and the other is a levanbiose. The newly detected levanbiose corresponds to the reaction product from LFTase lacking transferring activity. Two mutants (2-F8 and 2-G9) showed a high yield of levanbiose (38-40%) compared with the wild-type enzyme, and thus behaved as levanases. Sequence analysis of the individual mutants responsible for the enhanced hydrolytic activity indicated that Asn-85 was highly involved in the transfructosylation activity of LFTase.     

Amino acid substitutions in pilin of Pseudomonas aeruginosa. Effect on leader peptide cleavage, amino-terminal methylation, and pilus assembly.

A total of 37 separate mutants containing single and multiple amino acid substitutions in the leader and amino-terminal conserved region of the Type IV pilin from Pseudomonas aeruginosa were generated by oligonucleotide-directed mutagenesis. The effect of these substitutions on the secretion, processing, and assembly of the pilin monomers into mature pili was examined. The majority of substitutions in the highly conserved amino-terminal region of the pilin monomer had no effect on piliation. Likewise, substitution of several of the residues within the six amino acid leader sequence did not affect secretion and leader cleavage (processing), including replacement of one or both of the positively charged lysine residues with uncharged or negatively charged amino acids. One characteristic of the Type IV pili is the presence of an amino-terminal phenylalanine after leader peptide cleavage which is N-methylated prior to assembly of pilin monomers into pili. Substitution of the amino-terminal phenylalanine with a number of other amino acids, including polar, hydrophobic, and charged residues, did not affect proper leader cleavage and subsequent assembly into pili. Amino-terminal sequencing showed that the majority of substitute residues were also methylated. Substitution of the glycine residue at the -1 position to the cleavage site resulted in the inability to cleave the prepilin monomers and blocked the subsequent assembly of monomers into pili. These results indicate that despite the high degree of conservation in the amino-terminal sequences of the Type IV pili, N-methylphenylalanine at the +1 position relative to the leader peptide cleavage site is not strictly required for pilin assembly. N-Methylation of the amino acids substituted for phenylalanine was shown to have taken place in four of the five mutants tested, but it remains unclear as to whether pilin assembly is dependent on this modification. Recognition and proper cleavage of the prepilin by the leader peptidase appears to be dependent only on the glycine residue at the -1 position. Cell fractionation experiments demonstrated that pilin isolated from mutants deficient in prepilin processing and/or assembly was found in both inner and outer membrane fractions, indistinguishable from the results seen with the wild type.     

Investigation of the role of electrostatic charge in activation of the Escherichia coli response regulator CheY

In a two-component regulatory system, an important means of signal transduction in microorganisms, a sensor kinase phosphorylates a response regulator protein on an aspartyl residue, resulting in activation. The active site of the response regulator is highly charged (containing a lysine, the phosphorylatable aspartate, two additional aspartates involved in metal binding, and an Mg(2+) ion), and introduction of the dianionic phosphoryl group results in the repositioning of charged moieties. Furthermore, substitution of one of the Mg(2+)-coordinating aspartates with lysine or arginine in the Escherichia coli chemotaxis response regulator CheY results in phosphorylation-independent activation. In order to examine the consequences of altered charge distribution for response regulator activity and to identify possible additional amino acid substitutions that result in phosphorylation-independent activation, we made 61 CheY mutants in which residues close to the site of phosphorylation (Asp57) were replaced by various charged amino acids. Most substitutions (47 of 61) resulted in the complete loss of CheY activity, as measured by the inability to support clockwise flagellar rotation. However, 10 substitutions, all introducing a new positive charge, resulted in the loss of chemotaxis but in the retention of some clockwise flagellar rotation. Of the mutants in this set, only the previously identified CheY13DK and CheY13DR mutants displayed clockwise activity in the absence of the CheA sensor kinase. The absence of negatively charged substitution mutants with residual activity suggests that the introduction of additional negative charges into the active site is particularly deleterious for CheY function. Finally, the spatial distribution of positions at which amino acid substitutions are functionally tolerated or not tolerated is consistent with the presently accepted mechanism of response regulator activation and further suggests a possible role for Met17 in signal transduction by CheY.     

Studies on the active sites ofBacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis

Summary Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites ofBacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced inBacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G towardp-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP andp-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities.     

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately -20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about 20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately -50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.     

Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis

Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate.     

Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.     

In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly.

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.     

Evaluation of the role of conserved His and Met residues among lipoxygenases by site-directed mutagenesis of recombinant human 5-lipoxygenase

The 5-, 12-, and 15-lipoxygenases contain a highly conserved sequence of the form His-(X)4-His-(X)4-His-(X)17-His-(X)8-His which represents a potential binding site for non heme iron to the protein. The importance of selected amino acids within this His cluster for the activity of human 5-lipoxygenase was investigated by site-directed mutagenesis using bacteria and insect cells expression systems. After single mutation of each of the 5 His residues at positions 363, 368, 373, 391, and 400 by Ser, Cys, or Lys, measurable levels of 5-lipoxygenase activity could be recovered in Escherichia coli only for the Ser363 and Cys363 mutants, with most amino acid substitutions causing a decrease in the levels of expression of the soluble protein. In contrast, 25-80% of soluble 5-lipoxygenase activity was recovered after the replacement of several of the hydrophobic amino acids in this region: Tyr384 by Ser or Phe; Phe394 by Trp and Val375 by Ala. Met436 could be replaced by Leu with little effect on 5-lipoxygenase activity or turnover inactivation half-time. High levels of mutant 5-lipoxygenases containing a Ser residue instead of His at each of the five positions were also expressed in Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus. The specific activity (58-75% of control) and the reaction time course of the Ser363, Ser391, and Ser400 mutants were comparable with that of native 5-lipoxygenase whereas inactive proteins were obtained for the Ser368 and Ser373 mutants. These results show that His368 and His373 residues are important for 5-lipoxygenase activity and that the other conserved His363, His391, His400, and Met436 residues are not crucial for the catalytic cycle or for the mechanism of self-inactivation of 5-lipoxygenase.     

Mutations in the alpha8 loop of human APE1 alter binding and cleavage of DNA containing an abasic site

Recent crystallographic studies reveal loops in human AP endonuclease 1 (APE1) that interact with the major and minor grooves of DNA containing apurinic/apyrimidinic (AP) sites. These loops are postulated to stabilize the DNA helix and the flipped out AP residue. The loop alpha8 interacts with the major groove on the 3' side of the AP site. To determine the essentiality of the amino acids that constitute the alpha8 loop, we created a mutant library containing random nucleotides at codons 222-229 that, in wild-type APE1, specify the sequence NPKGNKKN. Upon expression of the library (2 x 10(6) different clones) in Escherichia coli and multiple rounds of selection with the alkylating agent methyl-methane sulfonate (MMS), we obtained approximately 2 x 10(5) active mutants that complemented the MMS sensitivity of AP endonuclease-deficient E. coli. DNA sequencing showed that active mutants tolerated amino acid substitutions at all eight randomized positions. Basic and uncharged polar amino acids together comprised the majority of substitutions, reflecting the positively charged, polar character of the wild-type loop. Asn-222, Asn-226, and Asn-229 exhibited the least mutability, consistent with x-ray data showing that each asparagine contacts a DNA phosphate. Substitutions at residues 226-229, located nearer to the AP site, that reduced basicity or hydrogen bonding potential, increased Km 2- to 6-fold and decreased AP site binding; substitutions at residues 222-225 exhibited lesser effects. This initial mutational analysis of the alpha8 loop supports and extends the conclusion of crystallographic studies that the loop is important for binding of AP.DNA and AP site incision.     

Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.

The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.     

Cytoplasmic regions adjacent to the M3 and M4 transmembrane segments influence expression and function of alpha7 nicotinic acetylcholine receptors. A study with single amino acid mutants

We studied the role of the cytoplasmic regions adjacent to the M3 and M4 transmembrane segments of alpha7 nicotinic receptors in the expression of functional channels. For this purpose, a total of 50 amino acids were mutated throughout the mentioned regions. Mutants close to M3, from Arg294 to Leu321, showed slight modifications in the levels of alpha-bungarotoxin binding sites and acetylcholine-evoked currents. Exceptions were mutants located at two clusters (His296 to Pro300 and Ile312 to Trp316), which exhibited low expression levels. In addition, some mutants showed altered functional responses. Many mutants close to M4 showed increased receptor expression, especially the ones located at the hydrophobic face of a putative amphipathic helix. This effect seems to be the consequence of a combination of increased receptor biosynthesis, higher transport efficiency and delayed degradation, such that we postulate that elements in the amphipathic domain strongly influence receptor stability. Finally, some mutants in this region showed altered functional responses: elimination of positively charged residues (Arg424 and Arg426) increased currents, whereas the opposite was observed upon suppression of negatively charged ones (Glu430 and Glu432). These results suggest that the cytoplasmic regions close to M3 and M4 play important structural and functional roles.     

Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity.

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N-terminal and C-terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C-terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N-terminal and C-terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C-terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C-terminus of this sequence. Interestingly, the inactive C-terminal mutants could be complemented by cotransfection with a wild-type C-terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C-terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C-terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.     

Differential mechanisms of recognition and activation of interleukin-8 receptor subtypes

We have probed an epitope sequence (His18-Pro19-Lys20-Phe21) in interleukin-8 (IL-8) by site-directed mutagenesis. This work shows that single and double Ala substitutions of His18 and Phe21 in IL-8 reduced up to 77-fold the binding affinity to IL-8 receptor subtypes A (CXCR1) and B (CXCR2) and to the Duffy antigen. These Ala mutants triggered neutrophil degranulation and induced calcium responses mediated by CXCR1 and CXCR2. Single Asp or Ser substitutions, H18D, F21D, F21S, and double substitutions, H18A/F21D, H18A/F21S, and H18D/F21D, reduced up to 431-fold the binding affinity to CXCR1, CXCR2, and the Duffy antigen. Interestingly, double mutants with charged residue substitutions failed to trigger degranulation or to induce wild-type calcium responses mediated by CXCR1. Except for the H18A and F21A mutants, all other IL-8 mutants failed to induce superoxide production in neutrophils. This study demonstrates that IL-8 recognizes and activates CXCR1, CXCR2, and the Duffy antigen by distinct mechanisms.