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J. Biol. Chem., Vol. 281, Issue 16, 11366-11373, April 21, 2006
The Structure of CodY, a GTP- and Isoleucine-responsive Regulator of Stationary Phase and Virulence in Gram-positive Bacteria*![]() ![]() 1![]() 2
From the
Received for publication, December 6, 2005 , and in revised form, January 20, 2006.
CodY is a global regulator of transcription in Gram-positive bacteria. It represses during growth genes required for adaptation to nutrient limitation, including virulence genes in some human pathogens. CodY activity is regulated by GTP and branched chain amino acids, metabolites whose intracellular concentrations drop as cells enter stationary phase. Although CodY has a highly conserved sequence, it has no significant similarity to proteins of known structure. Here we report crystal structures of two fragments of CodY from Bacillus subtilis that clearly constitute its cofactor and DNA binding domains and reveal that CodY is a chimera of previously observed folding units. The N-terminal cofactor-binding fragment adopts a fold reminiscent of the GAF domains found in cyclic nucleotide phosphodiesterases and adenylate cyclases. It is a dimer stabilized by an intermolecular six -helical bundle that buries an extensive apolar surface rich in residues invariant in CodY orthologues. The branched chain amino acid ligands reside in hydrophobic pockets of each monomer distal to the dimer-forming surface. The structure of the C-terminal DNA binding domain belongs to the winged helix-turn-helix family. The implications of the structure for DNA binding by CodY and its control by cofactor binding are discussed.
The chromosomes of bacteria are packed with genes that enable the organism to exploit diverse ecological niches and to survive adversity. The key to adaptation and survival is to interpret indicators of a changing environment and respond by modulating the repertoire of genes being expressed. A fundamental challenge to survival is posed by the depletion of nutrients and the onset of starvation. Bacillus subtilis has at its disposal an array of adaptations to poor growth conditions, including secretion of macromolecule-degrading enzymes to scavenge what nutrients are available, synthesis and secretion of antibiotics that allow more effective competition for these nutrients, motility to seek new sources of nutrients, development of competence to take up exogenous DNA (which may confer a genetic advantage), or, in extreme circumstances, abandoning growth altogether and forming a dormant spore. CodY was first discovered as a repressor of the dipeptide permease operon in B. subtilis (1). Subsequently it has been recognized as having a much wider role in controlling the expression of stationary phase genes. It is now known to regulate over 100 genes distributed across some 70 or so operons (2). The CodY regulon encodes extracellular degradative enzymes, transporter proteins, catabolic enzymes, factors involved in genetic competence, antibiotic synthesis pathways, chemotaxis proteins, and sporulation proteins. During rapid growth, CodY thus represses genes whose products allow adaptation to nutrient depletion. The repressor function of CodY is activated by two different effectors, GTP and isoleucine or valine, which may be viewed as sensors of the energetic and metabolic status of the cell, respectively (3-5). These co-repressors act independently and additively to increase the affinity of CodY for its target sites on DNA. CodY binds its ligands selectively but with moderate affinities (in the millimolar range), consistent with its need to monitor the concentrations of GTP and isoleucine/valine obtaining in rapidly growing cells. CodY is highly conserved in the low G + C Gram-positive bacteria. In Lactococcus lactis, CodY regulates the expression of genes encoding extracellular peptidases, peptide transport proteins, and intracellular enzymes involved in peptide and amino acid utilization (4). In pathogenic bacteria such as Streptococcus pyogenes, Listeria monocytogenes, Enterococcus faecalis, Bacillus anthracis, and Clostridium difficile, accumulating evidence points to a role for CodY in the regulation of virulence gene expression (6). CodY from B. subtilis is a dimer of 29-kDa subunits. Its 259-amino acid residue sequence is unrelated to that of any proteins other than CodY orthologues. A putative helix-turn-helix (HTH)3 motif has been identified close to the C terminus of the protein and specific residues in this motif are required for high affinity DNA binding (7, 8). The only other clue to structure provided by the sequence is a set of possible GTP binding motifs again located in the C-terminal half of the molecule (3). To investigate the structural basis of GTP- and branched chain amino acid (BCAA)-dependent regulation of stationary phase gene expression, we embarked on crystallographic studies of CodY. Although we have been able to crystallize the intact protein (9), the crystals were weakly diffracting and unsuitable for structure determination. We therefore sought to identify stable proteolytic fragments of CodY as alternative targets for structure determination. Here we report the crystal structures of N- and C-terminal fragments constituting residues 1-155 and 168-259, respectively.
Protein Preparation and CrystallizationCodY was purified as a C-terminal histidine-tagged protein (9) and partially digested with a series of proteases. Mass spectrometry of the digestion products led us to conclude that a region containing a cluster of 12 highly charged residues (156-167) was particularly sensitive to proteolysis. Using ligation-independent cloning methods, we prepared pET28a derivative constructs encoding CodY fragments spanning residues 1-155 and 168-259, each with an N-terminal His6 tag. The recombinant CodY fragments were overexpressed in Escherichia coli BL21 and purified by (i) Ni2+-chelation chromatography on a 5-ml high-performance chelating-Sepharose column (Amersham Biosciences) charged with nickel ions and (ii) gel filtration chromatography. For CodY-(1-155) a HiLoad Superdex 200 preparation grade column equilibrated in 50 mM Tris, pH7.5, 150 mM sodium chloride was used. For CodY-(168-259) a Superdex 75 preparation grade column equilibrated with 0.1 M sodium citrate, pH 5.6, 200 mM sodium chloride was used. SeMet derivatives were prepared by growth of E. coli B834 cells harboring the expression plasmids in ZYP-5052 autoinduction medium (10).
Crystals of CodY-(1-155) were grown in hanging drops containing a 1:1 volume ratio of 20 mg ml-1 protein in gel filtration buffer and 18% monomethyl ethyl polyethylene glycol 5000 in 0.1 M Tris-HCl, pH 7.0, 0.2 M calcium acetate, 10 mM Gpp(NH)p, and either 10 mM isoleucine (native protein) or 10 mM valine (SeMet protein). Crystals of CodY-(168-259) were grown similarly from 1:1 mixtures of 20 mg ml-1 protein in 100 mM sodium citrate, pH 5.6, 0.2 M NaCl and 32% saturated ammonium sulfate in 0.1 M bis-Tris, pH 6.5, 5% glycerol, and 5% Tacsimate (Hampton Research).
Partial Proteolysis of CodYCodY-His6 protein (84 µg, purified as described above) or bovine serum albumin, Fraction V (BSA; 84 µg; Serologicals Products, Inc.), were preincubated at 25 °C for 10 min with or without BCAAs (10 mM each of isoleucine, leucine, and valine). We then added trypsin (0.26µg, Sigma) or
Structure DeterminationNative data taken from crystals of CodY-(1-155) to 1.7-Å resolution were collected on beamline 10.1 (
The crystals of CodY-(168-259) were in space group P422. The crystals were weakly scattering and the x-ray diffraction data were limited to 2.8-3.0 Å spacing. The structure was solved using three wavelength MAD data and the model was built and refined as described above. The model contains a complete A chain and all but residues 234-235 and 257-259 in chains B and C. The temperature factors of chain C are significantly higher than for chains A and B. The final free R factor is 24.8%, Rwork is 20.0% and the geometry is satisfactory (Table 1).
Tertiary Structure of CodY-(1-155)CodY-(1-155) has a three-layered globular structure. At the base of the molecule, as shown in Fig. 1A, is a three-helix bundle. Two of the helices are at the N terminus of the chain, whereas the third is at the C terminus of the chain. Helices 2 and 5 pack against a central 5-stranded anti-parallel -pleated sheet, with a strand order 2- 1- 5- 4- 3 (Fig. 1, A and B). The top layer, which does not lend itself to such simple description, is formed by two extended loops that connect strands 2 and 3, and 3 and 4. The 2- 3 segment contains two -helices 3 and 4, whereas the 2- 3 loop lacks any recognizable secondary structure. These loops form the walls of a cavity whose base is formed by the -sheet itself.
Amino Acid BindingThe electron density maps clearly define the presence of an isoleucine ligand consistent with the inclusion of this amino acid in the crystallization drops (Fig. 1D). No evidence for the binding of the GTP analogue, Gpp(NH)p was seen, however. The isoleucine is bound above the The residues making up the isoleucine-binding pocket are strongly conserved in the Bacillus, Listeria, Enterococcus, Staphylococcus, Streptococcus, and Lactococcus spp., although surprisingly they are less well conserved in the Clostridium spp. (Fig. 2). Arg61, which binds the ligand carboxylate, is replaced by apolar side chains in CodY from C. difficile and Clostridium perfringens, whereas the apolar Met65, which contacts the apolar side chain of the ligand, is replaced by Glu or Lys in these species. This suggests differences in the mode of ligand binding.
Quaternary StructureAnalysis of the molecular packing suggests an obvious dimer consistent with observations made from gel filtration of CodY-(1-155) during purification, and with the knowledge that intact CodY is a dimer (9).4 In this dimer, the subunits are related by a 2-fold crystallographic symmetry axis. Intermolecular contacts are mediated principally through the first and the last
Highly conserved and/or invariant residues among CodY orthologues cluster at the dimer interface. There are two intermolecular salt bridges formed between the pairs of Arg8 and Glu144 residues, which engage in two-pronged interactions with each other. These interactions would appear to be crucial for CodY structure and/or function as both residues are invariant in the CodY orthologues (Fig. 2). There is an additional pair of hydrogen bonding interactions between Gln15 and Thr148, the former residue being strongly conserved whereas the latter is invariant.
In the dimer, the isoleucine ligands of the respective subunits are distal to the dimer interface and their C atoms are separated by 57 Å (Fig. 3). The C-terminal Arg155 residues that in the intact protein are connected to the DNA binding domains are close in space.
CodY Has a GAF DomainDespite the absence of any substantial sequence homology, a number of structures in the Protein Data Bank contain domains that can be superimposed onto CodY-(1-155) to yield positional root mean squared deviations of 2.5-4.0 Å for 80-120 matching C
The structure of the GAF domain was first defined in a study of the yeast protein YKG9 (Protein Data Bank entry code 1f5m (18)) and subsequently other GAF domain structures in complex with ligands have been determined (Protein Data Bank entry codes 1mc0 and 1ykd (20, 21)). The superposition of CodY and YKG9 shows clearly that CodY belongs to the GAF domain family (Fig. 4, A and B). The
Structure of the C-terminal DomainCodY-(168-259) has a compact globular structure with an anti-parallel
Structural comparisons reveal that the DNA binding domain of CodY belongs to the winged HTH family of nucleic acid-binding proteins (17, 22). These domains consist of three
BCAA-induced Conformational ChangeBecause crystals prepared in the absence of BCAAs did not diffract well, we sought an alternative way of testing whether interaction with BCAAs induces a conformational change in CodY. We observed that trypsin has a preferred cleavage site after Lys169. Cleavage at other lysine and arginine residues also occurs, albeit with much lower efficiency (Fig. 5A). In the presence of branched chain amino acids, the major cleavage site was unchanged but none of the minor sites was recognized by trypsin (Fig. 5B). Isolation of each of the bands in Fig. 5A allowed us to determine the N-terminal sequence of each fragment and thereby deduce the sites of cleavage. Based on this analysis, we determined that BCAAs had no effect on cleavage after Lys169, but almost completely inhibited cleavage at Lys64, Arg69, Arg130, and Arg156. As a control, we showed that the cleavage pattern of CodY was not affected by threonine or methionine (data not shown) and that cleavage of BSA was the same in the presence or absence of BCAAs (Fig. 5, C and D). Incubation of CodY with -chymotrypsin yielded at least seven fragments (Fig. 5E). Of these, only cleavages after Tyr51, after Phe80, and near Tyr181 occurred in the presence of BCAAs (Fig. 5F). Cleavage at other sites, such as Tyr95, Tyr145, and Met173 was completely inhibited by BCAAs. Again, cleavage of BSA was unaffected by BCAAs (Fig. 5, G and H). It is interesting to note that the presumed conformational change in CodY that accompanies binding of BCAAs can affect residues far removed in space from the co-repressor binding site.
DNA binding and transcriptional repression by CodY are activated by the binding of the effector molecule, be it GTP or a BCAA (3, 5). The proteolytic fragmentation of CodY and the resulting structural studies presented here lead to the conclusion that CodY is a modular protein made up of an N-terminal cofactor binding domain and a C-terminal DNA binding domain, belonging to the GAF and winged HTH domain families, respectively. These domains have been characterized in other systems, but their combination within a single polypeptide chain has not been reported before. The exact mechanism by which binding of co-repressors increases the affinity of CodY for its target sites remains mysterious (see below), but it seems clear that interaction with BCAAs alters the conformation of CodY enough to change the susceptibility of certain residues to proteolytic enzymes (Fig. 5). These residues include some that are outside the N-terminal domain, suggesting that binding of BCAAs induces a conformational change that is translated to the C-terminal, DNA-binding domain. Implications for GTP BindingThe GAF domain complexes described here define the BCAA binding site in CodY leaving open the question of how GTP is bound. In the absence of a structure of a GTP complex, we have examined other GAF domain proteins and CodY sequence alignments for other clues to nucleotide binding. Two of the known GAF domain protein structures contain nucleotide ligands, cyclic guanosine monophosphate (cGMP) in mouse phosphodiesterase 2A (PDE-2A) and a pair of cyclic adenosine monophosphate (cAMP) ligands in the tandem GAF domains of a cyanobacterial adenylyl cyclase (20, 21). The topologies of the ligand-binding sites are very similar and the ligands and the surrounding protein backbones are closely superimposable. Compared with isoleucine in CodY, the cGMP in PDE-A2 is more deeply embedded in the GAF domain (Fig. 4C). Following superposition, the guanine base lies close to a pair of buried water molecules that occupy the largest cavity in the CodY structure (Fig. 4D). These are flanked by the side chains of Phe40 and Phe98 waters, which are therefore well placed to make ring-stacking interactions with the guanine base of a putative GTP ligand. Phe98 is invariant, whereas Phe40 is conserved in all of the CodYs except those from L. lactis and the Streptococcus spp. Interestingly the CodYs from L. lactis and Streptococcus pneumoniae (and by inference respond the other Streptococcus spp.) do not to GTP (23, 24)5. The side chains of residues Gln38, Gln55, and Ser129 provide hydrogen-bonding opportunities for the buried waters and potentially for the polar groups of a guanine base. Ser129 is conserved as a small residue (Ser, Gly, or Ala) in the CodY orthologues in Fig. 2, with the exception of those from L. lactis and the Streptococcus spp., which may again relate to their failure to respond to GTP. It is tempting to suggest that the Trp replacements in these CodY proteins provide an indole group to fill the volume that would otherwise be available to the guanine base of GTP.
The cavity containing these waters is contiguous with the isoleucinebinding pocket although it tapers considerably before opening out into the latter. However, given that ligand binding is probably accompanied by closure of the
Implications for Cofactor Control in CodYIn other transcriptional regulatory systems, ligand binding promotes relative domain motions and/or quaternary structural changes. In CodY, the isoleucine-binding site is not linked to any obvious hinge that could mediate long-range conformational change. As the isoleucines are bound in pockets on the faces of the The organization of the CodY-(1-155) subunits in the dimer places the C termini in close apposition, allowing for the possibility that the emerging DNA binding domains reside side by side. However, 12 linker residues between 155 and 168, a high proportion of which have ionizable side chains (Fig. 2), are not present in either of the structures and an appreciation of the juxtaposition of the domains in intact CodY will have to await the solution of the structure of the intact protein.
A system with possible structural and mechanistic analogy to CodY is TraR. TraR in Agrobacterium tumefaciens is a quorum sensing protein that responds to a homoserine lactone pheromone. The structure of TraR has been solved in complex with its cofactor and with a tra box target DNA (Fig. 6A) (25, 26). The cofactor binding domain has a GAF domain fold. Like CodY, TraR is a dimer with the monomermonomer interface constituted by the helical regions of the subunits that are distal to the effector binding sites. The DNA binding domains emerge from corresponding locations, but instead of the winged HTH domain observed in CodY, the DNA binding domain in TraR belongs to the LuxR family and has a GerE-type fold (27). The homoserine lactone ligand is believed to stabilize TraR against intracellular proteolysis (28). Consistent with this idea, it is even more deeply recessed in the hydrophobic cavity of the GAF domain (25, 26) than the isoleucine in CodY (Fig. 4C). This suggests that CodY too may be susceptible to proteolysis as BCAA/GTP levels drop when the cells enter the stationary phase. Although BCAA binding alters the susceptibility to in vitro proteolysis (Fig. 5), in vivo measurements indicate that CodY protein is present at the same concentrations throughout growth and stationary phase (3). Implications for DNA BindingA winged HTH domain with similarity to that observed in CodY occurs in the fatty acid-responsive transcription factor FadR. The structure of FadR in its complex with FadR operator DNA is shown in Fig. 6B (29, 30). The winged HTH DNA binding domain is connected to a C-terminal seven-helix bundle domain containing a hydrophobic pocket for acyl-CoA binding. FadR binds to DNA as a dimer using its recognition helix to make contacts with the major groove of the DNA and one of its wings, W1, to penetrate the minor groove.
The winged HTH domains of FadR and CodY can be superposed on one another to give a positional root mean square deviation of 2.7 Å for the 55 matching C It is interesting to note that there is no tertiary structural similarity between CodY and the leucine-responsive regulatory protein, which performs in Gram-negative bacteria many of the functions attributed to CodY in B. subtilis (31, 32). It would appear that amino acid and guanine nucleotide regulation of stationary phase gene expression has evolved independently in these two systems. Finally in the process of manuscript review, a referee drew our attention to a bioinformatics analysis where it is shown that the GAF plus winged HTH domain composition of CodY is predictable by bioinformatics analysis.
The atomic coordinates and structure factors (codes 2B18 (CodY-(1-155)) and 2BOL (CodY-(168-259))) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
* This work was supported by the Biotechnology and Biological Science Research Council, United Kingdom, Grant BBS/B/1213X, European Union SPINE Contract QLG2-CT-2002-00988, and United States Public Health Service Grant GM042219. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Present address: Laboratoire de Microbiologie et Pathologie Cellulaire Infectieuse, INSERM U431, Universite de Montpellier II, Place E. Bataillon, 34095 Montpellier, France. 2 To whom correspondence should be addressed. Tel.: 44-1904-328261; Fax: 44-1904-328366; E-mail: ajw{at}ysbl.york.ac.uk.
3 The abbreviations used are: HTH, helix-turn-helix; BCAA, branched chain amino acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; SeMet, selenium methionine; BSA, bovine serum albumin; PDE-2A, phosphodiesterase 2A; Gpp(NH)p, guanosine 5'-(
4 K. Matsuno and A. L. Sonenshein, unpublished data.
5 R. P. Shivers and A. L. Sonenshein, unpublished data.
We thank the Tufts Protein and Nucleic Acid Core Facility for assistance with peptide sequencing, and the staff at the Synchrotron Radiation Source at Daresbury, UK, and the European Synchrotron Radiation Facility in Grenoble, France, for help with data collection, and Dr. James A. Brannigan for helpful comments and discussion.
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