The complete genome sequence of the largest known double-stranded DNA virus, mimivirus, reveals the presence of a gene (denoted R355) that potentially encodes a cysteine protease that is expressed late (after 6 h) in the infectious cycle of the virus. In order to verify a sequence-based functional prediction and understand its role during the infectious process, the R355 protein was produced to assay its proteolytic activity and solve its three-dimensional structure. Here, the preliminary crystallographic analysis of the recombinant viral protein is reported. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1), with a monomer in the asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal from a selenomethionine-substituted protein crystal.
Pandoraviridae is a rapidly growing family of giant viruses, all of which have been isolated using laboratory strains of Acanthamoeba The genomes of 10 distinct strains have been fully characterized, reaching up to 2.5 Mb in size. These double-stranded DNA genomes encode the largest of all known viral proteomes and are propagated in oblate virions that are among the largest ever described (1.2 μm long and 0.5 μm wide). The evolutionary origin of these atypical viruses is the object of numerous speculations. Applying the chaos game representation to the pandoravirus genome sequences, we discovered that the tetranucleotide (4-mer) "AGCT" is totally absent from the genomes of 2 strains (Pandoravirus dulcis and Pandoravirus quercus) and strongly underrepresented in others. Given the amazingly low probability of such an observation in the corresponding randomized sequences, we investigated its biological significance through a comprehensive study of the 4-mer compositions of all viral genomes. Our results indicate that AGCT was specifically eliminated during the evolution of the Pandoraviridae and that none of the previously proposed host-virus antagonistic relationships could explain this phenomenon. Unlike the three other families of giant viruses (Mimiviridae, Pithoviridae, and Molliviridae) infecting the same Acanthamoeba host, the pandoraviruses exhibit a puzzling genomic anomaly suggesting a highly specific DNA editing in response to a new kind of strong evolutionary pressure.IMPORTANCE Recent years have seen the discovery of several families of giant DNA viruses infecting the ubiquitous amoebozoa of the genus Acanthamoeba With double-stranded DNA (dsDNA) genomes reaching 2.5 Mb in length packaged in oblate particles the size of a bacterium, the pandoraviruses are currently the most complex and largest viruses known. In addition to their spectacular dimensions, the pandoraviruses encode the largest proportion of proteins without homologs in other organisms, which is thought to result from a de novo gene creation process. While using comparative genomics to investigate the evolutionary forces responsible for the emergence of such an unusual giant virus family, we discovered a unique bias in the tetranucleotide composition of the pandoravirus genomes that can result only from an undescribed evolutionary process not encountered in any other microorganism.
The complete sequence of the largest known double-stranded DNA virus, Acanthamoeba polyphaga mimivirus, has recently been determined [Raoult et al. (2004), Science, 306, 1344–1350] and revealed numerous genes not expected to be found in a virus. A comprehensive structural and functional study of these gene products was initiated [Abergel et al. (2005), Acta Cryst. F61, 212–215] both to better understand their role in the virus physiology and to obtain some clues to the origin of DNA viruses. Here, the preliminary crystallographic analysis of the viral nucleoside diphosphate kinase protein is reported. The crystal belongs to the cubic space group P213, with unit-cell parameter 99.425 Å. The self-rotation function confirms that there are two monomers per asymmetric unit related by a twofold non-crystallographic axis and that the unit cell thus contains four biological entities.
ABSTRACT The detailed proteomic analysis of Marseilleviridae icosahedral capsids revealed that the two most abundant protein components of the virions were the Major Capsid Protein (MCP) and the product of an ORFan gene conserved in all Marseilleviridae . The noumeavirus NMV_189 3D structure revealed a common fold with fiber head proteins used by a variety of viruses to recognize their cellular receptor. However, the trimeric structure of NMV_189 uniquely lacking a tail domain, presented a deep concave site suggesting it could be directly anchored to the pseudo-hexagonal capsomers of the virion. This was confirmed by the unambiguous fit of the structure in the melbournevirus 4.4 Å cryo-EM map. In parallel, our structural genomic study of zamilon vitis virophage capsid proteins revealed that Zav_19 shared the same trimeric fiber head fold, but presented an N-terminal tail with a unique β-prism fold. The fiber head fold thus appears to be conserved in all types of non-enveloped icosahedral virions independently of their genomic contents (dsDNA, ssRNA, dsRNA). This could be a testimony of a common origin or the result of convergent evolution for receptor binding function. IMPORTANCE Giant viruses and their associated virophages exhibit a large proportion (≥60%) of orphan genes, i.e . genes without homologs in databases, and thus a vast majority of their proteins are of unknown function. The structural characterization of two ORFans, NMV_189 and Zav_19, both major components of noumeavirus and zamilon virophage capsids, respectively, revealed that despite a total lack of sequence homology, the two proteins share a common trimeric fold typical of viral receptor binding proteins and could be responsible for host receptor recognition. These two structures extend the range of unrelated viruses using fiber head structures as common receptor binding fold.
Megavirus chilensis, a close relative of the Mimivirus giant virus, is able to replicate in Acanthamoeba castellanii. The first step of viral infection involves the internalization of the virions in host vacuoles. It has been experimentally demonstrated that Mimivirus particles contain many proteins capable of resisting oxidative stress, as encountered in the phagocytic process. These proteins are conserved in Megavirus, which has an additional gene (Mg277) encoding a putative superoxide dismutase. The Mg277 ORF product was overexpressed in Escherichia coli, purified and crystallized. A SAD data set was collected to 2.24 Å resolution at the selenium peak wavelength on the BM30 beamline at the ESRF from a single crystal of selenomethionine-substituted recombinant superoxide dismutase protein.
The analysis of the Acanthamoeba polyphaga mimivirus genome revealed the first virus-encoded nucleoside diphosphate kinase (NDK), an enzyme that is central to the synthesis of RNA and DNA, ubiquitous in cellular organisms, and well conserved among the three domains of life. In contrast with the broad specificity of cellular NDKs for all types of ribo- and deoxyribonucleotides, the mimivirus enzyme exhibits a strongly preferential affinity for deoxypyrimidines. In order to elucidate the molecular basis of this unique substrate specificity, we determined the three-dimensional (3D) structure of the Acanthamoeba polyphaga mimivirus NDK alone and in complex with various nucleotides. As predicted from a sequence comparison with cellular NDKs, the 3D structure of the mimivirus enzyme exhibits a shorter Kpn loop, previously recognized as a main feature of the NDK active site. The structure of the viral enzyme in complex with various nucleotides also pinpointed two residue changes, both located near the active site and specific to the viral NDK, which could explain its stronger affinity for deoxynucleotides and pyrimidine nucleotides. The role of these residues was explored by building a set of viral NDK variants, assaying their enzymatic activities, and determining their 3D structures in complex with various nucleotides. A total of 26 crystallographic structures were determined at resolutions ranging from 2.8 A to 1.5 A. Our results suggest that the mimivirus enzyme progressively evolved from an ancestral NDK under the constraints of optimizing its efficiency for the replication of an AT-rich (73%) viral genome in a thymidine-limited host environment.
The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression ofEscherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (Ki ≈ 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor ofvertebrate lysozyme) at the E. coligenetic stock center. The complete nucleotide sequences of over 37 microbial and three eukaryote genomes are already publicly available, and more sequencing is in progress. Despite this accumulation of data, newly sequenced microbial genomes continue to reveal up to 50% of functionally uncharacterized "anonymous" genes. A majority of these anonymous proteins have homologues in other organisms, whereas the rest exhibit no clear similarity to any other sequence in the data bases. This set of unique, apparently species-specific, sequences are referred to as ORFans. The biochemical and structural analysis of ORFan gene products is of both evolutionary and functional interest. Here we report the cloning and expression ofEscherichia coli ORFan ykfE gene and the functional characterization of the encoded protein. Under physiological conditions, the protein is a homodimer with a strong affinity for C-type lysozyme, as revealed by co-purification and co-crystallization. Activity measurements and fluorescence studies demonstrated that the YkfE gene product is a potent C-type lysozyme inhibitor (Ki ≈ 1 nm). To denote this newly assigned function, ykfE has now been registered under the new gene name Ivy (inhibitor ofvertebrate lysozyme) at the E. coligenetic stock center. open reading frame(s) hen egg white lysozyme isoelectrofocusing Despite the accumulation of sequence information from a large diversity of species and phyla, newly sequenced bacterial genomes continue to reveal a high proportion of genes of unknown function (1Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3366) Google Scholar), including a significant subset of "ORFans" (2Fischer D. Eisenberg D. Bioinformatics. 1999; 15: 759-762Crossref PubMed Scopus (177) Google Scholar),i.e. putative open reading frames (ORFs)1 without significant similarity to any previously encountered protein (or conceptual translation) sequences. Most genes found in data bases have only been predicted by computer methods and never experimentally validated. It is thus expected that some annotated ORFs, in particular among the ORFans, might not correspond to real genes. In a previous study, we verified the existence of a cognate transcript for 25 Escherichia coli ORFans with a surprising rate of success (92%) (3Alimi J.P. Poirot O. Lopez F. Claverie J.M. Genome Res. 2000; 10: 959-966Crossref PubMed Scopus (24) Google Scholar). Given that most ORFans appear to be transcribed, we have now initiated a systematic expression and structure determination program for the proteins encoded by these (apparently) unique genes. Because three-dimensional structures are more resilient to evolution and change than amino acid sequences, it is expected that some ORFans should exhibit structural similarity to previously described protein families, hence providing some functional hints. Alternatively, targeting ORFans for structure determination is also a suitable strategy to optimize the discovery of original protein folds, one of the goals of structural genomics. In a pilot study involving five ORFan genes, we succeeded in producing four of them in E. coli as soluble proteins, and we report here the most advanced project, ykfE. YkfE(Swiss-Prot accession number P45552; b0220 in the Blattner data base (4Blattner F.R. Plunkett G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. et al.Science. 1997; 277: 1453-1474Crossref PubMed Scopus (5987) Google Scholar)) is a 474-nucleotide-long uncharacterized ORF. It is part of a single gene operon and was found to exhibit a high level of expression during the exponential and stationary phases of E. coligrowth (3Alimi J.P. Poirot O. Lopez F. Claverie J.M. Genome Res. 2000; 10: 959-966Crossref PubMed Scopus (24) Google Scholar). The ykfE ORF exhibits an N-terminal signal peptide cleaved to produce the mature protein (5Wasinger V.C. Humphery-Smith I. FEMS Microbiol. Lett. 1998; 169: 375-382Crossref PubMed Google Scholar, 6Pasquali C. Frutiger S. Wilkins M.R. Hughes G.J. Appel R.D. Bairoch A. Schaller D. Sanchez J.C. Hochstrasser D.F. Electrophoresis. 1996; 17: 547-555Crossref PubMed Scopus (68) Google Scholar). Initial purification steps and biochemical analyses suggested a strong interaction between this protein and hen egg white lysozyme (HEWL). The existence of a stable complex was confirmed by biophysical analyses, and enzymatic studies revealed the capacity of ykfE to inhibit hen and human C-type lysozymes through a specific interaction. The x-ray structure determination of ykfE, both in isolation (7Abergel C. Monchois V. Chenivesse S. Jeudy S. Claverie J.M. Acta Crystallogr. 2000; 56: 1694-1695Google Scholar) and in a complex with HEWL, is currently in progress and should allow us to understand the molecular basis of the ykfE-lysozyme interaction at atomic resolution. To denote its newly assigned function, ykfE has been registered under the new gene name Ivy (forinhibitor of vertebrate lysozyme) at the E. coli genetic stock center. The 474-base pair ykfE ORF including its own signal peptide was polymerase chain reaction amplified from E. coli K-12 MG1655 genomic DNA using pwo DNA polymerase (Roche Molecular Biochemicals). Primer sequences, 5′-TTATA CCATGGGCAGGATAAGCTC-3′ (sense) and 5′-GCTAAAGATCTAAAATTAAAGCCATCCGGA-3′ (antisense) with NcoI (sense) and BglII (antisense) sites (underlined), were used. After digestion with NcoI + BglII, the polymerase chain reaction product was cloned into a pQE-60 vector (Qiagen) to expressykfE in phase with a C-terminal His6 tag (plasmid pQE-0220). The ykfE gene product (Ivy) was expressed by culturing E. coliXL1-Blue carrying the plasmid pQE-0220 in LB + Amp medium. After initial growth at 37 °C, temperature was set at 30 °C whenA600 reached 0.4, and Ivy expression was induced by adding 1 mmisopropyl-1-thio-β-d-galactopyranoside. Cells were harvested at A600 around 2–2.5 and resuspended in Buffer A (20 mm sodium phosphate, pH 8.0, 300 mm NaCl) containing 1.5% Triton X-100, 1.5% glycerol, and 1 mg·ml−1 HEWL before sonication. Protein extraction was also performed in the absence of exogenous lysozyme to obtain lysozyme-free ykfE protein after the existence of a complex had been recognized. In both cases, purification was achieved by nickel affinity chromatography. The cleared lysate was applied to a 5-ml HiTrap chelating column (Amersham Pharmacia Biotech) charged with Ni2+ and was washed with 10 column volumes of Buffer A, followed by 10 column volumes of Buffer A containing 25 mm imidazole, and 5 column volumes of Buffer A containing 70 mm imidazole at a flow rate of 1 ml·min−1. Elution was performed with a linear gradient over 8 column volumes from 70 to 500 mm imidazole. The recombinant protein was eluted with 150–200 mm imidazole, and fractions were pooled and desalted against 20 mm Tris, pH 8.0, on a fast desalting column HR 10/10 (Amersham Pharmacia Biotech) at a flow rate of 5 ml·min−1. Protein concentration was determined by UV absorption at 280 nm using extinction coefficients calculated on the basis of tyrosine and tryptophan contents (8Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5034) Google Scholar). Protein purity was assessed by SDS polyacrylamide gel electrophoresis and isoelectrofocusing (IEF) using 3 to 10 pH gradient pre-cast gels (Novex). Preliminary molecular weights for the purified proteins were estimated by gel filtration using a calibrated Superdex 75 HR10/30 column (Amersham Pharmacia Biotech) equilibrated with a 20 mm sodium citrate buffer, pH 6.5, at a flow rate of 0.5 ml·min−1. The purified proteins were characterized by mass spectroscopy (matrix-assisted laser desorption ionization/time of flight, Voyager DE-RP; PerSeptive Biosystems) and by N-terminal Edman sequencing (473A; Applied Biosystems). To assay the interaction between Ivy and HEWL, 50 mg of HEWL were loaded on a nickel column at a flow rate of 1 ml·min−1 of 20 mm sodium phosphate, pH 8.0, 300 mm NaCl in the presence or absence of 3 mg of pure Ivy protein. After extensive washing with 20 mm sodium phosphate, pH 8.0, 1 m NaCl, elution was performed with a linear gradient over 5 column volumes to 1m imidazole Intrinsic protein fluorescence was measured with a Spex Fluorolog3 photon-counting spectrofluorimeter (Jobin Yvon-Spex, Longjumeau, France) equipped with a 450-watt Xenon source and a cooled photomultiplier. Tryptophan fluorescence emission spectra were recorded between 290 and 450 nm from solutions containing the individual proteins and from solutions containing a mixture of proteins excited with 280 nm of light. The degree of protein-protein interaction was determined from the extent of fluorescence quenching observed at 344 nm when spectra of a mixture of proteins were compared with the sum of the individual protein spectra at the same concentration. Interaction-dependent fluorescence quenching was determined in 10 mm Tris-HCl buffer, pH 7.0, 8.0, or 9.0, containing 100 mm NaCl at protein concentrations varying from 1 μm to 0.5 nm. HEWL activity assay was performed at 25 °C in 100 mm potassium phosphate, pH 6.4, using 0.125 mg·ml−1 Micrococcus lysodeikticus(Sigma) as substrate. Inhibition studies were carried out by monitoring the change in turbidity associated with the lysis ofM. lysodeikticus cells as described previously (9Charlemagne D. Jollès P. C. R. Acad. Sci..Paris. 1970; : 2721-2723Google Scholar). One unit of HEWL activity was defined as the amount of enzyme causing a decrease in extinction of 0.001 per min at 450 nm. Ki value was determined according to the slow tight binding competitive inhibition model (with no conformational change) (10Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (715) Google Scholar, 11Morrison J.F. Walsh C. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). The following equation was used,(VIvy/V0)2+(VIvy/V0)*(It/Et+Ki/Et−1)−Ki/Et=0Equation 1 where Ki is the apparent dissociation constant, Et is the total enzyme (HEWL) concentration, It is the total inhibitor (Ivy) concentration, VIvy is the inhibited velocity for a given concentration of Ivy, andV0 is the velocity in the absence of inhibitor. HEWL (70 nm) was pre-incubated with Ivy (0–200 nm) at room temperature for 15 min prior to the addition of the M. lysodeikticus substrate (0.125 mg·ml−1). The Ki value was determined by fitting the experimental data onto theVIvy/V0 theoretical curves computed from the above equation (see Fig. 4). The effect of Ivy on C-type lysozyme activity was determined in the presence of 0.5 μg·ml−1 of HEWL (Sigma), with increasing concentrations of Ivy from 0 to 100 μg·ml−1. The effect on λ phage lysozyme activity was assayed in 20 mmTris, pH 8.0, at 25 °C, according to Soumillion et al. (12Soumillion P. Jespers L. Vervoors J. Fastrez J. Protein Eng. 1995; 8: 451-456Crossref PubMed Scopus (42) Google Scholar) and using chloroform-treated E. coli K-12 MG1655 cells as substrate. Activity was determined by measuring the decrease of turbidity over time at 570 nm in the presence of 0.04 μg·ml−1 of λ phage lysozyme and concentrations of Ivy ranging from 0 to 6 μg·ml−1. The chitinase assay was performed in a 200 mm potassium phosphate buffer, pH 6.0, and 2 mm CaCl2 using crab shell chitin covalently linked with remazol brilliant violet 5R (Sigma) as substrate, according to Hackman and Golberg (13Hackman R.H. Golberg M. Anal. Biochem. 1964; 8: 397-401Crossref PubMed Scopus (81) Google Scholar). The effect of Ivy on chitinase activity was determined in the presence of 0.5 μg·ml−1 of Streptomyces griseus chitinase (Fluka) at Ivy concentrations ranging from 0 to 5 μg·ml−1. The enzymatic activity was monitored at 575 nm. Human saliva was also used as a source of lysozyme. 10 μl of fresh saliva were mixed with 1 ml of 100 mm potassium phosphate, pH 6.4, containing 0.125 mg·ml−1 M. lysodeikticus. Concentrations of Ivy ranging from 0 to 20 μg·ml−1 were used for this assay. Exogenous HEWL is usually added prior to sonication to help the disruption of the E. coli cell wall according to the usual extraction protocol for recombinant proteins. HEWL is then removed during the subsequent purification steps. In the case of Ivy, a succession of anomalies led us to suspect a strong interaction between the two proteins. After the purification step by metal chelating chromatography on a nickel resin, SDS polyacrylamide gel electrophoresis analyses of the eluted proteins revealed the presence of two bands of nearly identical molecular mass, around 15 kDa, thus close to the predicted value for the mature form of Ivy (without signal peptide). Mass spectrometry and N-terminal sequencing clearly indicated that these fractions consisted of a mixture of two proteins present in equivalent quantities and identified one of them as the mature Ivy protein (molecular mass = 15.04 kDa) and the other as the exogenous HEWL (molecular mass = 14.3 kDa). Preliminary results suggested the existence of a specific interaction between the two proteins. During metal chelating chromatography on a nickel resin, HEWL could only be retained if Ivy (extracted in the absence of lysozyme) had first been trapped on the column (see "Experimental Procedures"). The SDS polyacrylamide gel electrophoresis analysis of the eluted fractions confirmed the co-elution of Ivy and HEWL. Finally, the effect of the increase of HEWL concentrations on the IEF behavior of Ivy also suggested a strong interaction (Fig. 1). In the absence of HEWL, the IEF migration of Ivy exhibited two close bands at pI = 7.0 and pI = 6.7 (for a theoretical pI of 6.74). These two bands most likely correspond to the dimeric and monomeric forms of Ivy in solution. HEWL alone was found to migrate at pI>10. The increase of HEWL concentrations resulted in a decrease of intensity of the Ivy bands at pI = 7.0 and pI = 6.7 (Fig. 1) and the appearance of an extra band of material reverse migrating into the gel wells, at pI ≃ 10. The HEWL-Ivy interaction was further studied by fluorescence spectroscopy. The fluorescence spectrum of a 1 μmlysozyme, 1 μm Ivy mixture was found to differ significantly from that expected when adding the fluorescence emission spectra of the individual proteins. An overall 20% quenching of the fluorescence was measured with maximal quenching at 344 nm (Fig.2). The shape and maximum of the spectrum are consistent with at least one relatively exposed tryptophan being quenched in the lysozyme-Ivy complex. The examination of the concentration dependence of the quenching spectrum showed that the shape of this spectrum was independent of the protein concentration between 1 nm and 1 μm. No reliableKd value measurement could be obtained because of the insufficient intrinsic fluorescence intensity in the nmconcentration range. Finally, co-crystallization experiments and the subsequent analysis of the crystal content demonstrated the presence of both proteins, thus suggesting a specific and stable interaction between the two molecules (data not shown). The determination of the complex three-dimensional structure is currently in progress. In addition to its specific physical interaction with HEWL, Ivy is also a potent inhibitor of HEWL enzymatic activity (Fig.3). Preliminary experiments showed that in the presence of 1 μg·ml−1 of Ivy, the addition of 1 μg·ml−1 of HEWL produced a nonlinear kinetic with an upward concavity (Fig. 3 a, curve b). In contrast, the pre-incubation of Ivy with HEWL for 15 min resulted in kinetic exhibiting a slight downward concavity (Fig. 3 a, curve c). These results suggest a slow binding kinetic model for the Ivy-HEWL interaction. In addition, near-complete inhibition is reached for a range of Ivy concentrations comparable with the concentration of HEWL (see Figs. 3 b and Fig.4), indicating that Ivy behaves as a slow tight binding inhibitor. A Ki value of about 1 nm was thus estimated by fitting the experimental data (Fig. 4) with Morrison's equation corresponding to this model (see Eq.1 and Refs. 10Morrison J.F. Biochim. Biophys. Acta. 1969; 185: 269-286Crossref PubMed Scopus (715) Google Scholar and 11Morrison J.F. Walsh C. Adv. Enzymol. Relat. Areas Mol. Biol. 1988; 61: 201-301PubMed Google Scholar). The previous experiments demonstrated the potent inhibitory activity of Ivy on hen egg white lysozyme. We then explored the effect of Ivy on the related proteins of increasing evolutionary divergence. We selected a set of lysozyme and lysozyme-like proteins based on structural similarity using the MMDB data base (14Wang Y. Addess K.J. Geer L. Madej T. Marchler-Bauer A. Zimmerman D. Bryant S.H. Nucleic Acids Res. 2000; 28: 243-245Crossref PubMed Scopus (40) Google Scholar). Using HEWL as initial query (MMDB accession number 1151), λ phage lysozyme (root mean square deviation, 1.3 Å; 21.1% identity), and chitinase (root mean square deviation, 1.9 Å; 11.1% identity) were selected as representatives of structural homologs with low sequence similarity. The inhibitory effect of Ivy was thus tested on the two proteins. Ivy was found to cause a weak inhibition of λ phage lysozyme (Fig.5). The activity was only reduced by 15% at a molar ratio of 200:1, Ivy:λ phage lysozyme. We found no inhibitory effect of Ivy on chitinase from S. griseus. We then investigated the capacity of Ivy to inhibit other C-type lysozymes and tested human saliva, because this secretion was reported to contain 30–55 μg·ml−1 of lysozyme (15Taylor D.C. Cripps A.W. Clancy R.L. J. Immunol. Methods. 1992; 146: 55-61Crossref PubMed Scopus (22) Google Scholar). Ivy was found to strongly inhibit the lysozyme activity in saliva (Fig.6). Around 50 μg·ml−1 of Ivy is sufficient to observe a decrease of 50% of the activity, which was fully abolished for an Ivy concentration of 0.5 mg per ml of saliva.Figure 6Inhibition of lysozyme activity of human saliva by increasing concentrations of ykfE/Ivy.View Large Image Figure ViewerDownload Hi-res image Download (PPT) On a gel filtration column, Ivy is eluted with an apparent molecular mass of about 30 kDa, indicating that the predominant form in solution is a homodimer, as already suggested by IEF experiments. Fluorescence studies confirmed this model. The fluorescence emission spectrum of HEWL exhibits a broad peak with a maximum at 342 nm and a long wavelength tail typical of relatively exposed tryptophan residues. In contrast, the spectrum of Ivy shows a peak at 334 nm ∼25% more intense on an absolute scale and 2.5 times more intense on a per tryptophan scale (Fig. 2). Such intense fluorescence and the relatively short wavelength of maximum emission both argue for tryptophans buried within apolar environments. Furthermore, the shape of the emission spectrum was found to be independent of the protein concentration in a broad 0.5 nm to 1 μm range and appears insensitive to change in pH between 7.0 and 9.0. The order of magnitude of the dimerization Kd appeared much lower than 10−9m, although no precise measurement could be made in this concentration range. Altogether, these biophysical results suggest that the Ivy homodimer is the physiologically active unit. C-type lysozyme is an ancient protein whose origin goes back about 500 million years (16Qasba P.K. Kumar S. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 255-306Crossref PubMed Scopus (148) Google Scholar). 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Infect. Immun. 1999; 67: 3267-3275Crossref PubMed Google Scholar). There is multiple evidence that lysozymes play a significant role in the control of the host microflora to prevent infection (32Kondo L.R. Hanna L. Keshishyan H. Proc. Soc. Exp. Biol. Med. 1973; 142: 131-132Crossref PubMed Scopus (6) Google Scholar, 33Walker W.A. Ciba Found. Symp. 1979; 70: 201-219PubMed Google Scholar, 34Ved'mina E.A. Pasternak N.A. Shenderovich V.A. Zhuravleva T.P. Andrusenko I.T. Antibiot. Khimioter. 1979; 24: 746-750Google Scholar, 35Hancock R.E. Scott M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8856-8861Crossref PubMed Scopus (818) Google Scholar, 36Vollmer W. Tomasz A. J. Biol. Chem. 2000; 275: 20496-20501Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Bacterial C-type lysozyme inhibitors might thus have emerged to balance the host defense. Indeed, an increase of anti-lysozyme activity has been linked to bacterial persistence in several systems (37Bukharin O.V. Zh. Mikrobiol. Epidemiol. Immunobiol. 1994; 1, (Suppl.): 4-13Google Scholar, 38Bondarenko V.M. Petrovskaia V.G. Iablochkov A.L. Zh. Mikrobiol. Epidemiol. Immunobiol. 1994; 1, (Suppl.): 22-28Google Scholar). However, lysozyme inhibitors could also be directed against lysozyme activities of other microorganisms and play a role in ecological competition (39Nemtseva N.V. Zh. Mikrobiol. Epidemiol. Immunobiol. 1997; 4: 123-126Google Scholar, 40Lentsner A.A. Lentsner Kh.P. Toom M.A. Zh. Mikrobiol. Epidemiol. Immunobiol. 1975; 8: 77-81Google Scholar). Finally, these inhibitors could also have emerged as a protection against bacteriophage-encoded lysozymes, the activity of which is essential to the release of mature virions (41Bukharin O.V. Deriabin D.G. Zh. Mikrobiol. Epidemiol. Immunobiol. 1989; 11: 16-19Google Scholar). It thus makes evolutionary sense that bacteria might have evolved a resistance mechanism against the bactericidal activity of various lysozymes found in their environment. However, it must be noted that for Gram-negative bacteria such as E. coli, the presence of an outer membrane impermeable to molecules larger than 0.6 kDa should be sufficient to protect the peptidoglycan moiety from the lysozymes present in the medium. We selected ykfE as an ORFan gene expressed byE. coli K12 (5Wasinger V.C. Humphery-Smith I. FEMS Microbiol. Lett. 1998; 169: 375-382Crossref PubMed Google Scholar). The presence of a signal peptide predicted a periplasmic location for its protein product, which is now consistent with its newly assigned function. Our biochemical and functional analyses of Ivy show that its predominant homodimeric form strongly interacts with hen and human, and probably all C-type lysozymes, thereby abrogating their activity in a stoichiometric manner. Ivy does inhibit C-type lysozymes under physiological conditions, as tested in human saliva, the secretion where lysozyme is naturally found at the highest concentration. It is thus likely that at least one purpose of Ivy is to protect E. coli from its natural host lysozyme bactericidal activity, for instance in cases where the integrity of the outer membrane might be compromised (e.g. by chemically aggressive compounds in the medium or at the time of cell division). As such a resistance mechanism against an ubiquitous bactericidal enzyme should be advantageous to all murein-containing bacteria, in particular Gram-positive bacteria, ykfE/Ivy-like genes are expected to exist in many bacterial genomes, making its ORFan nature a paradox. Indeed, we detected a putative ortholog of E. coli Ivy within the recently published genome of Pseudomonas aeruginosa (1Stover C.K. Pham X.Q. Erwin A.L. Mizoguchi S.D. Warrener P. Hickey M.J. Brinkman F.S. Hufnagle W.O. Kowalik D.J. Lagrou M. Garber R.L. Goltry L. Tolentino E. Westbrock-Wadman S. Yuan Y. Brody L.L. Coulter S.N. Folger K.R. Kas A. Larbig K. Lim R. Smith K. Spencer D. Wong G.K. Wu Z. Paulsen I.T. Nature. 2000; 406: 959-964Crossref PubMed Scopus (3366) Google Scholar). However, the two protein sequences only share 30% of identical residues in their most similar region, indicating a fast divergence rate (Fig. 7). It is thus likely that genes of the ykfE/Ivy family are not detected in other bacteria because of their low sequence conservation. It is our hope that the knowledge of the three-dimensional structure of Ivy will allow the discovery of other Ivy homologues by the identification of a set of critical positions in the sequence beyond the twilight zone of sequence similarity. We thank Dr. C. Cambillau for access to x-ray diffraction equipment and helpful discussions and Dr. Mary Berlin for quick validation of the Ivy name. We also acknowledge the helpful comments of anonymous referees concerning the analysis of the Ivy inhibition mechanism. We thank Dr. C. Evrard for the gift of λ phage lysozyme.
The Escherichia coli yeaZ gene encodes a 231-residue protein (Mr = 25 180) that belongs to a family of proteins that are conserved in various bacterial genomes. This protein of unknown function is predicted to be a hypothetical protease. The YeaZ protein was overexpressed in E. coli and crystallized at 298 K by the hanging-drop vapour-diffusion method. A MAD data set was collected using a gadolinium-derivative crystal that had been soaked with 0.1 M Gd-DOTMA. The data set contained data collected to a resolution of 2.7 Å at two wavelengths at the LIII absorption edge of gadolinium, while remote data were collected to a resolution of 2.28 Å. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 76.3, b = 97.6, c = 141.9 Å. Phasing using the MAD method confirmed there to be four monomers in the asymmetric unit related by two twofold axes as identified by the self-rotation function search.
