Báo cáo khoa học: Analysis of ancient sequence motifs in the H+-PPase family doc

Analysis of ancient sequence motifs in the H + -PPase family Joel Hedlund 1 *, Roberto Cantoni 2,3,4 *, Margareta Baltscheffsky 2 , Herrick Baltscheffsky 2 and Bengt Persson 1,4 1 IFM Bioinformatics, Linko ¨ ping University, Sweden 2 Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Sweden 3 Department of Physical Sciences, ‘Federico II’ University of Naples, Italy 4 Department of Cell and Molecular Biology (CMB), Programme for Genomics and Bioinformatics, Karolinska Institutet, Stockholm, Sweden Membrane-bound inorganic pyrophosphatase ⁄ pyro- phosphate synthase (H + -PPase ⁄ H + -PP i synthase) [1,2] activities were first described in chromatophores from the purple nonsulphur photosynthetic bacterium, Rho- dospirillum rubrum, where the enzyme functions as a proton pump [3]. The gene for H + -PPase ⁄ H + -PP i syn- thase, which is the enzyme involved in the photo- synthetic formation of pyrophosphate (PP i ) [2], was cloned in 1998 [4] and the primary structure and fur- ther properties have been deduced [5]. Moreover, 3D models of parts of the putative active site loop between transmembrane segments 5 and 6 of R. rubrum have been presented [6]. This 57 amino acid residue loop contains three sequence motifs, underlined in the following sequence: L GGGIFTKCADVGADLVGKV EAGIPEDDPRNPAVIA DNVGDNVGDCAGMAAD LFETY. In what apparently is a pyrophosphate-binding region and an essential part of the active site for the phosphorylation ⁄ phosphatase reaction [7], three differ- ent ‘primitive’ tetrapeptide motifs (DVGA, DLVG and DNVG) [5,6] have been located in two nonapeptide (enneapeptide) sequences (DVGADLVGK and DNVGDNVGD) which, with their charged amino acids at positions 1, 5 and 9, seem to be involved in binding the metal-phosphate substrates [7]. These seq- uence motifs are denoted ‘primitive’ because they have a high content of the four ‘very early’ proteinaceous amino acids G (glycine), A (alanine), D (aspartic acid) and V (valine). In 1978, Eigen & Schuster [8] proposed Keywords bioinformatics; hidden Markov models; molecular evolution; proteinaceous amino acids; pyrophosphatase Correspondence B. Persson, IFM Bioinformatics, Linko ¨ ping University, S-581 83 Linko ¨ ping, Sweden Fax: +46 13 137 568 Tel: +46 13 282 983 E-mail: bpn@ifm.liu.se *These authors contributed equally to this work (Received 9 August 2006, revised 26 Sep- tember 2006, accepted 27 September 2006) doi:10.1111/j.1742-4658.2006.05514.x The unique family of membrane-bound proton-pumping inorganic pyro- phosphatases, involving pyrophosphate as the alternative to ATP, was investigated by characterizing 166 members of the UniProtKB ⁄ Swiss- Prot + UniProtKB ⁄ TrEMBL databases and available completed genomes, using sequence comparisons and a hidden Markov model based upon a conserved 57-residue region in the loop between transmembrane segments 5 and 6. The hidden Markov model was also used to search the approxi- mately one million sequences recently reported from a large-scale sequen- cing project of organisms in the Sargasso Sea, resulting in additional 164 partial pyrophosphatase sequences. The strongly conserved 57-residue region was found to contain two nonapeptidyl sequences, mainly consisting of the four ‘very early’ proteinaceous amino acid residues Gly, Ala, Val and Asp, compatible with an ancient origin of the inorganic pyrophospha- tases. The nonapeptide patterns have charged amino acid residues at posi- tions 1, 5 and 9, are apparent binding sites for the substrate and parts of the active site, and were shown to be so specific for these enzymes that they can be used for functional assignments of unannotated genomes. Abbreviations H + -PPase ⁄ H + -PP i synthase, proton pumping inorganic pyrophosphatase ⁄ pyrophosphate synthase; HMM, hidden Markov model; P i , inorganic phosphate; PP i , pyrophosphate. FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS 5183 a detailed model for the evolution of RNA codons, which suggests that the RNA triplets GGC and GCC (coding for glycine and alanine, respectively) were the earliest codons. Transition mutations at the second base would then have given GAC and GUC, which code for aspartic acid and valine, respectively. The evi- dence supporting the role of these four proteinaceous amino acids as ‘very early’ [5] has been further con- firmed by Trifonov [9]. Notably, experiments by Miller [10], leading to the synthesis of amino acids under cer- tain ‘simulated prebiotic’ conditions, gave these four amino acids in the highest yield, and they all were among the amino acids found in the Murchison mete- orite [11]. Both the high content of the ‘very early’ amino acids in these sequence motifs, and the fact that the motifs have remained essentially unchanged during billions of years of biological evolution, from Archaea and Bacteria to Eukaryotes, provide a background for our fascination with various aspects of their evolution and function. As pointed out previously [5], the GGG motif of the R. rubrum H + -PPase may be of special functional significance in a possible conformational change mechanism for the physiological coupling between the light-induced pumping of protons and the photo- phosphorylation of inorganic phosphate (P i )toPP i [2] or its reversal, the dark hydrolysis of PP i to P i [1]. All three glycines are strictly conserved, except in very rare cases, where one, usually the first G, is substituted with an A. With this conservative substitution, and the additional fact that, just as glycine, alanine can serve as a versatile link in conformational changes, the sub- stitution of one G with one A should not cause any drastic change in the suggested function of the GGG motif. G and A are also seen elsewhere to have been interchanged, as will be exemplified in our discussion of the DVGADLVGK motif. Two notable points of the H + -PPase family exist at present. Unique simplicity of these homologous, inte- grally membrane-bound enzymes resides in both their substrates (P i and PP i ) and their single-subunit dimer- ized structure [12]. Its extreme hydrophobicity, with approximately half of the residues in the 16 ± 1 trans- membrane segments, has made all attempts to obtain high-resolution 3D information of the H + -PPase unsuccessful, to date. Bioinformatics may be used to provide new perspectives on the possible evolution of this ancient, widely spread and unusually highly con- served energy-transferring enzyme family. The possibility has been considered that PP i could have been a predecessor to ATP, and that H + -PPases could have been direct or indirect evolutionary ances- tors of ATPases [5,13]. From numerous genome projects, a large number of both prokaryotic and euk- aryotic H + -PPase sequences are now known. Our new overview of the high content of strongly conserved, very early amino acids in the above shown three puta- tive active site motifs in the loop between transmem- brane segments 5 and 6 deserves a closer look for existing sequence similarities in other known polypep- tides. We thus also evaluate the possibility that the putative active site may have evolved from an original enzyme structure involved in an emerging metabolism of energy-rich phosphate [6]. A recent, additional indi- cation in this direction was given by the discovery that shifting the growth conditions of R. rubrum from aerobic ⁄ dark to anaerobic ⁄ light resulted in concerted transcriptional activation of both H + -PPase and pho- tosynthetic genes, by the same anaerobic regulatory factor [14], pointing towards PP i as the energy-rich phosphate product of early bacterial photophosphory- lation. Furthermore, the unique property of the H + - PPase family, to use PP i rather than ATP for energy coupling and utilization in biological membranes, pro- vides an alternative angle to the study of the central, but notoriously elusive, coupling mechanism between energy-rich phosphates and proton pumps. Finally, the major problems encountered by several laboratories in obtaining H + -PPase samples for 3D studies have moti- vated detailed searches with various bioinformatic techniques in order to extend the characterization of the members of the H + -PPase family. Results and Discussion Characterization of H + -PPase members In order to detect members of the membrane-bound, proton pumping H + -PPase family, all completed genomes and the UniProtKB protein sequence data- base [15] were searched using fasta [16], resulting in characterization of over 100 different members. The sequences were multiply aligned, revealing a number of well-conserved regions, especially the highly conserved 57-residue segment in the loop between transmembrane segments 5 and 6. This segment was used to create a hidden Markov model (HMM), which subsequently was used to search for further homologues in the data- bases of UniProtKB and the currently available genomes, in order to identify further family members. Using this HMM, 166 sequences were found (supple- mentary Table S1). Remarkably, only other H + - PPases were found using this strategy, indicating high specificity of the model. The poorest scoring H + -PPase sequence had an E-value of 8.3e-25 (i.e. under the circumstances of the search, only 8.3e-25 unrelated Ancient sequence motifs in the H + -PPase family J. Hedlund et al. 5184 FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS sequences could be expected to attain the same match quality by chance alone) [17]. Thus, the E-value is highly statistically significant. Furthermore, the best scoring non-H + -PPase protein was detected at an E-value of 15, far below statistical significance. Thus, the model is well suited for detection of H + -PPases. The 57-residue segment seems to be unique for the H + -PPase family, which might be seen as an index of their early separation from other families, making H + -PPases a low-positioned branch in the genealogi- cal tree of protein families. Consequently, this seg- ment can be used as a fingerprint in the search for further members of this family from new sequence data. Analyses of the various primary structures of H + - PPases (PP i synthases) raise the possibility of explor- ing, in molecular detail, various properties of this ‘primitive’ alternative to ubiquitously occurring ATP synthases. Looking at the species distribution, H + - PPases are present in several archaeal and bacterial species [5,18]. In eukaryotes, these enzymes are found in plants and in a few blood parasites, such as Tetra- hymena and Plasmodium. The fact that four ‘very early’ amino acid residues have been retained indicates very early optimization. This may be a rather unusual situation compared with early motifs in other proteins, where stepwise evolution of the motifs may have provided further optimization, through introduction of later amino acids. Different H + -PPase subfamilies In order to characterize the interfamily relationships, a dendrogram was calculated (Fig. 1) based upon the multiple sequence alignment. The tree shows that H + - PPases form two subfamilies, corresponding to type 1 and type 2 [19,20]. Several species present multiple forms of H + -PPases, which in many cases are distantly related (37–39% pairwise residue identity) and belong to the separate types. For cress (Arabidopsis thaliana), there are five type 1 and six type 2 enzymes, whereas for rice (Oryza sativa) there exist 18 enzymes of type 1 and two enzymes of type 2. Among plants, the multi- plicity has so far only been seen in organisms for which the complete genomes are available, and this distantly related multiplicity can thus be expected to occur also in further plants. The blood parasites Tetra- hymena, Plasmodium falciparum and Plasmodium yoelii show a similar multiplicity. Moreover, some archaeal species (e.g. Methanosarcina mazei and Methanosarcina acetivorans) have multiple H + -PPases, whereas most bacterial species do not show any multiplicity, except for the bacterium Rhodopseudomonas palustris, which has two different pyrophosphatases, found on separate branches within type 2 (Fig. 1). This multiplicity contrasts to the situation for the family I soluble PPases, where two variants of different sizes are known, but they are strictly divided between different kingdoms – one in prokaryotes and the other in eukaryotes [21]. Surprisingly, two eukaryotic sequences from the frog Xenopus tropicalis are found (see Fig. 1) among the bacterial type 2 members. However, because this gen- ome project is not complete, it cannot yet be claimed that these sequences are correct. In order to characterize the differences between sequences of types 1 and 2, we compared the positions strictly conserved within each type, but differing between the types, as indicated in Fig. 2. In total, seven such differences were found – four in the trans- membrane segments and three on the cytosolic side – whereas none was found on the noncytosolic side. Two of the differences were found between residues with different physico-chemical properties, implying possible functional impact (Fig. 2, residues indicated by bullet symbols). Functional impact has already been confirmed for one of these differences (position 507 in HPPA_STRCO), as an Ala⁄ Lys mutation introduced at the corresponding position in Carboxydothermus hydrogenoformans type 1 H + -PPase has been shown to confer the potassium independence of type 2 H + - PPases to the enzyme [22]. At position 253 in the loop between transmembrane segments 5 and 6, close to one of the conserved nonapeptides, the type 1 enzymes have predominantly hydrophobic Ile or Val, while type 2 enzymes have polar Cys or Ser. Furthermore, there are two exchanges within weakly conserved clustalw groups of residues [23] (Fig. 2, open ring symbols). At position 266 in transmembrane segment 6, the type 1 residues are Glu or Gly, while the type 2 residues are Val or Ala. At position 510, in loop 11–12 close to the membrane, type 1 enzymes prefer Gly, while type 2 enzymes prefer Thr or Ala. The remaining three residue exchanges are within clustalw groups, and these are all located in trans- membrane segments (Fig. 2). Conserved regions within H + -PPases From the multiple sequence alignment of all H + -PPas- es, a limited number of strongly conserved regions are clearly visible. A conservation profile was calculated, showing the degree of conservation averaged over win- dows of 11 residues along the protein chain (Fig. 3). From the resulting plot it is clear that most of the con- served residues are located on the cytosolic side of the J. Hedlund et al. Ancient sequence motifs in the H + -PPase family FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS 5185 Ancient sequence motifs in the H + -PPase family J. Hedlund et al. 5186 FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS Fig. 2. Schematic view of the H + -PPase sequence with residue differences between type 1 and type 2 indicated. The black line symbolizes the amino acid chain, and the horizontal green lines denote membrane borders with the cytosolic side downwards and the noncytosolic side upwards. The transmembrane topology is from a previous publication [26]. The transmembrane segments are numbered from 1 to 17 and the loops are numbered 1–2 to 16–17. The letters N and C denote the N- and C-terminus, respectively. Positional numbers refer to the refer- ence sequence from Streptomyces coelicolor (UniProtKB ⁄ Swiss-Prot ID HPPA_STRCO, AC Q9X913). The green regions represent the five conserved regions in Fig. 3. The red boxes indicate the four nonapeptides (shown in red in Fig. 3). Thick lines denote positions where the two most common residue types together make up more than 95% of the residue type content. Positions with differential conservation are indicated by labels of the type ‘AB ⁄ CD’, which denotes that more than 90% of the Type 1 enzymes have residue A or B at this position, while 90% of the Type 2 enzymes have C or D, and that A and C are the most common ones. Boldface residue letters indicate that more than 85% of the sequences in the corresponding subtype have this residue. Conserved substitutions within a CLUSTALW ‘strongly conserved group of residues’ are shown by dash symbols on the backbone, and open ring symbols are similarly used for CLUSTALW ‘weakly conserved groups of residues’. Bullet symbols on the backbone denote conserved substitutions between two residues that do not occur together in any of the CLUSTALW groups. This latter form of substitution implies functional impact. Also, in order to count as a conserved substitution, none of the residue types A or B can be identical to the residue types C or D. Fig. 1. Dendrogram of H + -PPases. The dendrogram is based upon the multiple sequence alignment of all PPase sequences found in Uni- ProtKB ⁄ Swiss-Prot, UniProtKB ⁄ TrEMBL and GenomeLKPG databases, with removal of sequences showing 90% or more identity to any of the other sequences in the alignment. Two sequences – Q420R8_DESHA and Q4CED6_CLOTM – show unclear relationships and were excluded without affecting the general tree topology. Red marks at branch points indicate bootstrap values below 888 ⁄ 1000, the bootstrap value at the branch point separating Type 1 (K + -dependent) from Type 2 (K + -independent) enzymes. The horizontal bar shows the branch length corresponding to 5% residue differences. Each branch end-point is designated with identifiers from UniProtKB ⁄ Swiss-Prot, Uni- ProtKB ⁄ TrEMBL or the respective genome project, prefixed indicators of kingdom (A, archaeal; B, bacterial; E, eukarytoic), species group (A, Alveolata; E, Euglenozoa; P, Plant; V, Vertebrates), and PPase type (1 or 2). Archaea are labelled yellow, bacteria blue, and plants green. The red-labelled sequences originate from primitive eukaryotes (alveolates and euglenozoans). Two sequences from the unfinished Xen- opus tropicalis genome project are shown in purple. Accession numbers are given within parentheses after the UniProtKB ⁄ Swiss-Prot ID. For UniProtKB ⁄ TrEMBL IDs, the accession number forms the first part of the ID. All type 1 PPases are found in the upper part of the dendro- gram, while type 2 PPase are in the lower part. Several plants and protozoan organisms have both type 1 and type 2 PPases, whereas most of the bacterial forms only present one of the types. J. Hedlund et al. Ancient sequence motifs in the H + -PPase family FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS 5187 membrane, while only few residues on the noncytosolic side are conserved. Furthermore, there are five seg- ments that are seen as distinct peaks, indicating strong conservation. These sequences are listed in Fig. 3. The locations of these segments are schematically indicated in the topology plot in Fig. 2, shown as red-coloured segments. The first region corresponds to the previ- ously mentioned 57-residue conserved region in the loop between transmembrane segments 5 and 6, while the second and third regions are found in the loop between segments 11 and 12 and the fourth and fifth regions in the loop between segments 15 and 16. We developed HMMs for the regions 2+3 and 4+5, which were used to search UniProtKB ⁄ Swiss- Prot and UniProtKB ⁄ TrEMBL databases for poten- tially homologous proteins. However, also for these regions, we only found proteins of the H + -PPase fam- ily, thus emphasizing the uniqueness of this family. The 57-residue conserved region in the loop between transmembrane segments 5 and 6 From the multiple sequence alignment of all 145 H + - PPases, the consensus sequence for the well-conserved 57-residue region was calculated (Fig. 4A). The region contains the three conserved sequence motifs: GGG, DVGADLVGK and DNVGDNVGD, which have already received particular attention from the evolution- ary viewpoint [5,6] and appear to form functionally sig- nificant parts of the active site of the enzyme [7]. Both the second and the third motif contain nine amino acid residues, of which the first, fifth and ninth are charged. Fig. 3. Conservation plot for H + -PPases and conserved sequence motifs. From the multiple sequence alignment of H + -PPases, CLUSTALW col- umn scores were averaged for ungapped 11-residue windows of the reference sequence from Streptomyces coelicolor, HPPA_STRCO, and plotted in green. The predicted membrane topology is shown in blue, where high values indicate the noncytosolic side and low values the cytosolic side, whereas medium values correspond to transmembrane regions. The plot shows that the conserved regions coincide with cytoplasmic and transmembrane regions. There are five regions with distinct peaks above 55% column score (dotted line), indicating strong conservation. The sequences for these regions in S. coelicolor are shown below the plot. In region 1, the well-conserved patterns, including the two nonapeptides, are highlighted in red. Distantly related nonapeptides in regions 4 and 5 are also highlighted in red, together with a GGS pattern, possibly corresponding to the GGG pattern in region 1 (cf. text). Ancient sequence motifs in the H + -PPase family J. Hedlund et al. 5188 FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS The number of amino acid residues separating the three motifs is remarkably constant in all the H + -PPases. The nonapeptide DVGADLVGK was seen to have a partial counterpart in the P-loop [24] of the active b-subunit of ATP synthase. In an alignment of PP i synthase from R. rubrum with the P-loop in animal mitochondrial ATP synthase, four of eight amino acid residues were found to be identical [5]. In order to investigate further the evolutionary vari- ation of this 57-residue region, we applied the HMM to search the approximately one million sequences recently reported from a large-scale sequencing project of organisms in the Sargasso Sea [25]. With the model, we were able to extract an additional 164 pyrophos- phate sequences (supplementary Table S2), not over- lapping with the initial set of sequences. The consensus sequence of the Sargasso sequences is shown in Fig. 4B. Comparing all sequences detected in Uni- ProtKB ⁄ Swiss-Prot, UniProtKB ⁄ TrEMBL and Geno- meLKPG databases (Fig. 4A), it can be seen that the variability is smaller among the Sargasso sequences, but many of the residue variations are identical. Fur- thermore, the variable regions are generally located at the same sites as for the sequences in Fig. 4A. For a number of plant H + -PPases, the HMM finds distant similarity also to a second region of the B A Fig. 4. Consensus sequences of the conserved 57 amino acid residue region. (A) The consensus sequence derived from all sequences found in UniProtKB ⁄ Swiss-Prot, UniProtKB ⁄ TrEMBL and available genomes using the HMM search is shown. The sequence is shown using sequence logos [34], clearly showing the high conservation of this region. Residues are coloured according to chemical properties: green represents polar residues (G, S, T, Y, C, Q, N), blue basic (K, R, H), red acidic (D, E) and black hydrophobic residues (A, V, L, I, P, W, F, M). The amino acid residues at each position are also shown in plain text below the x-axis, where the top row represents the most common residue type with alternative residues ordered in decreasing frequency. (B) We also used the HMM to search the approximately one million sequences recently reported from the Sargasso Sea [25]. The sequence logos and positional residue variants are shown as in (A). J. Hedlund et al. Ancient sequence motifs in the H + -PPase family FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS 5189 enzymes, possibly being visible traces of an ancient gene duplication. This second region is located at resi- dues 738–785 (numbering according to the A. thaliana sequence with accession number Q9FWR2 (Uni- ProtKB Q9FWR2_ARATH)). This region forms loop 15–16, located at the cytosolic side according to experi- mental investigations [26] (cf. Fig. 3). The patterns of this second region are also seen in further species vari- ants, but are most clearly distinguishable in the plant sequences. According to the three well-conserved sequence segments of H + -PPases, the second nonapep- tide motif is well conserved, with all three aspartic acid residues unchanged (Fig. 3, marked residues in regions 1 and 5). For the first nonapeptide motif, charges are found at positions 1, 5 and 9 in the order Asp, Asp, Lys in region 1, whereas the order is Asp, Lys, Asp in region 4. Notably, the positional spacing between the two nonapeptides in region 1 and region 4+5 is identi- cal (26 residues). Furthermore, the GGG motif pre- ceding the nonapeptides in region 1 could correspond to a GGA or GGS motif, preceding the nonapeptide in region 4 (Fig. 3, marked residues in peptides 1 and 4). Thus, these observations, taken together, might reflect an ancient gene duplication event, as previously suggested [5]. Occurrence of the typical H + -PPase nonapeptides in other proteins In order to investigate the general occurrence of the two nonapeptides, characterized as typical of H + - PPases, they were compared with all sequences in the UniProtKB ⁄ Swiss-Prot and UniProtKB ⁄ TrEMBL databases. The sequence patterns used in the searches were based upon all positional variants occurring in any of the H + -PPases, excluding those with only a single observation, resulting in the patterns: D-[VIMT]-[GA]- [AGS]-D-[LI]-[VSMA]-G-K and D-[NCFL]-[VITA]- G-D-N-[VA]-G-D. In Table 1, these patterns are shown as DpppDppGK and DppGDNpGD, respectively. Remarkably, only 11 and 7, respectively, of the 192 and 32 possible combinations of different nonapeptides were found to have matches in UniProtKB ⁄ Swiss-Prot or UniProtKB ⁄ TrEMBL databases (Table 1). All but one of the sequences found by the first nonapeptide pattern are annotated as H + -PPases. For the second nonapep- tide pattern, all but two are H + -PPases. The exceptions are a putative DNA damage-inducible protein from Erythrobacter litoralis (Q4TQ38–9SPHN), presumably not related to the H + -PPases, and two hypothetical pro- teins from Neurospora crassa (Q871A9_NEUCR and Q7RZ15_NEUCR). Furthermore, as seen in Table 1, four H + -PPases are not detected by the first nonapep- tide pattern, because those proteins have one atypical amino acid residue in this pattern. Thus, the nonapep- tide patterns are, with these few exceptions, specific for the H + -PPases. As seen in Table 1, the number of non- H + -PPase hits increases dramatically when the patterns are extended to DXXXDXXGK and DXXGDNXGD, respectively, where X represents any amino acid residue. We extended the pattern search to screen the Uni- ProtKB ⁄ Swiss-Prot database for very simple motifs of possible ancestral significance, with an alternation of Asp and one of the other ‘very early’ amino acids (e.g. DADADADAD) (Table 2). It can be seen that the pat- tern VDVDV is under-represented compared with the patterns ADADA and GDGDG, even when considering the general frequencies of the residues (V, 6.7%; A, 7.9%; G, 7.0%). Similarly, the DGDGD pattern is over-represented compared with the patterns DADAD and DVDVD. This over-representation is still present Table 1. Number of proteins and occurrences of PP i -related sequence motifs in the UniProtKB ⁄ Swiss-Prot and Uni- ProtKB ⁄ TrEMBL databases. The peptides DpppDppGK and Dpp- GDNpGD denote patterns based upon all positional variants occurring in any of the H + -PPases, excluding those with only a sin- gle observation, corresponding to the patterns: D-[VIMT]-[GA]- [AGS]-D-[LI]-[VSMA]-G-K and D-[NCFL]-[VITA]-G-D-N-[VA]-G-D. In the peptides DXXXDXXGK and DXXGDNXGD, X represents any amino acid residue. Sequence motif UniProtKB ⁄ Swiss-Prot UniProtKB ⁄ TrEMBL Proteins Hits PPases Proteins Hits PPases First nonapeptide DVGADLVGK 20 20 20 81 81 81 DVGGDLVGK 8 8 8 4 4 4 DMAADLVGK 1 1 1 7 7 7 DVGADLSGK 0 0 0 7 7 7 DIGADLVGK 0 0 0 6 6 6 DVGADLAGK 0 0 0 4 4 4 DTGADIVGK 0 0 0 2 2 2 DMGADLVGK 0 0 0 2 2 2 DVAADLVGK 0 0 0 1 1 1 DVGADIAGK 0 0 0 1 1 1 DTGADLAGK 0 0 0 1 1 0 DpppDppGK 29 29 29 116 116 115 DXXXDXXGK 809 810 30 7332 7366 118 Second nonapeptide DNVGDNVGD 29 29 29 93 93 93 DLVGDNVGD 1 1 1 15 15 15 DCTGDNAGD 1 1 1 5 5 5 DCIGDNVGD 0 0 0 7 7 7 DFVGDNVGD 0 0 0 2 2 2 DCAGDNAGD 0 0 0 2 2 2 DLTGDNAGD 0 0 0 2 2 0 DppGDNpGD 31 31 31 126 126 124 DXXGDNXGD 31 31 31 160 175 125 Ancient sequence motifs in the H + -PPase family J. Hedlund et al. 5190 FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS after homology reduction (at the 80% and 60% levels) and might well reflect structural properties. We also searched the complete UniProtKB (Swiss- Prot + TrEMBL) database for patterns with altera- tions of any two ‘very early’ amino acid residues. The largest number of proteins was found for the sequences AGAGA (6185) and GAGAG (6203), in agreement with the assumption that G and A are both very early, flexible and frequent amino acids. Close to 6000 results were also reported for the pattern AVAVA (5865), while much smaller numbers were found for GVGVG (3386), ADADA (2383), DGDGD (2100) and DVDVD (852). The small difference in frequencies between V and D (6.7% and 5.3%, respectively) in known proteins does not fully explain the discrepancy between AVAVA and ADADA. Three of the sequences found had long segments consisting of repetitious patterns containing two of the four early amino acid residues A, G, D and V. The residues A and D, present in an alternation pattern (ADADAD ) were found in the surface protein, SdrI, from Staphylococcus saprophyticus and two putative peptidoglycan-bound proteins from Listeria innocua and Listeria monocytogenes. These proteins are suppo- sedly attached to the cell wall peptidoglycan by amide bonds. Such patterns are believed to be evolutionary relics of early sequence pattern formation by muta- tions and duplications, from early homo-oligomers (GGGGG and AAAAA ) [27]. In the conserved nonapeptides DVGADLVGK and DNVGDNVGD, 14 out of 18 residues belong to the four ‘primitive’ residue types G, A, V and D. In order to make a general assessment of frequency of ‘primitive’ and repetitive patterns including the ancient amino acid residues, we searched the protein databases for sequences including these four residues in various com- binations. Thus, we scanned UniProtKB ⁄ Swiss-Prot for the sequence motif D-[A ⁄ G ⁄ V] 3 -D-[A ⁄ G ⁄ V] 3 -D, and a similar motif, where alanine is replaced with asparagine, given the presence of asparagines in one of the two puta- tive active site motifs of R. rubrum H + -PPase. The only patterns found in the proteins were DNVGDNVGD, unique to the H + -PPase family, and DNNNDNNND, in the spindle assembly checkpoint component MAD1 from Saccharomyces cerevisiae (Mitotic arrest deficient protein 1; UniProtKB ⁄ Swiss-Prot ID MAD1_YEAST). We thus concluded that the charged residues (1, 5 and 9) of the two nonapeptides form a unique and unaltered pattern, presumably with critical function and charac- teristics of the H + -PPase family. Putative metal-binding patterns Asp residues are strictly conserved in the H + -PPase nonapeptide motifs DVGADLVGK and DNVGD NVG D. The residues aspartic acid (Asp) and glutamic acid (Glu) can act as metal ligands in various proteins [28,29]. UniProtKB ⁄ Swiss-Prot was screened for pat- terns of nine amino acid residues with either Asp or Glu at every fourth position (1, 5 and 9) and allowing any residue at the remaining positions. If the sequence formed an a-helix, with one turn every 3.6 residues, the charged residues would be facing the same side, to facili- tate metal-binding properties at the active site. In UniProtKB ⁄ Swiss-Prot, 11 648 proteins were found with the motif D-X 3 -D-X 3 -D, while over two- fold as many (26 389 proteins) were found with the motif E-X 3 -E-X 3 -E. We investigated protein family relationships based upon the Pfam annotations in the UniProtKB ⁄ Swiss-Prot entries. For the ‘E-motif’, the most frequent domain was elongation factor Tu (with 472 proteins), and the protein kinase domain (with 320 proteins) was the second most frequent. For the ‘D-motif’, the most frequent domain was the EF hand Ca 2+ ion-binding motif (297 occurrences), followed by S-adenosylmethionine synthases (187 proteins) and kinases (178 occurrences). Conclusions The rapidly expanding numbers of available amino acid sequences provided novel possibilities to explore early biological evolution, in the direction from ‘very early’ polypeptide synthesis to various known or putative active sites of enzymes, especially those of the H + -PPase family. Our analyses with bioinformatic methods have Table 2. Sequence patterns containing the four ‘primitive’ amino acid residues searched in the UniProtKB ⁄ Swiss-Prot database. Pattern Number of proteins Number of occurrences A-D-A-D-A 162 173 D-A-D-A-D 104 112 A-D-A-D-A-D-A-D-A 22 D-A-D-A-D-A-D-A-D 22 D-A-A-A-D-A-A-A-D 00 D-V-D-V-D 74 79 V-D-V-D-V 55 59 D-V-D-V-D-V-D-V-D 12 V-D-V-D-V-D-V-D-V 12 D-V-V-V-D-V-V-V-D 00 D-G-D-G-D 140 145 G-D-G-D-G 101 108 D-G-D-G-D-G-D-G-D 00 G-D-G-D-G-D-G-D-G 00 D-G-G-G-D-G-G-G-D 00 J. Hedlund et al. Ancient sequence motifs in the H + -PPase family FEBS Journal 273 (2006) 5183–5193 ª 2006 The Authors Journal compilation ª 2006 FEBS 5191 shown that the H + -PPases are unique in their sequence properties, with no close relatives detectable using the presently available sensitive methods. The analyses have also shown that the membrane-bound H + -PPases form a large family, divided into two subclasses – types 1 and 2 – where, notably, both types are found in plants, pro- tozoa, bacteria and archaea. No occurrences exist in ver- tebrates, with the possible exception of a reported sequence from the X. tropicalis early genome project. In soluble family I PPases, two structural types are known to be strictly divided – one in prokaryotes and the other in eukaryotes [21]. The well-conserved nonapeptides in the loop between transmembrane segments 5 and 6 show specific patterns that can be used for functional assignments of unanno- tated genomes. The distance between the two nonapep- tides is unchanged in all known sequences. We believe that our novel explorations on peptide motifs, both as such and as formed in apparent closeness to situations plausibly existing at the time of the origin and very early evolution of life on Earth, may be usefully extended when even more sequence data and, especially, when the first 3D structure of an H + -PPase, become available. Based on the 3D structure and the results presented here, rational selections of site-specific mutants may be expected to illuminate further both the evolutionary and the dynamic aspects of H + -PPase function. Experimental procedures Pyrophosphatase sequences were searched using blast [30] towards UniProtKB, version 6.3 (October 2005, http:// www.uniprot.org) [15], and an in-house database of all genomes in the public domain (ftp.ensemble.org; ftp.ncbi. nih.gov; ftp.tigr.org), denoted GenomeLKPG (A. Bresell and J. Hedlund, Linko ¨ ping University, Sweden, personal commu- nication). The searches were complemented by HMM-based screenings based upon the ‘H_PPase’ model from Pfam [31]. We also built and calibrated our own HMM, based upon 86 sequences. For the creation of HMM and screenings, the hmmer software (http://hmmer.wustl.edu) [17] was used with default parameters for building and calibrating (commands ‘hmmbuild’ and ‘hmmcalibrate’). General sequence comparisons were made using the pro- gram fasta [16] and pattern searches using the ps_scan utility from the Prosite database [32]. 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Both the high content of the ‘very early’ amino acids in these sequence motifs, and the. along the protein chain (Fig. 3). From the resulting plot it is clear that most of the con- served residues are located on the cytosolic side of the J. Hedlund et al. Ancient sequence motifs in the. where alanine is replaced with asparagine, given the presence of asparagines in one of the two puta- tive active site motifs of R. rubrum H + -PPase. The only patterns found in the proteins were