Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center
© Saikatendu et al; licensee BioMed Central Ltd. 2006
Received: 28 September 2006
Accepted: 23 December 2006
Published: 23 December 2006
The protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function.
The structure of SA1388 has been solved to 2.0Å resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site.
SA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric "lids" that cap the central cavity of the toroid on either side and provide only small openings to allow regulated entry of small molecules into the occluded chamber. The presence of the electron density of the bound ligand may provide important clues on the likely function of NIF3-like proteins.
Despite the increased sophistication of annotation tools, a significant number of protein sequences emerging from genome sequencing efforts continue to remain in the realm of "hypothetical proteins" with little or no functional annotation associated with them. Ultimately, a definite functional annotation would require experimental characterization, which is often time consuming and expensive. Careful remote homology detection and manual analysis has in many occasions helped to glean useful structural and functional insights into these so-called "hypothetical proteins". Typically, such studies involve a combination of profile based methods like transitive PSI-BLAST , COMPASS  and HMMer  as well as structure prediction and fold recognition methods [4–6].
One such study  had analyzed sequences of the ubiquitously found protein modules homologous to the nitrogen regulatory PII proteins as defined in the COG  and Pfam databases . This comprehensive analysis expanded the PII protein superfamily to include five very divergent groups of proteins, with below random (~1%) pairwise sequence identities between some members of distant groups. Nevertheless, each group has distinct patches of conserved local similarities and was predicted to retain the same overall structural fold as PII and a trimeric structure essential for ligand-binding site formation. The PII-like proteins are small protein modules of ferredoxin-like fold containing a core (βαβ)2 secondary structural pattern, and function as trimers. While the nitrogen regulatory PII proteins that belong to the Group II of the superfamily have been studied extensively , the functions of other groups in the superfamily are either poorly understood or completely unknown. One group of PII-like proteins, Group III, is significantly larger (~370 aa) than a typical PII protein domain (~112 aa). In these proteins, the PII domain is embedded within the central region of the polypeptide while the N- and C-terminal regions together belong to the NIF3 (NGG1p interacting factor 3)-like protein family. Presumably, the PII domain of these proteins would play some sort of ligand binding and signalling role analogous to that of classical PII proteins [10, 11], while the function of the NIF3-like domains is not known.
We chose the Group III proteins of the PII superfamily (represented by the Staphylococcus aureus protein SA1388) for structure determination with two primary objectives: to structurally characterize both the Group III PII-like domain and NIF3 domains which may provide clues to their potential function that is otherwise unattainable from sequence information alone. Apart from providing confirmation of our prediction of the central P-II domain, the structure of SA1388 would also be important for functional analysis of the NIF3 domains, which were recently highlighted in the top 10 list of important structural targets due to their broad phylogenetic distribution, sequence conservation patterns with putative "active-site like" features and their uncharacterized role as a putative regulatory molecules of eukaryotic transcription .
Description of SA1388 monomer
Overall, the protein is organized into three distinct structural domains with interconnecting topological connectivities (Figure 1B). The two NIF3 domains at the N- and C-terminus of the protein (henceforth denoted as NIF3-N and NIF3-C, respectively) have the same overall fold as canonical NIF3-like proteins [13, 14], with a central 5 stranded mixed β-sheet and a pair of helices on either side. NIF3-N and NIF3-C are structurally similar and are clearly superimposable as shown in Figure 1C, and therefore are probably a result of gene duplication.
The middle region of the polypeptide (residues 126–236) bulges out between the two NIF3 domains and is structured as a classical PII-like fold with a (β/α/β)2 architecture (Figure 1A), confirming our earlier prediction . This domain is relatively well ordered only in one of the two monomers in the asymmetric unit. In this monomer, although all the secondary structural elements are well defined, we do not observe electron density for residues 169–193 that connects the strands β7 and β8. In the other monomer, in addition to this loop, helix α6 and strand β7 are also disordered. Residues in this domain have substantially higher B factors, indicating a higher mobility compared to the rest of the protein.
Structure comparison with other NIF3 and PII proteins
The PII-like domain of SA1388 is topologically identical to several PII proteins with characterized structures (Figure 2B), such as GlnB  and GlnK , as well as PII-like protein CutA  and the C-terminal regulatory domain of ATP phosphoribosyltransferase (HisG) . This domain has a core (βαβ)2 secondary structural pattern described in SCOP as a ferredoxin-like fold . It remains to be determined whether the SA1388 PII domain binds ATP or other ligands either in vivo or in vitro.
The NIF3 domains are involved in dimerization
The PII-like domains are involved in trimerization
The PII-like domain juts out of the dimer on the side opposite the dimer interface and forms homotrimers by interacting with the PII-like domains of symmetry related molecules along the three-fold axes (Figure 3B). The trimeric arrangement, where the individual β-sheets of each PII domain pack orthogonally is similar to that seen in several PII and PII-like proteins such as glnB , glnK , CutA  and the C terminal regulatory domain of HisG among others. A superimposition of PII trimer of SA1388 and that of ATP bound complex of E. coli glnK is shown in Figure 3C.
SA1388 forms a capped toroidal hexamer
Description of the putative active site
Additionally, we observe a clear electron density for a ligand of unknown identity in close proximity to the two bound zinc atoms that would provide two additional metal ligands, one to each of the two metal ions to complete the octahedral metal center configuration (Figure 5B). By trial and error, we have ruled out all components in the crystallization solution that might have been inadvertently bound either during crystallization or during cryoprotection and therefore conclude that the observed ligand must in fact be an endogenously bound ligand that was co-purified after recombinant overexpression. The contours of the difference electron density map (Figure 5B) after final refinement suggests the presence of a head group directly ligated to the metal ions and a mostly aliphatic tail that has few specific polar interactions with the surrounding protein residues (Figure 5B). Two aromatic residues, a tyrosine (Y289, shown in Figure 5A) and a tryptophan (W22) lie in close proximity to the bound ligand and may be involved in ligand binding.
Although homologs of both PII and NIF3 proteins are found ubiquitously in all three kingdoms of life, the function of SA1388 and its homologs that contain both PII-like and NIF3-like domains remain completely unknown. The fusion of the two proteins in one peptide chain indicates a functional coupling of these two proteins. The structural features revealed in the present study, such as the cage-like hexameric toroid structure with its NIF3 domains as walls and the two PII-like domain trimers as lids, the dinuclear metal site, and the intrinsically bound ligand, may provide certain clues of its potential function. The primary role of the nitrogen regulatory PII proteins is to integrate various intracellular carbon and nitrogen signals by regulating enzymes involved in nitrogen assimilation . PII exerts its regulatory effects by undergoing different post translational modifications, such as uridylylation  and phosphorylation  by various modifying enzymes in response to the primary cellular nitrogen signal glutamine. Structures of several PII and PII-like proteins have been solved including glnB (from E. coli, T. thermophilus, Synecococcus and Herbaspirillum), GlnA (E. coli), glnK (E. coli), HisG (E. coli and M. tuberculosis), CutA1 (human, rat and T. maritima), and a hypothetical protein from COG1993. Effectors of PII include transcription factors [10, 11], signalling proteins e.g., histidine kinases , and metabolic enzymes like glutamine synthase . Apart from its primary signalling modulator glutamine, PII proteins are known to bind a range of small molecule effectors such as ATP, UMP, and 2-ketoglutarate, which affects its function antagonistically to glutamine (reviewed in ). It is highly likely that the trimeric PII domain of SA1388 also plays a ligand induced signalling role and probably regulates the function of the NIF3-like domains.
Unlike PII domains, the NIF3-like domains have only recently begun to be structurally characterized, and their structure-function relationship remains sketchy. NIF3-like proteins are ubiquitously conserved from bacteria to higher eukaryotes . They have been defined in uniprot database as the uncharacterized protein family UPF0135 that has 64 homologs . Several homologs of this family (e.g., human and mouse) have a similar overall three-domain organization as in SA1388, while others (e.g., E. coli and Methanococcus) have only the NIF3 domains with the PII-like domain being absent. Experiments on spermatogonia derived cell line GC-1 suggest that nif3L1 (Ngg1-interacting factor 3-like 1) gene, a homolog of sa1388, expresses copiously during embryonic development and participates in retinoic acid-primed neural differentiation by interacting with the transcriptional corepressor Trip15/CSN2 . Its primary role appears to be to inhibit Ngg1p from translocation to the nucleus, presumably by forming a binary complex in the cytoplasm . Yeast two hybrid studies have shown that NIF3 binds to the amino terminal region of NGG1, an interaction that was implicated as a means of limiting transcriptional activation of GAL4p in glucose rich medium . Furthermore, a genomewide two-hybrid analysis of yeast protein-protein interactions suggests that yeast NIF3p interacts with a nuclear import/export protein (Srp1p) and a ras-like GTPase (Temp1p), which are both required for proper exit from mitosis in the cell cycle . In E. coli, expression of NIF3 homolog ybgI increases dramatically upon genotoxic stress induced by DNA damage . This observation might be significant in light of the structure reported here because proteins that have been implicated in processes involving DNA metabolism often adopt toroidal structures [33–35]. However, the active site observed for SA1388 in this study, along with that seen in ybgI are not similar to the previously characterized DNA interacting toroids (e.g., the λ exonuclease ).
Although the precise biochemical function of the NIF3-like proteins are not known, the presence of a binuclear metal center similar to those found in hemerythrins, ribonucleotide reductases and purple acid phosphatase, as well as the endogenously bound ligand in the current structure strongly suggest that SA1388 NIF3 domains and E. coli Ybgl proteins likely bind and perform some form of catalysis on small molecule ligands, possibly metabolic substrates, and whose function may be regulated by the PII-like domains. In an attempt to determine the chemical identity of the endogenous ligand, we have extracted the ligand from the purified protein by trichloroacetic acid (TCA)/acetone precipitation. The preliminary high-resolution mass spectroscopic analysis of the TCA/acetone extract suggested that the ligand might be a compound of novel chemical composition. Crystallization of the compound has not been successful probably due to its intrinsic structural flexibility, which may not be susceptible for crystallization. Alternative approaches, such as NMR spectroscopy, may be needed in order to determine the chemical formulas and three-dimensional structure of the endogenous ligand.
The recent upsurge in structures of a large number of hypothetical proteins by structural genomics efforts continue to provide important leads in the eventual determination of their function. This success has been largely due to either similarity of the overall structure, or local relatedness of catalytic residues to proteins of known function. Proteins with novel, previously unknown folds however are often limited in providing functional clues because of lack of similarity to any experimentally characterized proteins. Sometimes, the fortuitous observation of endogenously bound ligands offers important clues in this direction. The dinuclear metal center and the presence of a bound ligand at the active site of the NIF3 domain of SA1388 combined with the structural analysis of the two NIF3 homologs, ybgI and SA1609 should aid in the functional assignment of this widespread protein family.
Cloning, purification and crystallization
The gene coding for SA1388 was PCR amplified from genomic DNA of Staphylococcus aureus (ATCC, 700699D, Manassas, VA) and was cloned into a pProEX (Invitrogen) prokaryotic expression vector containing a trc promoter, 6xHis tag, and a tobacco etch virus (TEV) protease cleavage site. The resulting plasmid was transformed into the E. coli strain C41(DE3) (Imaxio, Clermont-Ferrand, France) for protein expression. Expression was induced at OD600 of 0.4 and the cells were pelleted after overnight growth at 20°C. Protein was first purified with a nickel-nitrilotriacetic acid (NTA)-agarose column (Qiagen) followed by cleavage of the N-terminal His tag by TEV protease. The protein was further purified by anion exchange chromatography on a MonoQ column (Amersham Biosciences) using a linear gradient of salt (Equilibration buffer: 50 mM Tris pH 7.8, 1 mM DTT; Elution buffer 50 mM Tris pH 7.8, 1 M NaCl, 1 mM DTT) and finally by gel-filtration chromatography on a Superdex 200 column (Amersham Biosciences) that was pre-equilibrated with a buffer containing 50 mM Tris pH 7.8, 300 mM NaCl, 1 mM DTT. The selenomethionine-substituted SA1388 protein was expressed in the minimum medium supplemented with selenomethionine and other nutrients according to standard protocols  and purified using the same procedure described above. Pure fractions of native and SeMet proteins were verified by SDS-PAGE and concentrated to 20 mg/ml for crystallization in a buffer containing 50 mM HEPES pH 7.2, 2 mM DTT and 250 mM NaCl. Crystals of SA1388 were grown by hanging drop vapour diffusion method. Single crystals of sizes around 0.3 × 0.3 × 0.2 mm3 appeared within 3 days in the condition with 0.1 M Bis-Tris propane, pH 8.5, 0.2 M MgCl2 and 30% PEG 400 as the well solution. Crystals were directly frozen in liquid propane and stored in liquid nitrogen until data collection. All crystallographic data was processed using HKL2000 
Structure determination and refinement
Data collection and refinement statistics
0.9792 (peak λ)
a = b = 133.17 Å
c = 125.80 Å
a = b = 132.54Å
c = 125.67Å
Resolution range (Å)
Total number of observations
R merge 1
Resolution range (Å)
R cryst 2
R free 3
Bond length rmsd from ideal
Bond angle rmsd from ideal
Average isotropic B-value
ESU based on free R
Metal ion determination
X-ray fluorescence scans were performed at the Advanced Photon Source (APS) beam line 19-BM at Argonne National Laboratory on a native SA1388 crystal. Metal analysis was also conducted on a Perkin Elmer Optima 3300 DV Inductively Coupled Plasma (ICP) atomic emission spectrometer calibrated with AccuStandard ICP Multi-Element solutions. The protein was first dialyzed against a buffer containing 50 mM Hepes, pH7.5 and 1 mM DTT, and then diluted to appropriate concentrations for ICP atomic emission spectra analysis. The dialysis buffer was used as blank.
This work is supported by NIH grants GM65243 (to H.Z.) and in part by a Welch Foundation grant I-1505 (to N.V.G). We thank Mischa Machius for help with synchrotron data collection. The authors gratefully acknowledge the user support staff of the Structural Biology Center beamlines (SBC-CAT, 19-ID and 19BM). Use of the Argonne National Laboratory Structural Biology Center beamlines at the Advanced Photon Source, was supported by the U. S. Department of Energy, Office of Energy Research, under Contract No. W-31-109-ENG-38.
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