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Nickel trafficking: insights into the fold and function of UreE, a urease metallochaperone
JOURNAL OF
Inorganic Biochemistry Journal of Inorganic Biochemistry 98 (2004) 803–813 www.elsevier.com/locate/jinorgbio
Nickel trafficking: insights into the fold and function of UreE, a urease metallochaperone q Francesco Musiani, Barbara Zambelli, Massimiliano Stola, Stefano Ciurli
*
Department of Agro-Environmental Science and Technology, University of Bologna, Viale Giuseppe Fanin 40, 40127 Bologna, Italy Received 21 October 2003; received in revised form 2 December 2003; accepted 22 December 2003
Abstract UreE is a metallo-chaperone assisting the incorporation of two adjacent Ni2þ ions in the active site of urease. This study describes an attempt to distill general information on this protein using a computational post-genomic approach for the understanding of the structural details of the molecular function of UreE in nickel trafficking. The two crystal structures recently determined for UreE from Bacillus pasteurii (BpUreE) and Klebsiella aerogenes (KaUreE) were comparatively analyzed. This analysis provided insights into the protein structural and conformational features. A structural database of UreE proteins from a large number of different genomes was built using homology modeling. All available sequences of UreE were retrieved from protein and cDNA databases, and their structures were modeled on the crystal structures of BpUreE and KaUreE. A self-consistent iterative protocol was devised for multiple sequence alignment optimization involving secondary structure prediction and evaluation of the energy features of the obtained modeled structures. The quality of all models was tested using standard assessment procedures. The final optimized structure-based multiple alignment and the derived model structures provided insightful information on the evolutionary conservation of key residues in the protein sequence and surface patches presumably involved in protein recognition during the urease active site assembly. Ó 2004 Published by Elsevier Inc. Keywords: Structure modeling; Metal-binding chaperone; Bacillus pasteurii; Klebsiella aerogenes; Urease
1. Introduction 1.1. Ni trafficking and urease active site assembly Urease [EC 3.5.1.5] is a nickel-containing enzyme that catalyzes the hydrolysis of urea to produce ammonia and carbamate in the last step of nitrogen mineralization [1,2]. The increase of pH arising from this reaction causes negative effects in human and agro-environmental settings, providing significance for the discovery of the molecular details of this process in order to develop potential drugs. Studies of the chemistry and biochemistry of bacterial urease from Klebsiella aerogenes (Ka) q
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio.2003.12.012. * Corresponding author. Tel.: +39-051-209-6204; fax: +39-051-2096203. E-mail address: [email protected] (S. Ciurli). 0162-0134/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.jinorgbio.2003.12.012
[3–9], Bacillus pasteurii (Bp) [10–15], and Helicobacter pylori (Hp) [16] resulted in the elucidation of the mechanism of catalysis and inhibition of the enzyme at the molecular level. In particular, following the determination of the high-resolution active site solvent structure in B. pasteurii urease and its complex with diamidophosphate, a transition state analogue [11], together with kinetic studies on the enzyme inhibition by fluoride [9], a consensus has been reached on the role of the hydroxide ion bridging the two Ni ions present in the enzyme active site as the nucleophile acting on the Nibound urea during enzymatic hydrolysis [14]. Other steps of the mechanism, such as the substrate binding mode and the identity of the groups involved in the proton transfer, still require further investigation [15]. The new frontier in the chemistry of urease is represented by the in vivo assembly of urease, and in particular by the molecular mechanisms by which the Ni-containing active site is built. The solution of this
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problem depicts, more in general, the paradigm for the activation of a variety of enzymes that depend of the presence of metal ions for catalysis, in particular those containing Ni ions in the active site [1,17]. The biology of the transport and insertion of Ni2þ into the active site of urease has been extensively studied for Klebsiella aerogenes, and we will use this case to illustrate the general features of the process. The key players in this metabolic route are four accessory proteins [18–20], named KaUreD (30 kDa), KaUreF (25 kDa), KaUreG (22 kDa), and KaUreE (18 kDa), which are the biosynthetic products of four corresponding genes clustered in the urease operon. This operon also includes the ureA, ureB and ureC genes, encoding the three structural subunits of the a3 b3 c3 heteropolymeric urease enzyme [4,11]. The specific functions of these four accessory proteins are not fully understood. However, all evidences point to a timely ordered mechanism at the basis of the in vivo urease activation, in which each urease chaperone plays a precise role. Apourease can be partially activated in vitro by addition of Ni2þ in the presence of CO2 [21]. KaUreD may maintain a proper conformation of apourease by forming a specific complex [22,23]. KaUreF is required to facilitate carbamylation of the Ni-bridging Lys residue in the active site of urease, and to exclude Ni2þ ions from binding to the active site until the Lys has been carbamylated. KaUreG exhibits clear sequence similarity to nucleotide triphosphate-binding proteins, featuring a so-called P-loop motif, suggesting a possible role in an energy-dependent step during the in vivo urease assembly [24–27]. While UreD, UreF, and UreG are absolutely necessary for urease active site assembly, UreE is only required to facilitate this process [19]. KaUreE features Ni2þ -binding capability [28–32]. It possesses a His-rich C-terminus and binds ca. 6 Ni2þ per dimer in a coordination environment of 3–5 His, with an
average Kd of ca. 10 lM [28]. Not all UreE proteins feature such a His-rich tail. A genetically modified and truncated form of KaUreE, named H144* KaUreE, lacking the last 15 residues (10 of which are His), still displays properties and physiological activity comparable to that of the wild-type KaUreE [29], consistently with the existence of UreE proteins that do not possess the His-rich C-terminal tail. The truncated H144* KaUreE was reported to bind ca. 2 Ni2þ per dimer, and the two metal binding sites were shown to be spectroscopically distinguishable [30]: each different site appeared to feature a pseudo-octahedral Ni2þ ion in an N/ O coordination environment, differing in the number of His donors. Site-specific mutagenesis experiments have suggested the presence of His96, His112, Asp111 and Cys79 (K. aerogenes consensus sequence) in one of the two putative Ni2þ -binding sites, whereas His110 was proposed to take part in the second site [31]. However, only His96 and Asp111 are most critical for Ni2þ -incorporation into the urease active site, suggesting that only one Ni2þ -binding site is involved in the physiological action of KaUreE [27,31]. The crystal structure of UreE from two different species, Bacillus pasteurii (BpUreE, PDB codes: 1EB0 respectively) [33] and 1EAR, resolution 1.85 and 1.70 A, and Klebsiella aerogenes (KaUreE, PDB codes: 1GMU, 1GMV, and 1GMW, resolution 1.50, 2.80 and 1.50 A, respectively) [34], was reported recently (Fig. 1). The present paper describes a contribution towards the comprehension of the molecular role of UreE in Ni2þ trafficking and incorporation into the urease active site, involving a range of computational tools within the framework of a post-genomic approach. In the first part, the two available UreE crystal structures will be compared in detail, highlighting both the strikingly large similarity of the fold despite the low sequence homology, and the significant differences. Subsequently, the construction of a large structural database is described,
Fig. 1. Crystal structures of BpUreE (panel (a), PDB code 1EAR) and KaUreE (panel (b), PDB code 1GMW) reported as ribbon diagrams. The proteins are shown with the metal binding site toward the viewer (top panels) and rotated by 90° about the long horizontal axis (bottom panels). Metal ions are shown as spheres of arbitrary radius. The figures were made with MOLSCRIPT [50] and Raster3D [51].
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containing all UreE proteins for which the sequence is known. This database will prove to be useful to obtain information on conserved structural details that could lead to a mechanistic proposal for the interaction of UreE with the preformed UreDFG-apourease supercomplex, and more in general for the molecular mechanism of urease active site building.
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for the search. UreE sequences were retrieved from a non-redundant sum of different databases (SwissProt, TrEMBL, TrEMBLNew, GenBank CDS, PDB, PIR, PRF). Multiple sequence alignments were performed using ClustalW program [39], available at www.ebi. ac.uk/clustalw, and alignment optimization was carried out using information deriving from secondary structure predictions provided by the program JPRED [40], available at www.compbio.dundee.ac.uk/~www-jpred.
2. Materials and methods 2.2. Structure prediction and homology modeling 2.1. Sequence search and alignment Sequences of UreE were searched using sequence similarity criteria and the primary structure of BpUreE as template. The program FASTA3 [35,36] available at the address www.ebi.ac.uk/fasta33 and the program BLAST (Basic Local Alignment Search Tool) [37,38] available at www.ncbi.nlm.nih.gov/BLAST were utilized
Model structures were calculated using the program MODELLER 6.2 [41] with the model-default options. The dimer of BpUreE, built from the roto-translational matrix provided in 1EAR file, and the dimer of KaUreE, from chains A,C of 1GMW, were used as templates for all the modeling. Although three additional PDB files for these proteins are available (1EB0 for BpUreE, and
Table 1 Primary sequence index, code, biological source and identity respect to B. pasteurii UreE of all native non-redundant UreE sequences available in SWALL database on FASTA server and in NT database on PSI-BLAST server Sequence number
Code
Source
Identity (%)
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
UREE_BACPA Q9KG58 Q99RY1 UREE_BACSB Q9AQT2 Q9L419 UREE_ACTNA Q9S0Q3 Q93T80 Q9ETE2 Q9RFF1 UREE_UREPA Q98CZ2 O06708 UREE_ACTPL UREE_HAEIN Q8XXT2 AF347070 Q8YYW0 UREE_ALCEU UREE_LACFE O87398 Q93PJ2 UREE_KLEAE UREE_STRSL Q9HUS2 Q9L640 Q92MY3 AF417006_4 Q9RYJ8 UREE_PROMI BAB13789
Bacillus pasteurii Bacillus halodurans Staphylococcus aureus Bacillus sp. Rhodobacter capsulatus Corynebacterium glutamicum Actinomyces naeslundii Helicobacter pylori Brucella abortus Ureaplasma urealyticum Rhodobacter sphaeroides Ureaplasma parvum Rhizobium loti Bordetella bronchiseptica Actinobacillus pleuropneumoniae Haemophilus influenzae Ralstonia solanacearum Rhizobium leguminosarum Anabaena sp. Alcaligenes eutrophus Lactobacillus frementum Synechoccus sp. (str.WH7805) Helicobacter hepaticus Klesbiella aerogenes Streptococcus salivarius Pseudomonas aeruginosa Prochlorococcus sp. (str.PCC9511) Rhizobium meliloti Nitrosococcus oceani Deinococcus radiodurans Proteus mirabilis Vibrio parahaemolyticus
100.00 55.10 37.58 34.01 33.62 33.54 30.67 30.53 30.08 29.86 29.66 29.41 29.36 29.30 29.22 29.22 28.70 28.44 28.32 28.03 27.78 26.50 25.93 25.66 25.33 25.20 24.37 23.77 22.77 22.05 21.20 20.38
Q8YHZ9 UREE_YERPE AAK88135 AAA50997
Brucella melitensis Yersinia pestis – Y. pseudotubercolosis Agrobacterium tumefaciens Yersinia enterocolica
E01 E02 E03 E04
(*) (*) (*) (*)
(*) Excluded from the modeling due to low homology.
28.95 27.34 27.27 26.56
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1GMU and 1GMV for KaUreE) these were excluded either because of their lower resolution (as in the case of 1EB0 and 1GMV) or because they represent non-functional states of the protein (1GMU and 1GMV do not have bound metal ions). The PDB file 1EAR is relative to BpUreE mutated in residues 13–17 (LSHQI instead of YESSD), while 1GMW is the structure of a Ala91His mutant featuring additionally a truncation of the last 15 residues. The calculated models of BpUreE and KaUreE are related to the wild-type protein sequences. All modeled sequences are related to the protein fragments for which the template structure has been experimentally determined. Therefore, this procedure excluded, from the model, the modeling of the unstructured His-rich Cterminal tails of some of the selected sequences. The short loops found in sequences 6 and 7 (Table 1, UreE from Corynebacterium glutamicum and Actinomyces naeslundii, respectively) were modeled using an additional template composed of residues 309–336 of Enterococcus faecalis NadH peroxidase (PDB code: 1JOA). This expedient was necessary to avoid misfolding in this particular region of the cited model structures. The program PROSA II (Version 3.0, 1994) [42] was used for selecting the best models provided by MODELLER and for protein structure analysis, to test the coherency and validity of the model structures. The Z-scores reported in this work are derived through the standard ‘‘hide and seek’’ procedure of the program, by which the scores are correlated to the difference in potential energy, calculated using mean field potentials, between the input structure and other randomly assigned folds for its amino acid sequence. A lower Z-score corresponds to a more favorable potential energy associated with the structure under examination. The output PROSA Z-scores are reported in Table 1, Supplementary material. The obtained final alignment was employed to derive a phylogenetic tree as provided by the program PHYLIP [43], available at evolution.genetics.washington. edu/phylip.html. Structure validation was performed using PROCHECK [44] and WHATIF [45]. The calculated final structures were deposited in the www.postgenomicnmr.net site. The molecular surfaces and the electrostatic color-coding were generated by the pro and are gram GRASP [46] using a probe radius of 1.4 A, available as Supplementary material. 1 The electrostatic potential was calculated using a simple version of a Poisson–Boltzmann solver with the GRASP full charge set. All the histidine residues were considered neutral and the N- and C-terminal residues were charged. Dielectric constants of 80 and 2 were used for the solvent and protein interior, respectively. 1 Supplementary material is available in the online version of this paper.
3. Results 3.1. Comparison of protein architecture between BpUreE and KaUreE BpUreE and KaUreE reveal a unique tertiary structure, made up of two distinct domains (Fig. 1). The N-terminal is composed of two three-stranded mixed parallel and anti-parallel b-sheets stacked upon each other in a nearly perpendicular fashion, with a short helical region between the two sheets. In BpUreE this is a 310 helix, while it is a regular helix in KaUreE. The C-terminal domain is organized in a babbab-fold, notably similar to the Cu-chaperone Atx1 [47]. The functional dimers of both BpUreE and KaUreE are built by a head-to-head interaction, involving the hydrophobic face of an amphiphilic helix in the C-terminal domain. The root mean square deviation (RMSD) of the two (Fig. 2(a)). dimers is 1.50 A The two domains from each protein are compared independently. The backbone RMSD for the N-terminal respectively. and C-terminal domains is 1.41 and 1.12 A,
Fig. 2. (a) Crystal structures of BpUreE and KaUreE superimposed and reported as ribbon diagram. The proteins are shown with the metal binding site toward the viewer (top panel) and rotated by 90° about the long horizontal axis (bottom panel). BpUreE His100 and KaUreE His96 are reported using ‘‘sticks’’ representation and the metal ions are evidenced as spheres (color scheme: BpUreE, green; KaUreE, gold; copper, cyan; zinc, purple). (b) Details of the metal binding site of BpUreE (left panel) and KaUreE (right panel) (color scheme: copper, cyan; zinc, purple; carbon, gray; nitrogen, blue; oxygen, red). The proposed H-bonds are colored according to the donor– acceptor scheme. Panel (a) was made with MOLSCRIPT and Raster3D.
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The main difference observed is the conformation of the loop formed by residues 9–18 in BpUreE and 8–15 in KaUreE (visible in the lower part of the ribbon diagram). The structural conservation of each separate domain contrasts with the variability of the inter-monomer and inter-domain relative positions. A peculiar inter-monomer movement can be detected by considering (i) the different inter-chain angle formed between the axes of the two a-helices composing the dimer interface (33.26° and 19.29° in BpUreE and KaUreE, respectively) and (ii) the similar distance between the Ca atoms of the His100 residues (BpUreE consensus sequence, His96 for KaUreE) located at one end of the same helix (9.67 and in BpUreE and KaUreE, respectively). In addi9.16 A tion to this, the N-terminal domains feature different relative orientations with respect to the C-terminal domains in the two proteins. This movement apparently derives from a different conformation of the short linker connecting the two domains in each monomer, which results in a rotation around an ideal axis crossing the dimer from one N-terminal domain to the other. The bottom panel of Fig. 2(a) shows a schematic diagram indicating the rotation around the protein long axis, needed for the BpUreE N-terminal domains to superimpose onto the same domains of KaUreE. This protein flexibility around the short linker between the two domains was also detected for the single BpUreE and KaUreE proteins by comparing different structures of each protein obtained in various conditions and crystal forms [33,34]. The present analysis, however, further extends the awareness of the conformational freedom available for this protein, possibly related to induced-fit processes occurring during the formation of protein– protein complexes involving the other urease chaperones. This flexibility is also suggested by recent studies on the influence of solution ionic strength on the protein conformation and thermal stability [48]. 3.2. Comparison of the metal binding sites in BpUreE and KaUreE A single Zn2þ ion is bound on the surface of the BpUreE dimer (as also determined by mass spectrometry [49]) through the coordination of two His100 Ne (one from each monomer), while a Cu2þ ion is bound to the homologous His96 in KaUreE. The Zn2þ ion in BpUreE can be replaced by Ni2þ , as revealed by paramagnetic NMR [49] and X-ray anomalous diffraction difference maps [33]. The structures of the metal-binding site in BpUreE and KaUreE are very similar, with an RMSD of considering the metal ions and metal-bound His 0.57 A only. The position of the metal-bound His backbone is stabilized in both cases by an intra-chain H-bond between Gly97 O and His NH (Fig. 2(b)). In BpUreE an inter-chain H-bond, which also contributes to the dimer
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stability, is formed between His100 Nd1 and Asn98 Od, further stabilizing the His100 Ne-Zn2þ bond through a fully conserved Ôelec-His-ZnÕ motif. In KaUreE this Hbond is missing due to the different conformation of the Asn residue side chain, which experiences an O/NH2 amide group swap as compared to BpUreE. Given the peripheral position of the metal ion on the protein surface, it would be interesting to have information on the number and position of water molecules bound in the coordination sphere around Ni and Cu ions, in order to correlate the structural information to the available spectroscopic data. For different reasons, this cannot be done for either BpUreE or KaUreE. The chemical surrounding of the metal ion in BpUreE is perturbed by the presence of another dimer bound to the same Zn2þ ion through the same residues, causing the formation of two tetrameric forms pivoted around the metal ion and not involving additional protein surface patches [33]. The coordination environment of the Zn2þ ion in BpUreE differs in the two forms structurally characterized, being either tetrahedral (four His100 Ne, one from each monomer) or octahedral (two additional axial water molecules), depending on the stacking angle between the two dimers. Such tetrameric state of BpUreE was previously identified by size-exclusion chromatography and paramagnetic NMR only in concentrated protein samples containing Zn2þ or Ni2þ ions and is not believed to be functionally relevant [49]. No tetrameric state has ever been reported for KaUreE. The Cu2þ ion in KaUreE, located in the same position as Ni2þ /Zn2þ in BpUreE, is bound only to the two conserved His residues, and no solvent molecules are found close to the metal ion. 2 This is probably a consequence of the way the metal-bound form was obtained, that is by simply soaking a crystal of the apo-KaUreE in a copper-containing solution. In both BpUreE and KaUreE structures, no electron density for the last few residues at the C-termini is observed, presumably because of disorder caused, in the case of BpUreE, by the formation of the dimer of dimers, while in the case of KaUreE this disorder could be due to the non-natural form of the protein, featuring a truncation of the Hisrich C-term, in addition, again, to the fact that the starting structure prior to the copper soaking of the crystal is that of the apo-form. In the crystal structure of KaUreE a second metal binding site, constituted by His110 and His112 (KaUreE consensus sequence), is found on each monomer,
2
The apparent presence of three water molecules around the copper ion in the conserved site of KaUreE, as resulting from Fig. 4(a) of [34], is an artifact, as it results from the analysis of the related PDB files. The figure was made by superimposing the structures of the dimers from both the apo-form (PDB code 1GMU) and the copper-soaked crystal (1GMW). In the latter case, the water molecule is located at 3.9 from the copper ion. A
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resulting in the presence of two additional Cu2þ ions. These two residues were previously indicated as belonging to two different binding sites on KaUreE [34]. In BpUreE this second binding site is absent, due to the substitution of His110 and His112 by Tyr and Lys, respectively, suggesting that this additional binding site is not functionally relevant. Overall, this structural comparison clarified the relevant similarities and differences between the two UreE proteins, and further indicated features of the fold that could be important for the functional role in Ni trafficking. The exploration of sequence and structural conservation in UreE proteins from different biological sources allows the building of a structural database using homology modeling procedures, as described in the following section. 3.3. UreE multiple sequence alignment construction and analysis A UreE sequence search resulted in 36 hits (Table 1). Four sequences were detected in the initial multiple se-
quence alignment, which presented very large differences, such as long insertions in the middle of otherwise highly conserved secondary structure regions, and they were excluded from further analysis. These sequences are listed in the bottom part of Table 1. The alignment of the remaining 32 UreE proteins was optimized using information derived from secondary structure predictions. This optimized alignment was used as input to obtain a first set of model structures, which were then analyzed to identify local fold problems using PROSA. Whenever possible, such structural misfoldings were corrected by modifying the alignment in the interested regions and building new models. This procedure was repeated for each model until all possible local fold problems were solved. This iterative optimization procedure, which involved (i) model building, (ii) model quality assessment, and (iii) correction of the multiple alignment, finally yielded an aligned database of acceptable model structures. In the final multiple alignment, reported in Fig. 3, the sequences were found to feature an identity with respect to the sequence of BpUreE ranging between 55.10% and 20.38%.
Fig. 3. Optimized multiple sequence alignment of UreE from biological sources indicated in Table 1. The order of the alignment follows the identity percentage with respect to BpUreE. The predicted secondary structure elements are highlighted in yellow (helixes) and turquoise (sheets). The conserved residues are highlighted in green (100% conservancy) or magenta (conservative substitution).
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From such alignment it is possible to extract a whole set of considerations. The metal binding site motif GNXH (where X is R in 26 sequences or K, T or L in 6 sequences) is fully conserved. The four amino acids (Met86, Met89, Gly90, and Ala93) (BpUreE consensus sequence) found at the interface of the dimer are generally mutated with hydrophobic residues, indicating a conservancy of the nature of the interaction holding together the two monomers in the functional dimer. Two amino acids are conservatively mutated: Leu55, found in the random coil region of the N-terminal domain and constituting the center of the hydrophobic core of such domain, and Asp115, found in the C-terminal domain at the beginning of the a-helix not involved in the dimer formation. The final structure-based multiple sequence alignment of the UreE proteins allows the calculation of the conservancy of every single residue. As reported in Fig. 4(a), only few positions show conservation higher than 80% (i.e. 2r higher than average).
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These positions involve, in the N-terminal domain, (i) Val26 (BpUreE consensus sequence), more generally found as a Leu and located on the lateral surface of the protein, (ii) Gly42, that stabilizes a loop with an H-bond between its NH and Thr39 O, (iii) Leu49, Leu55, Gly58, and Leu61, composing a large part of the hydrophobic core of the N-terminal domain, and (iv) Asp59, whose Od is H-bonded to Arg56 NH, stabilizing a loop conformation. In the C-terminal domain the conserved residues are (i) Gly97, Asn98, and His100, composing the metal binding site, and (ii) Arg99 and Asp115, positioned on the surface of the protein. The importance of Asp115 in the functional role of UreE has been previously highlighted, but its detailed role not yet unveiled [31]. The residues constituting the second metal binding site in the crystal structure of KaUreE (His110 and His112, KaUreE consensus sequence) show a relatively low conservation (37.50% and 31.25%, respectively). This result confirms that this second metal binding site is
Fig. 4. (a) Residue conservancy percentage as a function of the residue number of the ‘‘ghost-UreE’’ sequence. The dashed line delimitates high conserved residues. (b) ‘‘Ghost-UreE’’ sequence compared with BpUreE and KaUreE sequences. Boxed residues present a conservation ratio higher than 80%.
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a characteristic feature of KaUreE and few other UreE proteins, but cannot be considered as a general character of the functional role for all UreE. On the basis of such conservation analysis, a prototypical UreE sequence was built using the most conserved amino acid in each position of the multiple alignment presented in Fig. 3. Such sequence was named ‘‘ghost-UreE’’ and is reported in Fig. 4(b) as compared with the sequences of BpUreE and KaUreE. In the same figure, the positions of the highly conserved residues discussed in the previous paragraphs are highlighted. 3.4. UreE model structures building and analysis The unrooted phylogenetic tree, generated on the basis of the final multiple sequence alignment, is reported in Fig. 5. This diagram can be divided in two sections, where the sequence indicated as 30 (from Deinococcus radiodurans) should be taken as an ideal border. The sequences of BpUreE and KaUreE are found in the two different regions of the phylogenetic tree. This is an ideal situation by which the modeling procedure relies on template structures representing essentially all the sequences considered in this work. Models were generated for all the aligned 32 sequences. Fig. 6(a) shows the superimposition of the backbones of all the model structures obtained. The protein tertiary structure is very well conserved in both domains of the models. The different orientation between the N- and C-terminal domains observed in BpUreE and KaUreE crystal structures is reflected in a
range of different domain relative orientations in the models, resulting in the bundle observed at the edges of the superimposed molecules (Fig. 6(a)). The secondary structure elements found in the experimental structures are also found in the models. In particular, it is important to notice that the a-helix forming the dimer interface is always observed. In most of the cases, such helix is formed by 14 amino acids and, in the first six residues, it features the same hydrophobic patch observed in the crystal structures. The metal binding His residues are always found at the edge of this helix, and the H-bond between Gly97 O and His100 NH (BpUreE consensus sequence) is conserved in all model structures. The structures used as templates feature a metal ion bound in this position, and therefore all models are relative to the metal-bound UreE. Eventual conformational changes occurring upon metal loss cannot be explored using this methodology. The backbone RMSD of all models with respect to the BpUreE and KaUreE crystal structures ranges from and from 0.82 to 1.82 A, respectively 0.63 to 2.17 A 1 (Table 1, Supplementary material). The backbone RMSD of model structures 1 and 24 (corresponding to native BpUreE and KaUreE sequences, respectively) calculated with respect to the relative crystal structures (which represent mutated forms, as indicated above) is respectively. The superimposition of 0.63 and 0.82 A, such structures is reported in Fig. 7, showing the good quality of the models, in particular at the C-terminal domains and at the dimer interface. However, the Nterminal domains are well reproduced in the fold but not exactly in the relative position with respect to the C-
Fig. 5. Unrooted phylogenetic tree of the UreE sequences that constituted the dataset analyzed in this work. Sequence numbers as in Table 1 for UreE whose structure has been experimentally determined the label is reported in bolded type.
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Fig. 6. (a) Backbone superimposition of modeled structures of UreE. The proteins are shown with the metal binding site toward the viewer. (b) GRASP solid surface representation of the electrostatic potential of the ghost UreE model structure. The protein is shown with the metal binding site toward the viewer. The surface is colored according to the calculated electrostatic potential contoured from )6.0 kT =e (intense red) to +6.0 (where k, Boltzmann constant; T, absolute temperature; e, electron charge) (intense blue). (c) GRASP solid surface colored according to residues conservation ratio from 20.0% (red) to 100.0% (green).
terminal domain. As observed for the crystal structures, the N-terminal domain presents a relatively high degree of conformational freedom that is reflected also in the models with propagation on the RMSD values. The percentage of the monomer surface involved in the dimer formation is rather constant among all models, oscillating around 11%. The distance between the Ca atoms of the metal-bound His residues ranges from 9.23 Such values are comparable with the corto 10.44 A. responding experimental data, giving a first indication about the reliability of the structures obtained from the modeling procedure, and indicate that all modeled structures can easily sustain metal binding at the same location of the protein surface as found in the crystal structures. Interestingly, the quality of the model structures from BpUreE and KaUreE, as judged by
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the distribution among the so-called allowed and notallowed regions in the Ramachandran plot, is slightly higher than the corresponding experimental structures (Table 2, Supplementary material). 1 This is probably a consequence of the fact that the Ramachandran plot may not consider as feasible all possible protein conformations that are, however, experimentally observed. All models present a good percentage of residues in the core region of the Ramachandran plot (at least 87%), while the few residues found in the disallowed region are localized in loops where the modeling process is knowingly more difficult. BpUreE and KaUreE models yield structural parameters and Z-scores that are not worse than for the crystallographic data (Table 2, Supplementary material). 1 The energy profiles calculated with PROSA II of the two pairs of model and experimental structures are almost identical (Fig. 7, bottom panels). In the BpUreE model structure the energy profile presents a region where the energy is considerably lower than for the crystal structure. This region, found between residues 13 and 17, is a small portion of the BpUreE sequence that featured the LSHQI sequence instead of YESSD, presumably as a result of a cloning artifact [33]. The model structure, built using the wildtype sequence, presents a better local fold in that region, probably a consequence of inter-tetramer interactions in the mutated BpUreE in the solid state involving the His residue of the LSHQI fragment with Met1 of another tetramer. The Z-scores obtained for all UreE model structures range from )4.32 to )8.89, with an average value of )6.65. Problems in protein packing are evidenced for model structures from sequences 14, 17, 31 and 32, which present a relatively high Z-score (higher than )5.70, i.e. the average Z-score plus one standard deviation). In the phylogenetic tree the sequences corresponding to these model structures are clustered close to the KaUreE sequence.
3.5. Protein surface electrostatic potential and residue conservation In order to gain information on the conserved protein surface features of UreE from different species, solid surface representations of the electrostatic potential were calculated for each model structure (Fig. 1, Supplementary material). In Fig. 6(b) the surface for the modeled ghost UreE sequence is reported, colored according to the electrostatic potential, while the surface of the same model colored according to residue conservation is shown in Fig. 6(c). This figure essentially contains all the information that can be generally distilled from the constructed model structure database. In particular, it is possible to observe that almost all the highly conserved residues found in the N-terminal domain are located in the protein core. The opposite is true
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Fig. 7. Superimposition of the crystal and model structures of BpUreE (a) and KaUreE (b). The proteins are shown with the metal binding site toward the viewer (top panels) and rotated by 90° about the long horizontal axis (central panels). Color scheme: crystal structures, blue; model structures, red. The PROSA II energy graphs of the crystallographic structure (blue line) and model (red line) structures from BpUreE and KaUreE are shown as bottom panels. Energies are represented in units of E=kT (E, energy; k, Boltzmann constant; T, absolute temperature).
for the C-terminal, where a large conserved patch is found on the surface of the protein in the close vicinity of the metal binding site (zone A in Fig. 6(c)). This region is characterized by the presence of a hydrophobic area, centered around the metal-binding neutral His residues. Another conserved region (zone B) corresponds to Asp115 (BpUreE consensus sequence), already indicated as somehow important for Ni2þ transport and release, and indeed the electrostatic potential in this region is negative. A third region (zone C) is characterized by a hydrophobic patch containing the highly conserved Gly42. A fourth noticeable region features a relatively high conservancy (zone D) and a positive charge, consistently with the presence of Arg29, Lys31, Arg32, and Arg33. The arrangement of these peculiar protein surface patches highlights their probable role in protein–protein complex formation concomitantly with the physiological metal ion transfer during the urease active site assembly.
process. In particular, a detailed comparison of the metal binding mode has clarified that the available structures do not clarify all the necessary details of the mechanism of nickel uptake and release, prompting for more investigations. The identification of highly conserved residues in the C-terminal domain and in the metal binding region of the protein suggest a possible recognizable surface and docking mode for the UreE binding to the UreDFG-apourease complex, a crucial aspect for the nickel incorporation in the urease activation mechanism. The calculated model structures are made available to all researchers interested in the structural biology of the urease metallochaperones. In particular, they can be used to fit models into low-resolution electron density maps and to perform molecular replacement in X-ray crystallography, to refine solution NMR structures, to support site-directed mutagenesis, and finally to perform docking experiments when the structures of the other urease chaperones will be revealed, a task that our laboratory is actively pursuing.
4. Conclusions
5. Abbreviations
The comparative analysis of the available crystal structures of UreE proteins, together with the structurebased multiple alignment and structural modeling of all available UreE sequences has provided novel information on the role of these protein in the urease activation
Bp BpUreE Ka KaUreE RMSD
Bacillus pasteurii Bacillus pasteurii UreE Klebisiella aerogenes Klebsiella aerogenes UreE root mean square deviation
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Acknowledgements This research was funded by the COFIN2001 project from the Italian MIUR. Funding from CIRMMP (Consorzio interuniversitario di risonanze magnetiche di metalloproteine paramagnetiche) is acknowledged for support to FM and MS. References [1] S. Ciurli, S. Mangani, in: I. Bertini, A. Sigel, H. Sigel (Eds.), Handbook on Metalloproteins, Nickel-containing Enzymes, Marcel Dekker, New York, 2001, pp. 669–708. [2] R.P. Hausinger, P.A. Karplus, in: A. Messerschmidt, R. Huber, T. Poulos, K. Wieghardt (Eds.), Handbook of Metalloproteins, Urease, Wiley, Chichester, 2001, pp. 867–879. [3] I.-S. Park, L.O. Michel, M.A. Pearson, E. Jabri, P.A. Karplus, S. Wang, J. Dong, R.A. Scott, B.P. Koehler, M.K. Johnson, R.P. Hausinger, J. Biol. Chem. 271 (1996) 18632–18637. [4] E. Jabri, M.B. Carr, R.P. Hausinger, P.A. Karplus, Science 268 (1995) 998–1004. [5] M.A. Pearson, L. Overbye Michel, R.P. Hausinger, P.A. Karplus, Biochemistry 36 (1997) 8164–8172. [6] P.A. Karplus, M.A. Pearson, R.P. Hausinger, Acc. Chem. Res. 30 (1997) 330–337. [7] K. Yamaguchi, N.J. Cosper, C. Stalhandske, R.A. Scott, M.A. Pearson, P.A. Karplus, R.P. Hausinger, J. Biol. Inorg. Chem. 4 (1999) 468–477. [8] M.A. Pearson, I.S. Park, R.A. Schaller, L.O. Michel, P.A. Karplus, R.P. Hausinger, Biochemistry 39 (2000) 8575–8584. [9] M.J. Todd, R.P. Hausinger, Biochemistry 39 (2000) 5389–5396. [10] S. Benini, W.R. Rypniewski, K.S. Wilson, S. Ciurli, S. Mangani, J. Biol. Inorg. Chem. 3 (1998) 268–273. [11] S. Benini, W.R. Rypniewski, K.S. Wilson, S. Miletti, S. Ciurli, S. Mangani, Structure Fold. Des. 7 (1999) 205–216. [12] S. Ciurli, S. Benini, W.R. Rypniewski, K.S. Wilson, S. Miletti, S. Mangani, Coord. Chem. Rev. 190–192 (1999) 331–355. [13] S. Benini, W.R. Rypniewski, K.S. Wilson, S. Miletti, S. Ciurli, S. Mangani, J. Biol. Inorg. Chem. 5 (2000) 110–118. [14] F. Musiani, E. Arnofi, R. Casadio, S. Ciurli, J. Biol. Inorg. Chem. 3 (2001) 300–314. [15] S. Benini, W.R. Rypniewski, K.S. Wilson, S. Ciurli, S. Mangani, J. Biol. Inorg. Chem. 6 (2001) 778–790. [16] N.-C. Ha, S.-T. Oh, J.Y. Sung, K.A. Cha, M.H. Lee, B.-H. Oh, Nat. Struct. Biol. 8 (2001) 505–509. [17] R.P. Hausinger, J. Biol. Inorg. Chem. 2 (1997) 279–286. [18] S.B. Mulrooney, R.P. Hausinger, J. Bacteriol. 172 (1990) 5837– 5843. [19] M.H. Lee, S.B. Mulrooney, M.J. Renner, Y. Markowicz, R.P. Hausinger, J. Bacteriol. 174 (1992) 4324–4330. [20] H.L.T. Mobley, M.D. Island, R.P. Hausinger, Microbiol. Rev. 59 (1995) 451–480. [21] I.S. Park, R.P. Hausinger, Science 267 (1995) 1156–1158. [22] I.S. Park, M.B. Carr, R.P. Hausinger, Proc. Natl. Acad. Sci. USA 91 (1994) 3233–3237.
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Inorganic Biochemistry Journal of Inorganic Biochemistry 98 (2004) 803–813 www.elsevier.com/locate/jinorgbio
Nickel trafficking: insights into the fold and function of UreE, a urease metallochaperone q Francesco Musiani, Barbara Zambelli, Massimiliano Stola, Stefano Ciurli
*
Department of Agro-Environmental Science and Technology, University of Bologna, Viale Giuseppe Fanin 40, 40127 Bologna, Italy Received 21 October 2003; received in revised form 2 December 2003; accepted 22 December 2003
Abstract UreE is a metallo-chaperone assisting the incorporation of two adjacent Ni2þ ions in the active site of urease. This study describes an attempt to distill general information on this protein using a computational post-genomic approach for the understanding of the structural details of the molecular function of UreE in nickel trafficking. The two crystal structures recently determined for UreE from Bacillus pasteurii (BpUreE) and Klebsiella aerogenes (KaUreE) were comparatively analyzed. This analysis provided insights into the protein structural and conformational features. A structural database of UreE proteins from a large number of different genomes was built using homology modeling. All available sequences of UreE were retrieved from protein and cDNA databases, and their structures were modeled on the crystal structures of BpUreE and KaUreE. A self-consistent iterative protocol was devised for multiple sequence alignment optimization involving secondary structure prediction and evaluation of the energy features of the obtained modeled structures. The quality of all models was tested using standard assessment procedures. The final optimized structure-based multiple alignment and the derived model structures provided insightful information on the evolutionary conservation of key residues in the protein sequence and surface patches presumably involved in protein recognition during the urease active site assembly. Ó 2004 Published by Elsevier Inc. Keywords: Structure modeling; Metal-binding chaperone; Bacillus pasteurii; Klebsiella aerogenes; Urease
1. Introduction 1.1. Ni trafficking and urease active site assembly Urease [EC 3.5.1.5] is a nickel-containing enzyme that catalyzes the hydrolysis of urea to produce ammonia and carbamate in the last step of nitrogen mineralization [1,2]. The increase of pH arising from this reaction causes negative effects in human and agro-environmental settings, providing significance for the discovery of the molecular details of this process in order to develop potential drugs. Studies of the chemistry and biochemistry of bacterial urease from Klebsiella aerogenes (Ka) q
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jinorgbio.2003.12.012. * Corresponding author. Tel.: +39-051-209-6204; fax: +39-051-2096203. E-mail address: [email protected] (S. Ciurli). 0162-0134/$ - see front matter Ó 2004 Published by Elsevier Inc. doi:10.1016/j.jinorgbio.2003.12.012
[3–9], Bacillus pasteurii (Bp) [10–15], and Helicobacter pylori (Hp) [16] resulted in the elucidation of the mechanism of catalysis and inhibition of the enzyme at the molecular level. In particular, following the determination of the high-resolution active site solvent structure in B. pasteurii urease and its complex with diamidophosphate, a transition state analogue [11], together with kinetic studies on the enzyme inhibition by fluoride [9], a consensus has been reached on the role of the hydroxide ion bridging the two Ni ions present in the enzyme active site as the nucleophile acting on the Nibound urea during enzymatic hydrolysis [14]. Other steps of the mechanism, such as the substrate binding mode and the identity of the groups involved in the proton transfer, still require further investigation [15]. The new frontier in the chemistry of urease is represented by the in vivo assembly of urease, and in particular by the molecular mechanisms by which the Ni-containing active site is built. The solution of this
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problem depicts, more in general, the paradigm for the activation of a variety of enzymes that depend of the presence of metal ions for catalysis, in particular those containing Ni ions in the active site [1,17]. The biology of the transport and insertion of Ni2þ into the active site of urease has been extensively studied for Klebsiella aerogenes, and we will use this case to illustrate the general features of the process. The key players in this metabolic route are four accessory proteins [18–20], named KaUreD (30 kDa), KaUreF (25 kDa), KaUreG (22 kDa), and KaUreE (18 kDa), which are the biosynthetic products of four corresponding genes clustered in the urease operon. This operon also includes the ureA, ureB and ureC genes, encoding the three structural subunits of the a3 b3 c3 heteropolymeric urease enzyme [4,11]. The specific functions of these four accessory proteins are not fully understood. However, all evidences point to a timely ordered mechanism at the basis of the in vivo urease activation, in which each urease chaperone plays a precise role. Apourease can be partially activated in vitro by addition of Ni2þ in the presence of CO2 [21]. KaUreD may maintain a proper conformation of apourease by forming a specific complex [22,23]. KaUreF is required to facilitate carbamylation of the Ni-bridging Lys residue in the active site of urease, and to exclude Ni2þ ions from binding to the active site until the Lys has been carbamylated. KaUreG exhibits clear sequence similarity to nucleotide triphosphate-binding proteins, featuring a so-called P-loop motif, suggesting a possible role in an energy-dependent step during the in vivo urease assembly [24–27]. While UreD, UreF, and UreG are absolutely necessary for urease active site assembly, UreE is only required to facilitate this process [19]. KaUreE features Ni2þ -binding capability [28–32]. It possesses a His-rich C-terminus and binds ca. 6 Ni2þ per dimer in a coordination environment of 3–5 His, with an
average Kd of ca. 10 lM [28]. Not all UreE proteins feature such a His-rich tail. A genetically modified and truncated form of KaUreE, named H144* KaUreE, lacking the last 15 residues (10 of which are His), still displays properties and physiological activity comparable to that of the wild-type KaUreE [29], consistently with the existence of UreE proteins that do not possess the His-rich C-terminal tail. The truncated H144* KaUreE was reported to bind ca. 2 Ni2þ per dimer, and the two metal binding sites were shown to be spectroscopically distinguishable [30]: each different site appeared to feature a pseudo-octahedral Ni2þ ion in an N/ O coordination environment, differing in the number of His donors. Site-specific mutagenesis experiments have suggested the presence of His96, His112, Asp111 and Cys79 (K. aerogenes consensus sequence) in one of the two putative Ni2þ -binding sites, whereas His110 was proposed to take part in the second site [31]. However, only His96 and Asp111 are most critical for Ni2þ -incorporation into the urease active site, suggesting that only one Ni2þ -binding site is involved in the physiological action of KaUreE [27,31]. The crystal structure of UreE from two different species, Bacillus pasteurii (BpUreE, PDB codes: 1EB0 respectively) [33] and 1EAR, resolution 1.85 and 1.70 A, and Klebsiella aerogenes (KaUreE, PDB codes: 1GMU, 1GMV, and 1GMW, resolution 1.50, 2.80 and 1.50 A, respectively) [34], was reported recently (Fig. 1). The present paper describes a contribution towards the comprehension of the molecular role of UreE in Ni2þ trafficking and incorporation into the urease active site, involving a range of computational tools within the framework of a post-genomic approach. In the first part, the two available UreE crystal structures will be compared in detail, highlighting both the strikingly large similarity of the fold despite the low sequence homology, and the significant differences. Subsequently, the construction of a large structural database is described,
Fig. 1. Crystal structures of BpUreE (panel (a), PDB code 1EAR) and KaUreE (panel (b), PDB code 1GMW) reported as ribbon diagrams. The proteins are shown with the metal binding site toward the viewer (top panels) and rotated by 90° about the long horizontal axis (bottom panels). Metal ions are shown as spheres of arbitrary radius. The figures were made with MOLSCRIPT [50] and Raster3D [51].
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containing all UreE proteins for which the sequence is known. This database will prove to be useful to obtain information on conserved structural details that could lead to a mechanistic proposal for the interaction of UreE with the preformed UreDFG-apourease supercomplex, and more in general for the molecular mechanism of urease active site building.
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for the search. UreE sequences were retrieved from a non-redundant sum of different databases (SwissProt, TrEMBL, TrEMBLNew, GenBank CDS, PDB, PIR, PRF). Multiple sequence alignments were performed using ClustalW program [39], available at www.ebi. ac.uk/clustalw, and alignment optimization was carried out using information deriving from secondary structure predictions provided by the program JPRED [40], available at www.compbio.dundee.ac.uk/~www-jpred.
2. Materials and methods 2.2. Structure prediction and homology modeling 2.1. Sequence search and alignment Sequences of UreE were searched using sequence similarity criteria and the primary structure of BpUreE as template. The program FASTA3 [35,36] available at the address www.ebi.ac.uk/fasta33 and the program BLAST (Basic Local Alignment Search Tool) [37,38] available at www.ncbi.nlm.nih.gov/BLAST were utilized
Model structures were calculated using the program MODELLER 6.2 [41] with the model-default options. The dimer of BpUreE, built from the roto-translational matrix provided in 1EAR file, and the dimer of KaUreE, from chains A,C of 1GMW, were used as templates for all the modeling. Although three additional PDB files for these proteins are available (1EB0 for BpUreE, and
Table 1 Primary sequence index, code, biological source and identity respect to B. pasteurii UreE of all native non-redundant UreE sequences available in SWALL database on FASTA server and in NT database on PSI-BLAST server Sequence number
Code
Source
Identity (%)
01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
UREE_BACPA Q9KG58 Q99RY1 UREE_BACSB Q9AQT2 Q9L419 UREE_ACTNA Q9S0Q3 Q93T80 Q9ETE2 Q9RFF1 UREE_UREPA Q98CZ2 O06708 UREE_ACTPL UREE_HAEIN Q8XXT2 AF347070 Q8YYW0 UREE_ALCEU UREE_LACFE O87398 Q93PJ2 UREE_KLEAE UREE_STRSL Q9HUS2 Q9L640 Q92MY3 AF417006_4 Q9RYJ8 UREE_PROMI BAB13789
Bacillus pasteurii Bacillus halodurans Staphylococcus aureus Bacillus sp. Rhodobacter capsulatus Corynebacterium glutamicum Actinomyces naeslundii Helicobacter pylori Brucella abortus Ureaplasma urealyticum Rhodobacter sphaeroides Ureaplasma parvum Rhizobium loti Bordetella bronchiseptica Actinobacillus pleuropneumoniae Haemophilus influenzae Ralstonia solanacearum Rhizobium leguminosarum Anabaena sp. Alcaligenes eutrophus Lactobacillus frementum Synechoccus sp. (str.WH7805) Helicobacter hepaticus Klesbiella aerogenes Streptococcus salivarius Pseudomonas aeruginosa Prochlorococcus sp. (str.PCC9511) Rhizobium meliloti Nitrosococcus oceani Deinococcus radiodurans Proteus mirabilis Vibrio parahaemolyticus
100.00 55.10 37.58 34.01 33.62 33.54 30.67 30.53 30.08 29.86 29.66 29.41 29.36 29.30 29.22 29.22 28.70 28.44 28.32 28.03 27.78 26.50 25.93 25.66 25.33 25.20 24.37 23.77 22.77 22.05 21.20 20.38
Q8YHZ9 UREE_YERPE AAK88135 AAA50997
Brucella melitensis Yersinia pestis – Y. pseudotubercolosis Agrobacterium tumefaciens Yersinia enterocolica
E01 E02 E03 E04
(*) (*) (*) (*)
(*) Excluded from the modeling due to low homology.
28.95 27.34 27.27 26.56
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1GMU and 1GMV for KaUreE) these were excluded either because of their lower resolution (as in the case of 1EB0 and 1GMV) or because they represent non-functional states of the protein (1GMU and 1GMV do not have bound metal ions). The PDB file 1EAR is relative to BpUreE mutated in residues 13–17 (LSHQI instead of YESSD), while 1GMW is the structure of a Ala91His mutant featuring additionally a truncation of the last 15 residues. The calculated models of BpUreE and KaUreE are related to the wild-type protein sequences. All modeled sequences are related to the protein fragments for which the template structure has been experimentally determined. Therefore, this procedure excluded, from the model, the modeling of the unstructured His-rich Cterminal tails of some of the selected sequences. The short loops found in sequences 6 and 7 (Table 1, UreE from Corynebacterium glutamicum and Actinomyces naeslundii, respectively) were modeled using an additional template composed of residues 309–336 of Enterococcus faecalis NadH peroxidase (PDB code: 1JOA). This expedient was necessary to avoid misfolding in this particular region of the cited model structures. The program PROSA II (Version 3.0, 1994) [42] was used for selecting the best models provided by MODELLER and for protein structure analysis, to test the coherency and validity of the model structures. The Z-scores reported in this work are derived through the standard ‘‘hide and seek’’ procedure of the program, by which the scores are correlated to the difference in potential energy, calculated using mean field potentials, between the input structure and other randomly assigned folds for its amino acid sequence. A lower Z-score corresponds to a more favorable potential energy associated with the structure under examination. The output PROSA Z-scores are reported in Table 1, Supplementary material. The obtained final alignment was employed to derive a phylogenetic tree as provided by the program PHYLIP [43], available at evolution.genetics.washington. edu/phylip.html. Structure validation was performed using PROCHECK [44] and WHATIF [45]. The calculated final structures were deposited in the www.postgenomicnmr.net site. The molecular surfaces and the electrostatic color-coding were generated by the pro and are gram GRASP [46] using a probe radius of 1.4 A, available as Supplementary material. 1 The electrostatic potential was calculated using a simple version of a Poisson–Boltzmann solver with the GRASP full charge set. All the histidine residues were considered neutral and the N- and C-terminal residues were charged. Dielectric constants of 80 and 2 were used for the solvent and protein interior, respectively. 1 Supplementary material is available in the online version of this paper.
3. Results 3.1. Comparison of protein architecture between BpUreE and KaUreE BpUreE and KaUreE reveal a unique tertiary structure, made up of two distinct domains (Fig. 1). The N-terminal is composed of two three-stranded mixed parallel and anti-parallel b-sheets stacked upon each other in a nearly perpendicular fashion, with a short helical region between the two sheets. In BpUreE this is a 310 helix, while it is a regular helix in KaUreE. The C-terminal domain is organized in a babbab-fold, notably similar to the Cu-chaperone Atx1 [47]. The functional dimers of both BpUreE and KaUreE are built by a head-to-head interaction, involving the hydrophobic face of an amphiphilic helix in the C-terminal domain. The root mean square deviation (RMSD) of the two (Fig. 2(a)). dimers is 1.50 A The two domains from each protein are compared independently. The backbone RMSD for the N-terminal respectively. and C-terminal domains is 1.41 and 1.12 A,
Fig. 2. (a) Crystal structures of BpUreE and KaUreE superimposed and reported as ribbon diagram. The proteins are shown with the metal binding site toward the viewer (top panel) and rotated by 90° about the long horizontal axis (bottom panel). BpUreE His100 and KaUreE His96 are reported using ‘‘sticks’’ representation and the metal ions are evidenced as spheres (color scheme: BpUreE, green; KaUreE, gold; copper, cyan; zinc, purple). (b) Details of the metal binding site of BpUreE (left panel) and KaUreE (right panel) (color scheme: copper, cyan; zinc, purple; carbon, gray; nitrogen, blue; oxygen, red). The proposed H-bonds are colored according to the donor– acceptor scheme. Panel (a) was made with MOLSCRIPT and Raster3D.
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The main difference observed is the conformation of the loop formed by residues 9–18 in BpUreE and 8–15 in KaUreE (visible in the lower part of the ribbon diagram). The structural conservation of each separate domain contrasts with the variability of the inter-monomer and inter-domain relative positions. A peculiar inter-monomer movement can be detected by considering (i) the different inter-chain angle formed between the axes of the two a-helices composing the dimer interface (33.26° and 19.29° in BpUreE and KaUreE, respectively) and (ii) the similar distance between the Ca atoms of the His100 residues (BpUreE consensus sequence, His96 for KaUreE) located at one end of the same helix (9.67 and in BpUreE and KaUreE, respectively). In addi9.16 A tion to this, the N-terminal domains feature different relative orientations with respect to the C-terminal domains in the two proteins. This movement apparently derives from a different conformation of the short linker connecting the two domains in each monomer, which results in a rotation around an ideal axis crossing the dimer from one N-terminal domain to the other. The bottom panel of Fig. 2(a) shows a schematic diagram indicating the rotation around the protein long axis, needed for the BpUreE N-terminal domains to superimpose onto the same domains of KaUreE. This protein flexibility around the short linker between the two domains was also detected for the single BpUreE and KaUreE proteins by comparing different structures of each protein obtained in various conditions and crystal forms [33,34]. The present analysis, however, further extends the awareness of the conformational freedom available for this protein, possibly related to induced-fit processes occurring during the formation of protein– protein complexes involving the other urease chaperones. This flexibility is also suggested by recent studies on the influence of solution ionic strength on the protein conformation and thermal stability [48]. 3.2. Comparison of the metal binding sites in BpUreE and KaUreE A single Zn2þ ion is bound on the surface of the BpUreE dimer (as also determined by mass spectrometry [49]) through the coordination of two His100 Ne (one from each monomer), while a Cu2þ ion is bound to the homologous His96 in KaUreE. The Zn2þ ion in BpUreE can be replaced by Ni2þ , as revealed by paramagnetic NMR [49] and X-ray anomalous diffraction difference maps [33]. The structures of the metal-binding site in BpUreE and KaUreE are very similar, with an RMSD of considering the metal ions and metal-bound His 0.57 A only. The position of the metal-bound His backbone is stabilized in both cases by an intra-chain H-bond between Gly97 O and His NH (Fig. 2(b)). In BpUreE an inter-chain H-bond, which also contributes to the dimer
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stability, is formed between His100 Nd1 and Asn98 Od, further stabilizing the His100 Ne-Zn2þ bond through a fully conserved Ôelec-His-ZnÕ motif. In KaUreE this Hbond is missing due to the different conformation of the Asn residue side chain, which experiences an O/NH2 amide group swap as compared to BpUreE. Given the peripheral position of the metal ion on the protein surface, it would be interesting to have information on the number and position of water molecules bound in the coordination sphere around Ni and Cu ions, in order to correlate the structural information to the available spectroscopic data. For different reasons, this cannot be done for either BpUreE or KaUreE. The chemical surrounding of the metal ion in BpUreE is perturbed by the presence of another dimer bound to the same Zn2þ ion through the same residues, causing the formation of two tetrameric forms pivoted around the metal ion and not involving additional protein surface patches [33]. The coordination environment of the Zn2þ ion in BpUreE differs in the two forms structurally characterized, being either tetrahedral (four His100 Ne, one from each monomer) or octahedral (two additional axial water molecules), depending on the stacking angle between the two dimers. Such tetrameric state of BpUreE was previously identified by size-exclusion chromatography and paramagnetic NMR only in concentrated protein samples containing Zn2þ or Ni2þ ions and is not believed to be functionally relevant [49]. No tetrameric state has ever been reported for KaUreE. The Cu2þ ion in KaUreE, located in the same position as Ni2þ /Zn2þ in BpUreE, is bound only to the two conserved His residues, and no solvent molecules are found close to the metal ion. 2 This is probably a consequence of the way the metal-bound form was obtained, that is by simply soaking a crystal of the apo-KaUreE in a copper-containing solution. In both BpUreE and KaUreE structures, no electron density for the last few residues at the C-termini is observed, presumably because of disorder caused, in the case of BpUreE, by the formation of the dimer of dimers, while in the case of KaUreE this disorder could be due to the non-natural form of the protein, featuring a truncation of the Hisrich C-term, in addition, again, to the fact that the starting structure prior to the copper soaking of the crystal is that of the apo-form. In the crystal structure of KaUreE a second metal binding site, constituted by His110 and His112 (KaUreE consensus sequence), is found on each monomer,
2
The apparent presence of three water molecules around the copper ion in the conserved site of KaUreE, as resulting from Fig. 4(a) of [34], is an artifact, as it results from the analysis of the related PDB files. The figure was made by superimposing the structures of the dimers from both the apo-form (PDB code 1GMU) and the copper-soaked crystal (1GMW). In the latter case, the water molecule is located at 3.9 from the copper ion. A
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resulting in the presence of two additional Cu2þ ions. These two residues were previously indicated as belonging to two different binding sites on KaUreE [34]. In BpUreE this second binding site is absent, due to the substitution of His110 and His112 by Tyr and Lys, respectively, suggesting that this additional binding site is not functionally relevant. Overall, this structural comparison clarified the relevant similarities and differences between the two UreE proteins, and further indicated features of the fold that could be important for the functional role in Ni trafficking. The exploration of sequence and structural conservation in UreE proteins from different biological sources allows the building of a structural database using homology modeling procedures, as described in the following section. 3.3. UreE multiple sequence alignment construction and analysis A UreE sequence search resulted in 36 hits (Table 1). Four sequences were detected in the initial multiple se-
quence alignment, which presented very large differences, such as long insertions in the middle of otherwise highly conserved secondary structure regions, and they were excluded from further analysis. These sequences are listed in the bottom part of Table 1. The alignment of the remaining 32 UreE proteins was optimized using information derived from secondary structure predictions. This optimized alignment was used as input to obtain a first set of model structures, which were then analyzed to identify local fold problems using PROSA. Whenever possible, such structural misfoldings were corrected by modifying the alignment in the interested regions and building new models. This procedure was repeated for each model until all possible local fold problems were solved. This iterative optimization procedure, which involved (i) model building, (ii) model quality assessment, and (iii) correction of the multiple alignment, finally yielded an aligned database of acceptable model structures. In the final multiple alignment, reported in Fig. 3, the sequences were found to feature an identity with respect to the sequence of BpUreE ranging between 55.10% and 20.38%.
Fig. 3. Optimized multiple sequence alignment of UreE from biological sources indicated in Table 1. The order of the alignment follows the identity percentage with respect to BpUreE. The predicted secondary structure elements are highlighted in yellow (helixes) and turquoise (sheets). The conserved residues are highlighted in green (100% conservancy) or magenta (conservative substitution).
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From such alignment it is possible to extract a whole set of considerations. The metal binding site motif GNXH (where X is R in 26 sequences or K, T or L in 6 sequences) is fully conserved. The four amino acids (Met86, Met89, Gly90, and Ala93) (BpUreE consensus sequence) found at the interface of the dimer are generally mutated with hydrophobic residues, indicating a conservancy of the nature of the interaction holding together the two monomers in the functional dimer. Two amino acids are conservatively mutated: Leu55, found in the random coil region of the N-terminal domain and constituting the center of the hydrophobic core of such domain, and Asp115, found in the C-terminal domain at the beginning of the a-helix not involved in the dimer formation. The final structure-based multiple sequence alignment of the UreE proteins allows the calculation of the conservancy of every single residue. As reported in Fig. 4(a), only few positions show conservation higher than 80% (i.e. 2r higher than average).
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These positions involve, in the N-terminal domain, (i) Val26 (BpUreE consensus sequence), more generally found as a Leu and located on the lateral surface of the protein, (ii) Gly42, that stabilizes a loop with an H-bond between its NH and Thr39 O, (iii) Leu49, Leu55, Gly58, and Leu61, composing a large part of the hydrophobic core of the N-terminal domain, and (iv) Asp59, whose Od is H-bonded to Arg56 NH, stabilizing a loop conformation. In the C-terminal domain the conserved residues are (i) Gly97, Asn98, and His100, composing the metal binding site, and (ii) Arg99 and Asp115, positioned on the surface of the protein. The importance of Asp115 in the functional role of UreE has been previously highlighted, but its detailed role not yet unveiled [31]. The residues constituting the second metal binding site in the crystal structure of KaUreE (His110 and His112, KaUreE consensus sequence) show a relatively low conservation (37.50% and 31.25%, respectively). This result confirms that this second metal binding site is
Fig. 4. (a) Residue conservancy percentage as a function of the residue number of the ‘‘ghost-UreE’’ sequence. The dashed line delimitates high conserved residues. (b) ‘‘Ghost-UreE’’ sequence compared with BpUreE and KaUreE sequences. Boxed residues present a conservation ratio higher than 80%.
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a characteristic feature of KaUreE and few other UreE proteins, but cannot be considered as a general character of the functional role for all UreE. On the basis of such conservation analysis, a prototypical UreE sequence was built using the most conserved amino acid in each position of the multiple alignment presented in Fig. 3. Such sequence was named ‘‘ghost-UreE’’ and is reported in Fig. 4(b) as compared with the sequences of BpUreE and KaUreE. In the same figure, the positions of the highly conserved residues discussed in the previous paragraphs are highlighted. 3.4. UreE model structures building and analysis The unrooted phylogenetic tree, generated on the basis of the final multiple sequence alignment, is reported in Fig. 5. This diagram can be divided in two sections, where the sequence indicated as 30 (from Deinococcus radiodurans) should be taken as an ideal border. The sequences of BpUreE and KaUreE are found in the two different regions of the phylogenetic tree. This is an ideal situation by which the modeling procedure relies on template structures representing essentially all the sequences considered in this work. Models were generated for all the aligned 32 sequences. Fig. 6(a) shows the superimposition of the backbones of all the model structures obtained. The protein tertiary structure is very well conserved in both domains of the models. The different orientation between the N- and C-terminal domains observed in BpUreE and KaUreE crystal structures is reflected in a
range of different domain relative orientations in the models, resulting in the bundle observed at the edges of the superimposed molecules (Fig. 6(a)). The secondary structure elements found in the experimental structures are also found in the models. In particular, it is important to notice that the a-helix forming the dimer interface is always observed. In most of the cases, such helix is formed by 14 amino acids and, in the first six residues, it features the same hydrophobic patch observed in the crystal structures. The metal binding His residues are always found at the edge of this helix, and the H-bond between Gly97 O and His100 NH (BpUreE consensus sequence) is conserved in all model structures. The structures used as templates feature a metal ion bound in this position, and therefore all models are relative to the metal-bound UreE. Eventual conformational changes occurring upon metal loss cannot be explored using this methodology. The backbone RMSD of all models with respect to the BpUreE and KaUreE crystal structures ranges from and from 0.82 to 1.82 A, respectively 0.63 to 2.17 A 1 (Table 1, Supplementary material). The backbone RMSD of model structures 1 and 24 (corresponding to native BpUreE and KaUreE sequences, respectively) calculated with respect to the relative crystal structures (which represent mutated forms, as indicated above) is respectively. The superimposition of 0.63 and 0.82 A, such structures is reported in Fig. 7, showing the good quality of the models, in particular at the C-terminal domains and at the dimer interface. However, the Nterminal domains are well reproduced in the fold but not exactly in the relative position with respect to the C-
Fig. 5. Unrooted phylogenetic tree of the UreE sequences that constituted the dataset analyzed in this work. Sequence numbers as in Table 1 for UreE whose structure has been experimentally determined the label is reported in bolded type.
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Fig. 6. (a) Backbone superimposition of modeled structures of UreE. The proteins are shown with the metal binding site toward the viewer. (b) GRASP solid surface representation of the electrostatic potential of the ghost UreE model structure. The protein is shown with the metal binding site toward the viewer. The surface is colored according to the calculated electrostatic potential contoured from )6.0 kT =e (intense red) to +6.0 (where k, Boltzmann constant; T, absolute temperature; e, electron charge) (intense blue). (c) GRASP solid surface colored according to residues conservation ratio from 20.0% (red) to 100.0% (green).
terminal domain. As observed for the crystal structures, the N-terminal domain presents a relatively high degree of conformational freedom that is reflected also in the models with propagation on the RMSD values. The percentage of the monomer surface involved in the dimer formation is rather constant among all models, oscillating around 11%. The distance between the Ca atoms of the metal-bound His residues ranges from 9.23 Such values are comparable with the corto 10.44 A. responding experimental data, giving a first indication about the reliability of the structures obtained from the modeling procedure, and indicate that all modeled structures can easily sustain metal binding at the same location of the protein surface as found in the crystal structures. Interestingly, the quality of the model structures from BpUreE and KaUreE, as judged by
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the distribution among the so-called allowed and notallowed regions in the Ramachandran plot, is slightly higher than the corresponding experimental structures (Table 2, Supplementary material). 1 This is probably a consequence of the fact that the Ramachandran plot may not consider as feasible all possible protein conformations that are, however, experimentally observed. All models present a good percentage of residues in the core region of the Ramachandran plot (at least 87%), while the few residues found in the disallowed region are localized in loops where the modeling process is knowingly more difficult. BpUreE and KaUreE models yield structural parameters and Z-scores that are not worse than for the crystallographic data (Table 2, Supplementary material). 1 The energy profiles calculated with PROSA II of the two pairs of model and experimental structures are almost identical (Fig. 7, bottom panels). In the BpUreE model structure the energy profile presents a region where the energy is considerably lower than for the crystal structure. This region, found between residues 13 and 17, is a small portion of the BpUreE sequence that featured the LSHQI sequence instead of YESSD, presumably as a result of a cloning artifact [33]. The model structure, built using the wildtype sequence, presents a better local fold in that region, probably a consequence of inter-tetramer interactions in the mutated BpUreE in the solid state involving the His residue of the LSHQI fragment with Met1 of another tetramer. The Z-scores obtained for all UreE model structures range from )4.32 to )8.89, with an average value of )6.65. Problems in protein packing are evidenced for model structures from sequences 14, 17, 31 and 32, which present a relatively high Z-score (higher than )5.70, i.e. the average Z-score plus one standard deviation). In the phylogenetic tree the sequences corresponding to these model structures are clustered close to the KaUreE sequence.
3.5. Protein surface electrostatic potential and residue conservation In order to gain information on the conserved protein surface features of UreE from different species, solid surface representations of the electrostatic potential were calculated for each model structure (Fig. 1, Supplementary material). In Fig. 6(b) the surface for the modeled ghost UreE sequence is reported, colored according to the electrostatic potential, while the surface of the same model colored according to residue conservation is shown in Fig. 6(c). This figure essentially contains all the information that can be generally distilled from the constructed model structure database. In particular, it is possible to observe that almost all the highly conserved residues found in the N-terminal domain are located in the protein core. The opposite is true
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Fig. 7. Superimposition of the crystal and model structures of BpUreE (a) and KaUreE (b). The proteins are shown with the metal binding site toward the viewer (top panels) and rotated by 90° about the long horizontal axis (central panels). Color scheme: crystal structures, blue; model structures, red. The PROSA II energy graphs of the crystallographic structure (blue line) and model (red line) structures from BpUreE and KaUreE are shown as bottom panels. Energies are represented in units of E=kT (E, energy; k, Boltzmann constant; T, absolute temperature).
for the C-terminal, where a large conserved patch is found on the surface of the protein in the close vicinity of the metal binding site (zone A in Fig. 6(c)). This region is characterized by the presence of a hydrophobic area, centered around the metal-binding neutral His residues. Another conserved region (zone B) corresponds to Asp115 (BpUreE consensus sequence), already indicated as somehow important for Ni2þ transport and release, and indeed the electrostatic potential in this region is negative. A third region (zone C) is characterized by a hydrophobic patch containing the highly conserved Gly42. A fourth noticeable region features a relatively high conservancy (zone D) and a positive charge, consistently with the presence of Arg29, Lys31, Arg32, and Arg33. The arrangement of these peculiar protein surface patches highlights their probable role in protein–protein complex formation concomitantly with the physiological metal ion transfer during the urease active site assembly.
process. In particular, a detailed comparison of the metal binding mode has clarified that the available structures do not clarify all the necessary details of the mechanism of nickel uptake and release, prompting for more investigations. The identification of highly conserved residues in the C-terminal domain and in the metal binding region of the protein suggest a possible recognizable surface and docking mode for the UreE binding to the UreDFG-apourease complex, a crucial aspect for the nickel incorporation in the urease activation mechanism. The calculated model structures are made available to all researchers interested in the structural biology of the urease metallochaperones. In particular, they can be used to fit models into low-resolution electron density maps and to perform molecular replacement in X-ray crystallography, to refine solution NMR structures, to support site-directed mutagenesis, and finally to perform docking experiments when the structures of the other urease chaperones will be revealed, a task that our laboratory is actively pursuing.
4. Conclusions
5. Abbreviations
The comparative analysis of the available crystal structures of UreE proteins, together with the structurebased multiple alignment and structural modeling of all available UreE sequences has provided novel information on the role of these protein in the urease activation
Bp BpUreE Ka KaUreE RMSD
Bacillus pasteurii Bacillus pasteurii UreE Klebisiella aerogenes Klebsiella aerogenes UreE root mean square deviation
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