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Proline Iminopeptidase v Proline Aminopeptidase

Proline Iminopeptidase v Proline Aminopeptidase


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We're an undergraduate independent research team and we are having trouble purchasing commercial proline iminopeptidase as it is unavailable on Sigma Aldrich and very expensive on other websites. We need to test that this peptidase will cut a series of tripeptides we are synthesizing in e coli.

Could we purchase proline aminopeptidase instead of proline iminopeptidase; will the peptidase function the same way?

This paper says that they are the same, but this paper says that they are different. Does anyone have any experience with this commercial peptidase?


There are two related enzymes that cleave an N-terminal residue. Enzyme 3.4.11.9 cleaves any residue from a peptide when the next residue is proline. Enzyme 3.4.11.5 cleaves an N-terminal proline from a peptide. Unfortunately, it appears that both are sometimes referred to as proline aminopeptidase. However, only 3.4.11.5 is referred to as proline iminopeptidase.

The vendor should certainly specify which enzyme they are offering.


Aminopeptidase

SPP, a resident ER protein with 7 transmembrane domains, contains two conserved aspartate residues, each within adjacent transmembrane helices. As discussed above, such motifs are characteristic of the presenilin-type of aspartic protease. Similar to S2P, cleavage by SPP requires helix-breaking residues within the transmembrane domain of its signal peptide substrates.

The best-studied example of Rip mediated by SPP is the proteolytic processing of signal peptides from MHC class I molecules such as HLA-A, -B, -C, and -G. These molecules are expressed with a typical signal sequence for targeting to the secretory pathway. During their translocation through the ER membrane, the signal sequences are cleaved off from the pre-protein by signal peptidase. The cleaved signal peptides, which remain membrane-bound with a type 2 orientation, are then cleaved by SPP in the middle of the membrane to liberate the NH2-terminal half of the signal peptides into the cytosol ( Figure 1 ). These cytosolic fragments are then transported into the ER lumen where they bind to HLA-E, a nonclassical MHC class I molecule. The HLA-E/peptide complexes travel to the cell surface where they bind to CD94/NKG2A receptors on natural killer (NK) cells and inhibit NK cell-mediated lysis. This pathway protects cells expressing normal MHC class I molecules from killing by NK cells.


Xaa-Pro Aminopeptidase (Prokaryote)

Distinguishing Features

The structurally related Xaa-Pro dipeptidase (prolidase) ( Chapter 337 ) has been found in bacteria and is specific for Xaa-Pro dipeptides. In contrast, XProAP cleaves Xaa-Pro-Xbb peptides more readily than Xaa-Pro dipeptides. Prolyl aminopeptidase ( Chapter 760 ), like prokaryote XProAP, is capable of cleaving poly- l -proline, but is specific for an N-terminal Pro-Xaa bond.

A genomic analysis showed that an ortholog of E. coli XProXP is present in only 65% of other γ-proteobacteria species and in none of the α-proteobacteria species analyzed [42] . Most of the species without an E. coli XProAP ortholog have, instead, an ortholog of mammalian aminopeptidase P1 (APP1), which has an extra (third) domain at the N-terminus ( Chapter 342 ). This form has not yet been characterized in prokaryotes. Some Pseudomonas species have both orthologs. Interestingly, humans also have an E. coli XProAP ortholog, referred to as aminopeptidase P3 (APP3), in addition to APP1 and the membrane-bound APP2 ( Chapter 343 ) [42] . APP3 has a 33% amino acid sequence identity with E. coli XProAP, but only 12 and 16% identity over the last two domains of human APP1 and APP2, respectively. The APP3 transcript is subject to alternative splicing, producing mitochondrial and cytosolic isoforms [24,41] . Although APP3 has not yet been isolated and characterized, specific mutations in its gene have been found to result in cystic kidney disease [41,43] .


Structure of proline iminopeptidase from Xanthomonas campestris pv. citri: a prototype for the prolyl oligopeptidase family.

The proline iminopeptidase from Xanthomonas campestris pv. citri is a serine peptidase that catalyses the removal of N-terminal proline residues from peptides with high specificity. We have solved its three-dimensional structure by multiple isomorphous replacement and refined it to a crystallographic R-factor of 19.2% using X-ray data to 2.7 A resolution. The protein is folded into two contiguous domains. The larger domain shows the general topology of the alpha/beta hydrolase fold, with a central eight-stranded beta-sheet flanked by two helices and the 11 N-terminal residues on one side, and by four helices on the other side. The smaller domain is placed on top of the larger domain and essentially consists of six helices. The active site, located at the end of a deep pocket at the interface between both domains, includes a catalytic triad of Ser110, Asp266 and His294. Cys269, located at the bottom of the active site very close to the catalytic triad, presumably accounts for the inhibition by thiol-specific reagents. The overall topology of this iminopeptidase is very similar to that of yeast serine carboxypeptidase. The striking secondary structure similarity to human lymphocytic prolyl oligopeptidase and dipeptidyl peptidase IV makes this proline iminopeptidase structure a suitable model for the three-dimensional structure of other peptidases of this family.


Over-Expression of a Proline Specific Aminopeptidase from Aspergillus oryzae JN-412 and Its Application in Collagen Degradation

A strain that exhibited intracellular proline-specific aminopeptidase (PAP) activity was isolated from soy sauce koji and identified as Aspergillus oryzae JN-412. The gene coding PAP was cloned and efficiently expressed in Escherichia coli BL21 in a biologically active form. The highest specific activity reached 52.28 U mg −1 at optimum cultivation conditions. The recombinant enzyme was purified 3.3-fold to homogeneity with a recovery of 36.7 % from cell-free extract using Ni-affinity column chromatography. It appeared as a single protein band on SDS-PAGE with molecular mass of approximately 50 kDa. The purified enzyme exhibited the highest activity at 60 °C and pH 7.5. The enzyme activity was inhibited by PMSF and ions like Zn 2+ and Cu 2+ . DTT, β-mercaptoethanol, EDTA, and ions like Co 2+ , Mg 2+ , Mn 2+ , and Ca 2+ had no influence on enzyme activity, whereas Ni 2+ enhanced the enzyme activity. By using collagen as a substrate, the purified recombinant prolyl aminopeptidase contributed to the hydrolysis of collagen when used in combination with neutral protease, and free amino acids in collagen hydrolysates was significantly increased.

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Characterization of a unique proline iminopeptidase from white-rot basidiomycetes Phanerochaete chrysosporium

A putative gene encoding proline iminopeptidase (PchPiPA) was cloned from Phanerochaete chrysosporium BKM-F-1767 by RT-PCR and expressed successfully in Escherichia coli. The cDNA is 942 bp in length and encodes 313 amino acids. The recombinant enzyme was only able to hydrolyze Pro-pNA among the tested synthetic substrates. There is no activity detected toward Leu-pNA, Phe-pNA and Tyr-pNA, as well as GGG-pNA, SGR-pNA, AAV-pNA, AAPL-pNA, AAVA-pNA. And the recombinant enzyme could cleave the peptides derived from enzyme-hydrolytic natural proteins to release free lysine, which was confirmed using synthetic oligopeptides with lysine at N termini as substrate. The optimal pH and temperature for this enzyme were 8.0 and 45 degrees C, respectively. The catalytic activity was inhibited slightly by Mg(2+), Al(3+), Ca(2+), Fe(3+), Fe(2+) and Ba(2+) strongly by Ni(2+), Mn(2+) and Co(2+), and almost inactivated by Zn(2+), Cu(2+) and Hg(2+). In addition, the enzyme was not sensitive to EDTA-Na(2), as well as redoxes of DTT, beta-ME and H(2)O(2). The protease inhibitors of benzamidine hydrochloride and phenylmethyl sulfonyfluoride caused a moderate inhibition. The V(max), K(m) and k(cat) toward Pro-pNA were 347.86 mumol min(-1) mg(-1), 2.15 mM and 218.10 S(-1), respectively. The deduced catalytic triad of Ser(107), Asp(264) and His(292) was confirmed by site-directed mutagenesis because the individual replacement of Ser(107) to Asp, Asp(264) to Ala or His(292) to Leu led complete inactivation. Transcriptional analysis by RT-PCR showed that PchPiPA could be expressed under ligninolytic and non-ligninolytic conditions. Conclusively, it was suggested that the proline iminopeptidase may be a member of the proteolytic system in this fungus. The availability of recombinant protein may be potentially used in certain proteolytic processing.


Proline-Specific Extracellular Aminopeptidase Purified from Streptomyces lavendulae

Aminopeptidases catalyze the cleavage of specific amino acids from the amino terminus of protein or peptide substrates. A proline-specific aminopeptidase was purified to homogeneity from the culture-free extract of Streptomyces lavendulae ATCC 14162 in sequential steps comprising ammonium sulfate precipitation, ultra-filtration, and column chromatography on Q-sepharose and Sephadex G-100. The purified protein showed approximately 60 kDa in SDS-PAGE and was optimally active at pH 6.5 and 40 °C. Kinetic studies showed a K m and V max of 0.23 mM and 0.087 μmol/min, respectively, using Pro-p-NA, the substrate with maximum specificity. Enzyme activity was inhibited by PMSF and ions like Zn 2+ , Co 2+ , and Ni 2+ . However, unlike other aminopeptidases, the activity was enhanced in the presence of DTT, 1,10-phenanthroline, EDTA, amastatin, and bestatin. Ions like Ca 2+ , Mg 2+ , and Mn 2+ also enhanced the activity.

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Results

Sequence analysis of the xccR/pip locus

Search of the sequenced genomes of Xcc ATCC 33913 ( da Silva et al., 2002 ) and Xcc 8004 ( Qian et al., 2005 ) by tblastn revealed that there is only one copy of the xccR/pip locus or each of its components. In the locus, the xccR gene is upstream of and in the same orientation to pip and the intergenic sequence is 438 bp long (Fig. 1A).

Sequence analysis of the xccR/pip locus. A. The genetic architecture of the xccR/pip locus and the pip promoter sequence. The 2.1 kb DNA sequence of the xccR/pip locus of Xcc 8004 consists of two directly orientated ORFs (XC_1295 and XC_1296) encoding XccR and PIP respectively. A 438 bp intergenic sequence, containing the putative promoter of pip, harbours a 20 bp palindromic sequence (luxXc box), a −35 element and a −10 element. B. Alignment of the Xcc luxXc box with the lux boxes identified in promoter regions of other bacterial genes. The names of the genes are indicated to the right of the sequences. C. xccR/pip-like loci found in other bacterial species.

The deduced amino acid sequence of PIP consists of 313 amino acid residues with a calculated molecular mass of 34 kDa. blast search ( Altschul et al., 1997 ) against the databases revealed that PIP of Xcc shared significant sequence similarities with PIPs from Lactobacillus delbrueckii (CAA81556, E = 2 × 10 −40 ) ( Klein et al., 1994 ), Thermoplasma acidophilum (1MU0_A, E = 2 × 10 −39 ) ( Goettig et al., 2002 ) and Bacillus coagulans (BAA01792, E = 2 × 10 −39 ) ( Kitazono et al., 1992 ). Pfam analysis ( Bateman et al., 2004 ) indicated that Xcc PIP, like all reported PIPs, contained an α/β-hydrolase fold with Ser106, Asp246 and His273 residues constituting the typical catalytic triad. The sequence of Xcc PIP is more similar to that of LaaA ( l -amino acid amidase) from Pseudomonas azotoformans IAM 1603 (BAD15092 E = 2 × 10 −104 ) ( Komeda et al., 2004 ). It is interesting to note that the substrate-specificity analysis showed that LaaA could not hydrolyse peptides but l -amino acid amides.

The predicted XccR protein contains 254 amino acids. blast analysis demonstrated that XccR shared significant sequence similarities with functional QS regulators including AhyR from Aeromonas hydrophila (CAA61654 E = 2 × 10 −13 ) ( Swift et al., 1997 ), BviR from Burkholderia cepacia (AAK35156 E = 3 × 10 −11 ) ( Lutter et al., 2001 ) and LasR from Pseudomonas aeruginosa (BAA06489 E = 1 × 10 −10 ) ( Gambello and Iglewski, 1991 ). Analyses with Pfam and SMART ( Letunic et al., 2004 ) indicated that XccR had an N-terminal autoinducer-binding domain and a C-terminal helix–turn–helix DNA-binding domain. However, among the nine residues that are identical in at least 95% of LuxR-type proteins ( Zhang et al., 2002 ), Trp57 and Tyr61 (numbered according to TraR) were not conserved in XccR. Both residues were shown to participate in the interaction between TraR and AHLs ( Zhang et al., 2002 ), suggesting that XccR might lack the ability to bind the AHL-type molecules. Indeed, we found XccR could not be activated by any of AHLs tested in our experiments to turn on the pip promoter-driven GUS expression (data not shown). Genome search also revealed that Xcc did not encode any homologue of AHL synthases including LuxI ( Fuqua et al., 1996 ), LuxM ( Bassler et al., 1993 ) and HdtS ( Laue et al., 2000 ), consistent with the documented result that Xcc produced no compounds with AHL activity ( Cha et al., 1998 ).

Within the 438 bp intergenic sequence, a 20 bp palindromic sequence is found centred at −70.5 from the translation start site of pip (Fig. 1A) and highly similar to the lux box sequences ( Gray et al., 1994 Schuster et al., 2004 Urbanowski et al., 2004 Weingart et al., 2005 ) (Fig. 1B). In this study, we termed this sequence as luxXc box. In the intergenic sequence, there are also a −35 element-like TTATCC sequence downstream of and two nucleotides overlapping with the luxXc box, and a −10 element-like TAGGCT sequence (Fig. 1A). The location of these sequence motifs are consistent with the previous documentation that the lux boxes always overlap with the −35 elements of σ 70 -type promoters by one or two nucleotides ( Fuqua et al., 1996 ). The overall structure of the Xcc xccR/pip locus prompted us to predict that XccR or its activated form might bind to the luxXc box within the pip promoter located in the intergenic region to regulate the expression of pip.

We then used tblastx to compare the 2.1 kb DNA sequence covering the xccR/pip locus with other bacterial genomes and similar loci were found in several other plant pathogenic and symbiotic species (Fig. 1C). For most of the pip genes in those xccR/pip-like loci, a lux box-like element is located in the putative pip promoter region (Fig. 1C). The biological function and expression regulation of these loci have not been investigated.

The xccR/pip locus is required for pathogenicity of Xcc on the host

To determine whether the xccR/pip locus affected the pathogenicity phenotype of Xcc, we constructed the xccR- or pip-disrupted Xcc strain, named Xcc 8515 and 8702 respectively. The virulence of the mutant strains as well as the wild-type strain was tested each on 20 cabbage leaves by the leaf-clipping method, and the symptoms were rated according to the severity scale (0–4) at 9 days post inoculation. As shown in Table 1, the average virulence level caused by Xcc 8702 was significantly lower than that of the wild-type strain (0.40 versus 3.80, P < 0.01). When Xcc 8702 was transformed with pFR419 carrying a lac promoter-driven pip gene (lac-P/pip), the complemented strain exhibited a virulence level similar to that of the wild-type strain (3.70 versus 3.80, P > 0.05). As a control, introduction of the empty vector pLAFR3 to Xcc 8702 did not increase the mutant's virulence. Thus, we conclude that the pip gene is required for pathogenicity of Xcc on the host cabbage.

Strains Average virulence score (SD) a
Xcc 8004 3.80 (0.51)
Xcc 8702 0.40 (0.58)
Xcc 8702/pFR419 3.70 (0.71)
Xcc 8515 0.55 (0.67)
Xcc 8515/pFR420 3.60 (0.80)
  • a. Mean and standard deviation (SD) for each strain were calculated from the virulence scores of 20 leaves on eight different individual plants at 9 days post inoculation.

Disruption of xccR reduced the virulence of Xcc to a score of 0.55 (Table 1, Xcc 8515), similar to that of Xcc 8702. Complementation with an xccR gene by a plasmid pFR420 carrying a lac-P/xccR chimeric gene restored the virulence level to 3.60, close to that of Xcc 8004 (P > 0.05). Again, introduction of pLAFR3 to Xcc 8515 did not increase the mutant's virulence. These data supported that the xccR gene is also indispensable for full virulence of Xcc.

Many bacterial mutants are auxotrophic, and they often caused a reduced pathogenicity phenotype just by affecting their survival due to the auxotrophy. These mutants could not grow on minimal media. To examine whether the severely attenuated virulence of Xcc 8702 and Xcc 8515 was caused by the effect of auxotrophy, we assayed the growth of both mutants on the minimal medium. We found that the growth rate of either mutant strain was similar to that of the wild-type Xcc 8004 or the corresponding complemented strains (data not shown), suggesting that pip or xccR per se is required for the virulence of Xcc on the host.

Overexpression of XccR activates the pip promoter

To examine whether XccR activates the pip promoter, we introduced the XccR-overexpressing plasmid pFR420 into Xcc 8004 and PIP activities of Xcc 8004/pFR420 at various bacterial growth stages including early and mid-exponential, transition and stationary phases were assayed and compared with those of the wild-type strain Xcc 8004. We found that PIP activity of Xcc 8004 remained very low throughout the bacterial growth phases, while the plasmid-expressed XccR significantly increased the PIP activity at each measuring time point (data not shown). These results indicate that poor expression of Xcc PIP in the wild-type background is not altered along with the bacterial growth in the culture medium and XccR is a potential activator of the pip promoter. To further confirm the induction of the pip promoter (pip-P) by the plasmid-expressed XccR, we constructed Xcc strain 8008 that contained both chromosomal pip-P/pip and pip-P/gusA fusions (Fig. 2). As was expected, in Xcc 8008, both PIP and GUS activities were at very low levels, while introduction of pFR420 into the strain greatly increased the expression levels of both PIP and GUS by 20- and 400-fold respectively (Fig. 3A). Whole-genome search of Xcc had identified two proteins (GenBank Accession No. XC_0928 and XC_3394) which have predicted protein topology and enzymatic active site residues similar to those of PIP but share only 15–17% of overall sequence similarity with PIP. To examine whether these two proteins were involved in the above-mentioned XccR overexpression experiments, PIP activity in Xcc 8702 with or without the plasmid pFR420 was measured. Only neglectable PIP activity was detected in Xcc 8702 or Xcc 8702/pFR420 (data not shown), implying these two proteins made little contribution to the increased PIP activity observed in Xcc 8004/pFR420 and Xcc 8008/pFR420 strains.

Construction of Xcc 8008 and Xcc 8116. By homologous recombination via the pip promoter sequence cloned (together with gusA) in the suicide vector pKnockout-Ω3 and present in the Xcc chromosome, the whole vector sequence was integrated into Xcc chromosome. The resultant Xcc 8008 contains both the pip-P/gusA chimeric gene and a native pip-P/pip gene. To construct Xcc 8116, a pip promoter lacking the luxXc box [pip-P(Δbox)] was used as a recombination sequence so that expression of gusA would be controlled by pip-P(Δbox), while the wild-type pip and its promoter were maintained intact. Position of the deleted luxXc box is indicated by a grey rectangle.

Plasmid-encoded xccR induces expression from the pip promoter carrying an intact luxXc box. All the strains were cultured in liquid NYG medium and the assays were carried out at a cell density of OD600 = 1.5. A. Expression levels of both PIP and GUS in different Xcc strains assayed by enzymatic activities. B. Levels of the pip and gusA transcripts in different Xcc strains measured by real-time RT-PCR. The abundance of transcripts in other strains is presented relative to that in Xcc 8008. The mean and SD in (A) and (B) were calculated from the data derived from five independent cultures, each with duplicated determinations.

Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to determine whether overexpression of XccR increased pip or gusA expression at the transcriptional level. Total RNA was extracted from the culture of Xcc strain 8008 or 8008/pFR420 at OD600 of 1.5. The gusA and pip mRNA levels in Xcc 8008/pFR420 were 70- and 11-fold higher, respectively, than those in Xcc 8008 (Fig. 3B). These results were consistent with those of enzymatic activity assays and further indicated that overexpression of XccR had activated the pip promoter.

The luxXc box is essential for XccR to induce the pip promoter

As the luxXc box was predicted to be the binding site of XccR, the role of the luxXc box played in the induction of the pip promoter by XccR was investigated. We constructed the Xcc strain 8116 in which the expression of pip was under the control of a native pip promoter, while the gusA gene was fused to a variant of the pip promoter lacking the luxXc box [pip-P(Δbox)] (Fig. 2). Plasmid pFR420 was then introduced into the strain. Enzymatic assay showed that the PIP activity in Xcc 8116/pFR420 was 18-fold higher than that in Xcc 8116, similar to an enhancement observed in Xcc 8008/pFR420, while the level of the GUS activity remained unchanged after introduction of pFR420 into Xcc 8116, in sharp contrast to that observed in Xcc 8008/pFR420 (Fig. 3A). The influence of XccR on expression of both pip and gusA genes in Xcc 8116 was also analysed at the transcriptional level by real-time RT-PCR. Again, overexpression of XccR increased the transcript level of pip with the intact promoter by 11-fold but had little influence on that of gusA with the mutated promoter lacking the luxXc box (Fig. 3B). It is clear from these results that the luxXc box is a necessary cis-element in the induction of the pip promoter by the potential transactivator XccR. This proposition was supported by a demonstration of an in vitro binding of XccR to the luxXc box as shown by the gel-mobility shift experiments. When the total protein of Xcc 8004/pFR420 was incubated with a 32 P-labelled 46 bp DNA probe containing an intact luxXc box sequence, a protein–DNA complex was formed as a shifted band (Fig. 4A, lane 2). This shifted band could be competed by the unlabelled probe (Fig. 4A, lanes 3 and 4) but not by a DNA fragment without the luxXc box sequence (Fig. 4A, lane 5). The involvement of XccR in the observed protein–DNA complex was confirmed by the absence of the shifted band when a total protein of the xccR-disrupted strain 8515 was used in the probe-binding experiment (Fig. 4A, lane 6), and by appearance of a supershifted band only after the anti-XccR antiserum, not the pre-immune serum, was co-incubated with the total protein of Xcc 8004/pFR420 and the labelled probe (Fig. 4B, lanes 1 and 2). Furthermore, the specific association of XccR with the luxXc box sequence in Xcc cells was shown by chromatin immunoprecipitation (ChIP) experiments which were performed on Xcc 8116 transformed with the XccR-overexpressing plasmid pFR420. The anti-XccR antiserum was used to precipitate DNA sequences cross-linked to XccR. Real-time PCR analyses of the precipitated DNA sequences indicated that the abundance of the pip promoter containing an intact luxXc box in front of the pip gene was threefold higher than that of the luxXc box-deleted pip promoter sequence upstream of gusA as well as that of a gusA-coding region which was served as a control of apparently not bound by XccR (data not shown).

Gel-mobility shift experiments characterizing in vitro binding of XccR to the luxXc box. A. A 32 P-labelled 46 bp DNA fragment containing the luxXc box sequence (Probe) was used in the protein-binding assays (lane 1). A protein–DNA complex (Shift) was formed when the total protein of Xcc 8004/pFR420 was incubated with the probe (lane 2). The shifted band could be competed by 50- (lane 3) or 100-fold (lane 4) excess of the unlabelled probe, but not by 200-fold excess of a DNA fragment (Δbox) without the luxXc box sequence (lane 5). There was no such a shifted band when the total protein of the xccR-disrupted strain Xcc 8515 was incubated with probe (lane 6). The dried gel was exposed to X-ray film at −20°C for 12 h. B. Supershift assays. Involvement of XccR in the shifted band was revealed by a supershifted band (Super shift) when the anti-XccR antiserum (lane 2), not the rabbit pre-immune serum (lane 1), was co-incubated with the total protein of Xcc 8004/pFR420 and the labelled probe. The supershifted band was significantly enhanced when Xcc 8004/pFR420 was pre-incubated in the cabbage extract for 12 h before the total protein was extracted (lane 4). The dried gel was exposed to X-ray film at −20°C for 10 days.

As the sequence of the luxXc box overlaps with the predicted −35 element of the pip promoter by two nucleotides, deletion of the luxXc box may change the basal activity of the pip promoter. To clear this, we constructed Xcc strain 8173 in which gusA was under the control of a truncated pip promoter lacking only the upstream half of the luxXc box. When plasmid pFR420 was introduced into this strain, the GUS activity was not increased (Wang, L., unpublished data). This result further confirmed the importance of an intact luxXc box in activation of the pip promoter by XccR. In addition, as half-site of the palindromic sequence was not enough for the induction by XccR, we predicted that XccR may be an ambidextrous activator, similar to most of LuxRs which functioned as dimers.

The pip expression is induced by the host plant via the cis-element luxXc box

We have shown that in NYG medium culture, the pip promoter was induced by the overexpressed XccR and the luxXc box was essential for this induction. As pip was shown to be required for Xcc's pathogenicity, we were interested to see if pip expression in Xcc is also enhanced by the host in the Xcc-infected plants and the function of the luxXc box in the enhancement. For this purpose, we first assayed the expression level of gusA in Xcc 8008 grown in planta. The bacterial cells re-suspended in sterile water (OD600 = 0.5) were vacuum-infiltrated into 2- to 3-week-old cabbage seedlings. After 33 h, the bacterial cells were recovered from the plant leaves and GUS activity was assayed. The level of GUS activity in Xcc 8008 grown in planta was eightfold higher than that in the bacterium cultured either in NYG medium (Fig. 5A) or in minimal medium MMX (data not shown), indicating that the pip promoter was induced by the host cabbage. On the other hand, when Xcc 8116 was vacuum-infiltrated into the cabbage seedlings, the in planta growth of Xcc 8116 did not enhance the GUS expression compared with the GUS activity in this strain grown in NYG medium (Fig. 5A), indicating that the luxXc box was required for the in planta induction of the pip promoter.

Expression of the chromosomal pip-P/gusA fusion gene is induced in planta. A. In planta cultivation significantly increased the GUS activity from Xcc 8008 while had no influence on that of Xcc 8116. The bacteria were recovered from infiltrated cabbage leaves 33 h post infiltration and GUS activity was assayed. GUS activity in the bacteria grown in NYG medium was assayed at an OD600 of 1.5. The mean and SD were calculated from the data derived from 10 independent experiments. B. The growth rates of Xcc 8008 and 8116 after infiltrating into cabbage seedlings. The experiments were repeated four times with similar results. The figure shows a representative set of data.

We also examined the growth rate of Xcc strains 8008 and 8116 in planta. Both strains exhibited similar growth curves (Fig. 5B), thus excluding the possibility that the different responses of GUS expression to the host induction between strains 8008 and 8116 was caused by the difference in their growth in planta.

To further confirm the induction of the pip promoter by the host plant and through the luxXc box, we constructed GUS reporter plasmids pFR421 that carried a pip-P/gusA fusion and pFR422 carrying a gusA gene fused to pip-P(Δbox). The two plasmids were separately introduced into Xcc 8004, and the resultant strains were infiltrated into the cabbage seedlings. As predicted, the in planta cultivation increased the GUS activity in Xcc 8004/pFR421 by eightfold (Fig. 6, A), while had no influence on that in Xcc 8004/pFR422 (Fig. 6, B). The results were consistent with those observed by using Xcc strains containing chromosomal pip-P/gusA (8008) or pip-P(Δbox)/gusA (8116) (Fig. 5A). We then conclude from these results that the expression of pip was induced by the host plant, and the luxXc box was required for this in planta induction.

Induction of the pip promoter by the host plant monitored by reporter plasmids. The wild-type Xcc 8004 carrying a plasmid-borne pip-P/gusA fusion construction (Xcc 8004/pFR421) exhibited increased GUS activity when grown in planta (A). Deletion of the luxXc box in the pip-P/gusA fusion (Xcc 8004/pFR422) (B) or disruption of xccR in the Xcc chromosome (Xcc 8515/pFR421) (C) resulted in a failure in in planta induction of gusA expression. Disruption of the chromosomal pip gene (Xcc 8702/pFR421) enhanced the induction of the pip promoter by the host plant (D). GUS activity was assayed on the bacteria grown in NYG medium at an OD600 of 0.5, or cultured in the host 28 h post infiltration. The mean and SD were calculated from the data derived from five independent cultures.

The in planta induction of the pip promoter is dependent on XccR

We have shown that overexpression of XccR could activate the pip promoter in medium culture and that the pip promoter was induced in planta in the absence of the plasmid-expressed XccR but in the presence of the chromosomal xccR gene. To explore whether XccR expressed from the chromosomal xccR contributes to the induced expression of pip in the host plant, we constructed an xccR-disrupted strain 8515 from Xcc 8004 to determine the effect of vanishing of XccR on the induction of the pip promoter by the host. Plasmid pFR421 was introduced into Xcc 8515 and then Xcc 8515/pFR421 was vacuum-infiltrated into the cabbage seedlings. Bacterial cells were recovered 28 h post infiltration, and GUS activity was assayed. The result showed that the GUS activity in recovered Xcc 8515/pFR421 was much lower (Fig. 6, C) than that in Xcc 8004/pFR421 cultured in planta (Fig. 6, A), suggesting that the chromosomally expressed XccR was indeed necessary for the in planta induction of the pip promoter. We also compared the gusA expression level in Xcc 8004/pFR421 with that in Xcc 8515/pFR421 when they were cultured in NYG medium. No obvious difference in GUS activity was found between the two strains (Fig. 6, A and C), suggesting that when Xcc was grown in NYG medium, the chromosomal xccR expressed at a very low level or yielded a dysfunctional product unable to activate the pip promoter. We assume that the plant environment or some specific plant factors may help XccR become an active transcription regulator. This assumption was partly substantiated by the result of a supershift experiment in which a total protein of Xcc 8004/pFR420 that had been incubated in a cabbage extract for 12 h was used for formation of the ternary complex with the anti-XccR antiserum and the luxXc box-containing probe. The formed supershift band (Fig. 4B, lane 4) was significantly intensified compared with that formed with the total protein of the same strain cultured in NYG medium (Fig. 4B, lane 2), indicating that the host plant extract can enhance the activity of XccR to bind to the pip promoter.

PIP negatively regulates the pip promoter in planta

In a classic QS regulation system, LuxR, LuxI and AHL constitute a positive feedback circuit ( Engebrecht et al., 1983 ). Disruption of either gene can result in a failed induction of the luxI promoter by the LuxR/AHL complex. We were interested to see whether PIP also had some influence on its own expression. Plasmid pFR421 which carries a pip-P/gusA fusion was introduced into the pip-disrupted strain Xcc 8702. GUS activity in Xcc 8702/pFR421 grown in medium or recovered from the cabbage leaves was assayed and compared with that in Xcc 8004/pFR421 from the same growing condition. When cultured in planta, GUS activity in Xcc 8702/pFR421 (Fig. 6, D) was 2.3-fold higher than that in Xcc 8004/pFR421 (Fig. 6, A). However, when grown in medium, the two strains showed a similar low level of gusA expression (Fig. 6, A and D). We can conclude from these results that PIP negatively regulated the activity of its own promoter only when Xcc was grown in planta. Apparently, when Xcc was grown in planta, the pip promoter was subject to both positive and negative regulations by XccR and PIP respectively. The physiological implications of such a counteraction on Xcc pip expression remain to be elucidated.

PIP is a periplasmic protein

Information about subcellular localization of bacterial proteins may provide useful clues to the elucidation of their biological functions. To examine the subcellular localization of the Xcc PIP, total cellular protein from Xcc cells cultured in NYG medium was fractionated into periplasmic and cytoplasmic fractions by osmotic shock and PIP activities in each fraction were assayed. Because the GUS protein is known to accumulate in the cytoplasm, it was used as a marker to monitor the leakage of proteins from the cytoplasmic space. We used several strains to analyse the distribution of PIP and GUS in periplasm and cytoplasm, including Xcc 8008 in which both gusA and pip expressed at low levels, Xcc 8008/pFR419 in which PIP is overexpressed from the plasmid pFR419 carrying a lac-P/pip fusion gene, and Xcc 8008/pFR420 in which both genes expressed at high levels. Typically in Xcc 8008/pFR420, 93.4% of the total cellular PIP activity was found in the periplasmic fraction, while only 5.6% of the total GUS activity was in this fraction (Fig. 7). Similar distribution patterns of PIP and GUS were found in Xcc 8008 and Xcc 8008/pFR419 (data not shown). Moreover, only neglectable PIP activity was detected in the cell–free culture supernatants (data not shown), indicating that PIP was not secreted extracellularly. From these results, the Xcc PIP proved to be localized predominantly in the periplasm. Because PIP was shown to be capable of regulating its own expression, it is very likely that the self-regulation of PIP is through an indirect way.

Xcc PIP is localized in the periplasmic space. Xcc 8008/pFR420 was cultured in NYG medium and the total cellular protein was fractionated into periplasmic and cytoplasmic fractions by osmotic shock. PIP and GUS activities in each fraction were determined and are expressed as percentages of the total cellular activities of PIP and GUS respectively. The mean and SD were calculated from the data derived from three independent cultures harvested at OD600 of 1.5.


Materials and methods

Crystal data

XCPIP was purified and crystallized using NaCl as precipitating agent as previously described ( Medrano et al., 1997 ). These crystals belong to the orthorhombic space group C222, contain one homodimer per asymmetric unit and have cell constants a = 147.2 Å, b = 167.8 Å and c = 85.6 Å. These crystals diffract beyond 2.7 Å resolution.

Heavy-atom derivatives of the enzyme were prepared at room temperature by soaking crystals in heavy-atom solutions (Table I). One native and several derivative X-ray diffraction data sets were collected on a 300 mm MAR-Research image plate detector attached to a Rigaku RU200 rotating anode generator providing graphite monochromatized CuKα radiation at −15 to −20°C. Data were processed with the MOSFLM package ( Leslie, 1991 ), and loaded, scaled and merged with the CCP4 package ( CCP4, 1994 ). Statistics for native and heavy-atom derivative data are given in Table I.

Compound Concentration Time Limiting resolution (Å) No. of collected/unique reflections Completeness of data (%) Rmerge d d Rmerge = Σ|I−<I>|/ΣI, where I is the measured intensity and <I> is the average intensity obtained from multiple measurements of symmetry-related reflections.
Riso e e Riso = Σ‖FP|−|FPH‖/Σ|FP|, where |FP| is the protein structure-factor amplitude and |FPH| is the heavy-atom derivative structure-factor amplitude.
Phasing power f f Phasing power = r.m.s. (|FH|/E), where |FH| is the heavy-atom structure-factor amplitude and E is the residual lack of closure calculated for acentric data (20-3.0 Å).
Sites
Native 2.7 154 900/29 854 93.9 9.6 - - -
HG1 a a 0.1 M sodium citrate, pH 6.0 and 4.0 M NaCl.
HgS Saturated 3 days 3.6 27 028/9781 78.4 7.4 0.311 1.54 3
HG2 b b 0.2 M Tris-HCl, pH 8.1 and 15% polyethylene glycol monomethyl ether 5000.
1-(4-chloromercuriphenylazo)-2-naphthol (C16H11ClHgNO2) Saturated 2 days 3.0 64 607/18 837 84.6 12.8 0.256 0.16 2
PT1 b b 0.2 M Tris-HCl, pH 8.1 and 15% polyethylene glycol monomethyl ether 5000.
Pt(II)-(2,2′-6′,2′′-terpyridine)-chloride Saturated 2 days 3.0 65 116/20 036 93.8 14.7 0.177 0.88 2
PT2 a a 0.1 M sodium citrate, pH 6.0 and 4.0 M NaCl.
K2PtCl4 10 mM 16 h 3.0 52 678/19 170 88.7 8.9 0.242 0.29 3
URA c c 0.1 M sodium citrate, pH 5.0 and 4.0 M NaCl.
Na2U2O7 Saturated 16 h 3.0 71 979/14 608 68.4 12.9 0.101 0.12 1
  • a 0.1 M sodium citrate, pH 6.0 and 4.0 M NaCl.
  • b 0.2 M Tris-HCl, pH 8.1 and 15% polyethylene glycol monomethyl ether 5000.
  • c 0.1 M sodium citrate, pH 5.0 and 4.0 M NaCl.
  • d Rmerge = Σ|I−<I>|/ΣI, where I is the measured intensity and <I> is the average intensity obtained from multiple measurements of symmetry-related reflections.
  • e Riso = Σ‖FP|−|FPH‖/Σ|FP|, where |FP| is the protein structure-factor amplitude and |FPH| is the heavy-atom derivative structure-factor amplitude.
  • f Phasing power = r.m.s. (|FH|/E), where |FH| is the heavy-atom structure-factor amplitude and E is the residual lack of closure calculated for acentric data (20-3.0 Å).

Structure determination and refinement

The structure was solved by multiple isomorphous replacement (MIR). Heavy-atom positions were localized by difference Patterson maps, vector verification calculations and difference Fourier maps using the program PROTEIN ( Steigemann, 1974 ). The correct handedness was established using the procedure described by Hoeffken (1988) . Heavy-atom positions, occupancies and isotropic B-factors were refined with the program MLPHARE as implemented in the CCP4 package. The mean figure of merit at 3.0 Å resolution was 0.33 (see Table I for heavy-atom refinement and phasing statistics).

The initial 3.0 Å Fourier map was improved by density modification, including solvent flattening, solvent flip and skeletonization in two cycles with DM as implemented in the CCP4 package. After this step, the average figure of merit increased to 0.80, in particular due to the high solvent content (65%). The boundaries of both crystallographically independent molecules were clearly visible. An initial discontinuous poly-alanine polypeptide model was built into this modified electron density using the program TURBO-FRODO ( Roussel and Cambilleau, 1992 ). Atomic models were refined applying NCS restraints using the program X-PLOR ( Brünger, 1992 ). The electron density was additionally improved performing 2-fold averaging with the RAVE package ( Kleywegt and Jones, 1994 ).

Calculated phases, obtained from intermediate models in successive cycles of manual rebuilding, were combined with the phases obtained after density modification using the program SIGMAA as implemented in the CCP4 package these combined phases were used for calculation of 2FoFc and FoFc maps. The amino acid sequence ( Alonso and García, 1996 ) was successively built in to completion of the final model containing residues 1-313 for each crystallographically independent molecule. Additionally, 199 solvent molecules placed at stereochemically reasonable positions were added with the help of the program WATPEAK of CCP4 and checked visually. Positional and individual constrained isotropic B-factor refinement was performed with X-PLOR. A summary of the refinement statistics is shown in Table II. A Ramachandran plot of the main chain torsion angle pairs, calculated with the program PROCHECK ( Laskowski et al., 1993 ), show all residues but Ser110 situated in allowed regions, with only the latter in a generously allowed position. Pro44 and Pro76 exhibit a cis conformation. No significant differences are observed between both crystallographically independent molecules, reflected by the low r.m.s. deviation of 0.11 Å for all 313 common Cα atoms. The polypeptide chains of both molecules could be entirely traced only six side chains of molecule A and five of molecule B had to be inactivated due to partially discontinuous density.

Space group C222
Cell constants
a 147.2 Å
b 167.8 Å
c 85.6 Å
Limiting resolution 2.7 Å
Non-hydrogen protein atoms 6140
Solvent molecules 199
Reflections used for refinement 26 672
Resolution range 7.0-2.7 Å
R-factor 19.2%
Rfree 25.3%
R.m.s deviations from target values
bond length 0.010 Å
bond angles 1.530°
bonded B-factors 2.613 Å 2

Comparison of the XCPIP structure with other known three-dimensional structures was carried out with the program DALI ( Holm and Sander, 1993 ).

The X-ray coordinates of XCPIP will be deposited at the Brookhaven Protein Data Bank.