*Department of Biochemistry and Molecular Biology, Monash University, Melbourne;
Victorian Bioinformatics Consortium, Monash University, Melbourne;
Department of Microbiology, University of Nijmegen, The Netherlands;
Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge Clinical School, U.K
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Abstract |
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Introduction |
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In contrast to small "rigid" proteinase inhibitors such as those of the Kazal or Kunitz family, inhibitory serpins rely on a complex conformational change to inhibit the target proteinase (Huntington, Read, and Carrell 2000
). This mechanism requires precise timing to capture the target proteinase: the serpin fold has evolved to function as a finely tuned molecular machine. Some mutations that alter the conformational mobility of the serpin scaffold can result in the spontaneous transition to inactive forms, such as polymers or the latent conformation (see Stein and Carrell 1995
). Other mutations interfere with the process of conformational change and impair serpin function by promoting substrate-like rather than inhibitory behavior (Hopkins, Carrell, and Stone 1993
; Stein and Carrell 1995
).
A fundamental, unanswered question in the field is the evolutionary origin of the serpin fold, which is closely related to the origin of the serpin mechanism itself. In a previous study, we performed an extensive phylogenetic investigation of the serpin superfamily (Irving et al. 2000
). Despite using sensitive database searching methods such as PSI-BLAST and Hidden Markov Models, we were able to identify serpins only in viruses and higher (multicellular) eukaryotes and were unable to identify any putative prokaryotic or fungal serpin. This observation was somewhat surprising because the presence of serpins in both the animal and plant kingdoms suggests that they should also be found in a common ancestor (e.g., fungi or prokaryotes). Information bearing on the question of whether the ancestral serpin was a proteinase inhibitor would provide insight into the origin of the inhibitory mechanism: did this mechanism develop with the serpin fold or did the first serpin fulfill a noninhibitory function? Current biochemical evidence does not provide a simple answer. The most common serpin targets, trypsin-like serine proteinases, have bacterial homologs (HtrA; Lipinska, Zylicz, and Georgopoulos 1990
), and serpins have shown activity against bacterial subtilisin (Komiyama et al. 1996
; Dahlen, Foster, and Kisiel 1997
) and the caspase-like gingipain K enzyme from Porphyromonas gingivalis (Snipas et al. 2001
). These enzymes are present in organisms that lack recognizable serpin sequences.
In this study we have identified, from recently released genomic data, the first examples of prokaryotic serpins. Two closely related cyanobacteria, Nostoc punctiforme and Anabaena sp. PCC 7120, the firmicutes Thermobifida fusca, Desulfitobacterium hafniense, Ruminococcus albus, and Thermoanaerobacter tengcongensis, the green nonsulfur bacteria Dehalococcoides ethenogenes, the crenarchaeote Pyrobaculum aerophilum, and the euryarchaeotes Methanosarcina acetivorans and Methanosarcina mazei all contain putative serpin sequences. Sequence motifs and patterns of conservation suggest that they adopt a serpin-like fold and are capable of proteinase inhibition. The distribution of prokaryotic serpin sequences is sporadic. Although the serpin-bearing prokaryotes occur in markedly different environments, it remains unclear as to whether these genes have arisen through lateral gene transfer from eukaryotes or through inheritance of an ancient, prokaryotic serpin ancestor. The existence of a serpin in a hyperthermophilic organism is of particular interest. Using molecular modeling with reference to experimental evidence, we suggest a means by which resistance to polymerization can be obtained without compromising inhibitory activity.
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Materials and Methods |
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Sequence Analysis and Phylogenetic Relationships
The putative serpins were aligned and phylogenetic trees constructed as described previously (Irving et al. 2000
). Briefly, a structure-based alignment was generated of human
1-antitrypsin (pdb accession 1qlp), human antithrombin (1azx), Manduca sexta serpin 1K (1sek), and chicken ovalbumin (1ova). The bacterial serpins were aligned to this "seed" alignment with reference to secondary structure-specific gap penalties using CLUSTALW (Higgins, Thompson, and Gibson 1996
). Sites in the alignment were compared with sites strictly conserved in >70% of the serpin superfamily (identified in Irving et al. 2000
).
A profile alignment was used to incorporate these sequences into an alignment of 219 sequences from the superfamily as a whole, which in turn was used to derive bootstrapped neighbor-joining distance-based trees (500 replicates, JTT substitution model, using MOLPHY; Adachi and Hasegawa 1996
). Clusters of evolutionarily related sequences were obtained after analysis of the bootstrapped trees using the majority consensus tree approach (Felsenstein 1985
), the comparison consensus method, and the tree division method (Irving et al. 2000
). For the tree division method, the "clade-defining" proteins were from (1) vertebrates: human
1-antitrypsin (GenPept accession P01009), heat shock protein 47 (P29043), neuroserpin (Q99574), heparin cofactor II (AAC16324), alpha-2-antiplasmin (Q95121), angiotensinogen (P11859), and ovalbumin (P01012); (2) arthropods: Limulus intracellular coagulation inhibitor-1 (BAA06909) and M. sexta serpin 1K (AAB58491); (3) nematodes: Caenorhabditis elegans (AAB37049); (4) plants: barley protein Z (CAA66232), Triticum aestivum (CAA72274) and Arabidopsis thaliana (AAC27146).
Local Sequence Clusters
Predicted genes at genome positions 892,000912,000 (Anabaena sp. PCC 7120), 15,00035,000 (P. aerophilum), 2,750,7572,773,734; 3,235,9483,255,573; and 4,189,8204,190,826 (M. acetivorans), 1,508,8681,529,055 (T. tengcongensis), and sections 295297 (accessions AE013513AE013515, M. mazei) were obtained from the GenBank database. Nucleotide sequence from the region surrounding each putative serpin was obtained for T. fusca (within 10 kb upstream and downstream, on contig 64), N. punctiforme (contig 423), and D. hafniense (contig 3204). Each region was then compared with the nonredundant database using the standalone blastx program (genetic code 11; maximum expect 1 x 10-3; otherwise default parameters) and potential genes were thereby inferred.
Search for Proteinases Correlating with the Presence of Serpins
Sequence alignments representative of all proteinase families in the MEROPS database (http://www.merops.co.uk; Rawlings, O'Brien, and Barrett 2002
) were obtained. A consensus sequence was derived from each alignment, and in conjunction with tblastn it was used to identify homologs in the prokaryotic genomes.
Molecular Modeling
The serpin from P. aerophilum was modeled using the MODELLER program (Sali and Blundell 1993
) within the QUANTA package (Accelrys, San Diego, Calif.) using the structure of antithrombin (PDB accession, 1azx) as a template. The alignment between the two proteins was obtained using PSI-BLAST and manually adjusted to take account of elements of secondary structure. Where necessary, manual side-chain refinement was performed using CHARMm. A Ramachandran plot revealed that all residues in the model were in allowed conformations.
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Results |
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It has been noted that coexpressed genes are frequently situated as contiguous clusters within an operon. But an analysis of regions adjacent to the serpin sequences did not reveal, from one organism to the next, a consistent pattern of neighboring genes that might indicate the presence of a conserved operon. Five of the sequences were taken from unassembled contig data; thus whether they are present in transposable elements or plasmids is not yet clear. The genes from Anabaena sp. PCC 7120, P. aerophilum, T. tengcongensis, M. mazei, and M. acetivorans are localized to the single chromosome of each organism.
Recognizable serpin sequences are not ubiquitous or even widely distributed in the prokaryotic kingdom. Serpins appear to be absent from all other sequenced archaeal organisms (13 finished and two unfinished), from proteobacteria (26 finished and 43 unfinished), and from several other branches less well represented in genome sequencing efforts. Furthermore, one finished and four unfinished cyanobacterial genomes (including N. punctiforme) and 21 complete firmicute genomes (a family which includes T. fusca) also lack serpins.
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Discussion |
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The actinomycete T. fusca (formerly Thermomonospora fusca) is a thermophilic soil bacterium with an optimal growth temperature of 55°C that plays an important role in the degradation of plant detritus, for example in compost heaps (Crawford 1975
). A related organism, the firmicute T. tengcongensis, grows at 75°C and is found in hot springs (Xue et al. 2001
). The crenarchaeon P. aerophilum, a member of which has been obtained from a boiling marine water hole, is a hyperthermophile with an optimal growth temperature of 100°C (Fitz-Gibbon et al. 2002
). The existence of serpins in thermophilic organisms is of particular interest because serpins are metastable in their native state and susceptible to heat-induced polymerization (Lomas et al. 1992
). Thermophilic organisms' euryarchaeote neighbors, M. acetivorans and M. mazei, are methane-producing organisms that grow at moderate temperatures (3540°C) and were first isolated from marine sediments (Sowers, Baron, and Ferry 1984
). Desulfitobacterium hafniense is a gram-positive, endospore-forming, strictly anaerobic bacterium initially isolated from municipal sludge that is capable of dechlorinating both aromatic and alkyl chlorinated compounds (for a review see El-Fantroussi, Naveau, and Agathos 1998
). The filamentous nitrogen-fixing cyanobacterium N. punctiforme is able to form symbiotic relationships with a variety of terrestrial plants. Furthermore this organism is able to form a partnership with an obligate symbiotic fungus (Zygomycotina) to form the organism Geosiphon pyriforme, the only known example of endocytobiosis (intracellular association of two cells) between a fungus and cyanobacteria (Gehrig, Schussler, and Kluge 1996
). Dehalococcoides ethenogenes is a eubacterium capable of reducing tetrachloroethene to ethane (Maymo-Gatell et al. 1997
). Dehalococcoides ethenogenes, along with strain CBDB1 and several uncultivated bacteria, forms part of a clade phylogenetically removed from other bacterial families (Adrian et al. 2000
). Finally, R. albus is a gram-positive, cellulolytic anaerobe that inhabits the gut of herbivores (Leatherwood 1965
). Thus serpins appear to be present in a wide variety of prokaryotes that live in diverse environments.
Bootstrapped neighbor-joining trees were constructed on the basis of the alignment of the prokaryotic serpins with other members of the serpin superfamily (identified in Irving et al. 2000
). The majority consensus tree revealed three strictly conserved associations: the two cyanobacteria, N. punctiforme and Anabaena sp. PCC 7120, clustered together in 100% of the time; the firmicutes D. hafniense, R. albans and T. tengcongensis coincided in 100% of trees; and the euryarchaeote serpins from Methanosarcina spp. also formed a clade with 100% support (fig. 2 ). These associations are not unexpected: they reflect the close evolutionary origin of these prokaryotic species. The comparison consensus method, which identifies underlying relationships that may be obscured by other poorly resolved species, revealed a significant (100%) association between the cyanobacterial and firmicute serpins. It was not possible from these analyses to identify a well-supported relationship between these five bacterial serpins and those from T. fusca, D. ethenogenes, P. aerophilum, the Methanosarcina clade or any other subfamily. These five sequences therefore represent a novel bacterial clade in the serpin superfamily, the four Methanosarcina proteins form an archaeal clade, and the other three serpins are, at present, "orphans" (fig. 2
).
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Of 77 finished genomes, 67 did not contain any identifiable serpin homolog. Three scenarios seem feasible:
The latter two scenarios seem more favorable in light of the sporadic distribution of serpin genes in prokaryotic genomes; however, there is no evidence that strongly favors one over the other. With the elucidation of further sequences of prokaryotic organisms living at the boundaries of life, it should be possible to elucidate the evolutionary origin of serpins with greater certainty.
Sequence alignment of the 12 serpins with serpins of known structure reveals that the bacterial sequences, and all but one of the archaeal sequences, are predicted to contain the full complement of elements of secondary structure (fig. 1
). This is in contrast to viral serpins, several of which contain deletion of entire secondary structure elements (Renatus et al. 2000
; Simonovic, Gettins, and Volz 2000
; Guerin et al. 2001
). As with the viral serpins, the protein from the archaeon P. aerophilum appears to have lost its D-helix; however, we predict that P. aerophilum retains all other structural elements. All 12 prokaryotic serpins contain the pattern of small amino acids in the hinge region of the reactive center loop (RCL) (see fig. 1
), characteristic of serpins that function as proteinase inhibitors; hence we suggest that the most likely function of these proteins is the inhibition of proteinases.
One striking feature of four of the bacterial serpins (the two Nostoc species, D. ethenogenes and D. hafniense) is that the P1 and P1' residues (refer to abbreviations for Pn notation) are identical (T-S). The RCL has been noted to be variable in duplicated serpin genes undergoing functional diversification (Inglis et al. 1991
; Kaiserman et al. 2002
), and this is indicative of negative (purifying) selection. Two observations further support this hypothesis. First, these serpins are substantially different from one another overall, with 34%69% identity. Second, three of the sequences share a strong evolutionary relationship (Nostoc spp. and D. hafniense; see fig. 2
), but R. albus and T. tengcongensis, which are also predicted to be part of this clade, do not have a T-S at the P1-P1'. Furthermore, D. ethenogenes (which does not share a significant evolutionary relationship with Nostoc spp. or D. hafniense) does have a T-S at this site. It therefore appears likely that the four predicted inhibitors from Nostoc spp., D. hafniense, and D. ethenogenes are under selective pressure to target proteinases of similar or identical specificity. Similarly, T. fusca and T. tengcongensis both have the residues A-G; R. albus and M. acetivorans 1 share a P1E; and P. aerophilum and M. acetivorans share P1V.
The sequences A-G and T-S do not fit the substrate profile of any known serine proteinase. But examination of serpins that are known to inhibit papain-like cysteine proteinases reveal that these inhibitors often contain small residues such as Thr at P1 (Schick et al. 1998
; Irving et al. 2002
). Furthermore, the P2 position (usually of greater importance in targeting a serpin to a cysteine proteinase) is usually occupied by a large hydrophobic residue that interacts with the S2 specificity pocket. The inhibitors from T. fusca and D. ethenogenes both have a P2 leucine, and we hypothesize that these serpins may be able to inhibit papain-like cysteine proteinases.
The only serpins known to have an acidic P1 residue are the granzyme B inhibitor PI-9 (Sun et al. 2001
) and the caspase inhibitor crmA (Ray et al. 1992
). Nevertheless, it seems unlikely that the R. albus serpin (P1-P1' sequence E-A) would interact with granzyme B or caspase-1 in a physiological context such as the milieu of the rumen. We note that several bacteria have been shown to express enzymes that can cleave substrates with acidic P1 residues (Barbosa, Saldanha, and Garratt 1996
), including the V8 serine proteinase of the bacterium Staphylococcus aureus, which has a homolog in R. albus.
Methanosarcina acetivorans is unique among the prokaryotes in that it possesses three serpin genes, each of which has a unique P1-P1' sequence, suggesting that each targets a different enzyme. Methanosarcina mazei appears to have inherited one of these inhibitors. The close predicted evolutionary relationship between M. acetivorans serpin 3 and the M. mazei serpin and the similarity in their RCL sequence (P1-P1' of G-V and G-M) suggests the two may interact with the same class of proteinase.
We were unable to find a direct correlation between any of the current proteinase family classifications and serpins in the prokaryotic genomes. Therefore, it remains to be determined whether the serpins have evolved to inhibit endogenous proteinases or to target proteinases in the local environment.
The presence of a serpin in P. aerophilum is particularly intriguing because its optimum growth temperature (100°C) is well in excess of the temperature that a typical serpin would be expected to remain in the active inhibitory conformation. Numerous studies have demonstrated that serpins are susceptible to heat-induced polymerization, as a consequence of the metastability of the native state (Lomas et al. 1992
). This metastability is crucial for serpin inhibitory function: conformational change is required to trap the target proteinase in a distorted, inactive state (Huntington, Read, and Carrell 2000
). Certain members of the serpin family do demonstrate enhanced stabilityfor example, the noninhibitory serpin ovalbumin denatures at 73°C (Dong et al. 2000
). Therefore, while it is conceivable that the serpin from T. fusca serpin may remain active at 55°C through a dramatic increase in stability of the native form, we suggest that the serpin from P. aerophilum must demonstrate some special feature in order for it to use conformational change for inhibition of target proteinases at 100°C. Although it is possible that the P. aerophilum protein no longer undergoes conformational change to inhibit target proteinases, the presence of a typical inhibitory "hinge" in this serpin suggests that this is not the case. To investigate the P. aerophilum serpin further, we built a molecular model using the structure of antithrombin as a template. These data reveal that the predicted P. aerophilum serpin is able to adopt the serpin fold and that all essential elements of the serpin "core" are present (fig. 3
). We predict that the P. aerophilum serpin lacks the D-helix, however, this is not unprecedentedthe X-ray crystal structure of the viral serpin crmA reveals that the D-helix can be "lost" without disrupting the serpin fold (Renatus et al. 2000
; Simonovic, Gettins, and Volz 2000
). In comparison with typical serpins, the P. aerophilum serpin is also predicted to contain a significant insertion at the base of the E-helix. Most strikingly, however, we note that the RCL of the P. aerophilum serpin contains a cysteine residue at the P1' position that we predict would be able to form a disulfide bond with a second cysteine on strand s3C of the C ß-sheet. Stabilizing disulfides have been noted in the crystal structure of an intracellular P. aerophilum enzyme, adenylosuccinate lyase (Toth et al. 2000
). Such an interaction would be predicted to "tie down" the RCL and prevent inappropriate conformational change (such as polymerization). In a study based on antitrypsin, in which a disulfide bond was introduced between the RCL and strand s1C of the C ß-sheet, the presence of a covalent linkage prevented the serpin from polymerizing (Chang et al. 1997
). It was hypothesized that the introduction of this disulfide bond prevented polymerization by restricting conformational change in the RCL and the first strand of the C ß-sheet. We predict that a similar situation exists in the P. aerophilum serpin (fig. 4A
). A related study showed that the presence of the introduced disulfide bond in antitrypsin did not affect inhibitory activity (Hopkins et al. 1997
). Similarly, the predicted disulfide bond in P. aerophilum serpin would not be expected to affect the inhibitory mechanism because the RCL would still be able to rapidly insert into the A ß-sheet after cleavage at the P1-P1' (fig. 4B
). Thus we predict that nature may have used disulfide bonds as a method of stabilizing serpins in the most primitive and extreme environments.
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Acknowledgements |
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Footnotes |
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Abbreviations: Pn notation, according to the convention of Schecter and Berger (1967), residues of a peptide substrate N-terminal to the scissile bond are denoted Pn, ..., P2, P1 and those C-terminal to the scissile bond are denoted P1', P2', ..., Pm' RCL, reactive center loop.
Keywords: serpin
prokaryote
comparative genomics
proteinase inhibitor
phylogeny
proteinase
Address for correspondence and reprints: James C. Whisstock, Department of Biochemistry and Molecular Biology, Monash University, Clayton Campus, Melbourne, Victoria 3800, Australia. james.whisstock{at}med.monash.edu.au
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