Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis.

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (cGMP-stimulated phosphodiesterases, adenylate cyclases, FhlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown. Characterization of human Lin28's interaction with RNA has been complicated by difficulties in producing stable RNA-free protein. We have engineered a maltose binding protein fusion with Lin28, which binds let-7 miRNA with a Kd of 54.1 ± 4.2 nM, in agreement with previous data on a murine homologue. We show that human Lin28A binds miR-363 with a 1:1 stoichiometry and with a similar, if not higher, affinity (Kd = 16.6 ± 1.9 nM). Further analysis suggests that the interaction of the N-terminal cold shock domain of Lin28A with RNA is salt-dependent, supporting a model in which the cold shock domain allows the protein to sample RNA substrates through transient electrostatic interactions.

Structure of components of an intercellular channel complex in sporulating Bacillus subtilis.

Following asymmetric cell division during spore formation in Bacillus subtilis, a forespore expressed membrane protein SpoIIQ, interacts across an intercellular space with a mother cell-expressed membrane protein, SpoIIIAH. Their interaction can serve as a molecular "ratchet" contributing to the migration of the mother cell membrane around that of the forespore in a phagocytosis-like process termed engulfment. Upon completion of engulfment, SpoIIQ and SpoIIIAH are integral components of a recently proposed intercellular channel allowing passage from the mother cell into the forespore of factors required for late gene expression in this compartment. Here we show that the extracellular domains of SpoIIQ and SpoIIIAH form a heterodimeric complex in solution. The crystal structure of this complex reveals that SpoIIQ has a LytM-like zinc-metalloprotease fold but with an incomplete zinc coordination sphere and no metal. SpoIIIAH has an α-helical subdomain and a protruding ß-sheet subdomain, which mediates interactions with SpoIIQ. SpoIIIAH has sequence and structural homology to EscJ, a type III secretion system protein that forms a 24-fold symmetric ring. Superposition of the structures of SpoIIIAH and EscJ reveals that the SpoIIIAH protomer overlaps with two adjacent protomers of EscJ, allowing us to generate a dodecameric SpoIIIAH ring by using structural homology. Following this superposition, the SpoIIQ chains also form a closed dodecameric ring abutting the SpoIIIAH ring, producing an assembly surrounding a 60 Å channel. The dimensions and organization of the proposed complex suggest it is a plausible model for the extracellular component of a gap junction-like intercellular channel.

12-Fold symmetry of the putative portal protein from the Thermus thermophilus bacteriophage G20C determined by X-ray analysis.

In tailed bacteriophages and several animal viruses, the portal protein forms the gateway through which viral DNA is translocated into the head structure during viral particle assembly. In the mature virion the portal protein exists as a dodecamer, while recombinant portal proteins from several phages, including SPP1 and CNPH82, have been shown to form 13-subunit assemblies. A putative portal protein from the thermostable bacteriophage G20C has been cloned, overexpressed and purified. Crystals of the protein diffracted to 2.1 Šresolution and belonged to space group P42(1)2, with unit-cell parameters a = b = 155.3, c = 115.4 Å. The unit-cell content and self-rotation function calculations indicate that the protein forms a circular 12-subunit assembly.

The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer.

The assembly of double-stranded DNA bacteriophages is dependent on a small terminase protein that normally plays two important roles. Firstly, the small terminase protein specifically recognizes viral DNA and recruits the large terminase protein, which makes the initial cut in the dsDNA. Secondly, once the complex of the small terminase, the large terminase and the DNA has docked to the portal protein, and DNA translocation into a preformed empty procapsid has begun, the small terminase modulates the ATPase activity of the large terminase. Here, the putative small terminase protein from the thermostable bacteriophage G20C, which infects the Gram-negative eubacterium Thermus thermophilus, has been produced, purified and crystallized. Size-exclusion chromatography-multi-angle laser light scattering data indicate that the protein forms oligomers containing nine subunits. Crystals diffracting to 2.8 Å resolution have been obtained. These belonged to space group P212121, with unit-cell parameters a = 94.31, b = 125.6, c = 162.8 Å. The self-rotation function and Matthews coefficient calculations are consistent with the presence of a nine-subunit oligomer in the asymmetric unit.

Compensating stereochemical changes allow murein tripeptide to be accommodated in a conventional peptide-binding protein.

The oligopeptide permease (Opp) of Escherichia coli is an ATP-binding cassette transporter that uses the substrate-binding protein (SBP) OppA to bind peptides and deliver them to the membrane components (OppBCDF) for transport. OppA binds conventional peptides 2-5 residues in length regardless of their sequence, but does not facilitate transport of the cell wall component murein tripeptide (Mtp, L-Ala-γ-D-Glu-meso-Dap), which contains a D-amino acid and a γ-peptide linkage. Instead, MppA, a homologous substrate-binding protein, forms a functional transporter with OppBCDF for uptake of this unusual tripeptide. Here we have purified MppA and demonstrated biochemically that it binds Mtp with high affinity (K(D) ∼ 250 nM). The crystal structure of MppA in complex with Mtp has revealed that Mtp is bound in a relatively extended conformation with its three carboxylates projecting from one side of the molecule and its two amino groups projecting from the opposite face. Specificity for Mtp is conferred by charge-charge and dipole-charge interactions with ionic and polar residues of MppA. Comparison of the structure of MppA-Mtp with structures of conventional tripeptides bound to OppA, reveals that the peptide ligands superimpose remarkably closely given the profound differences in their structures. Strikingly, the effect of the D-stereochemistry, which projects the side chain of the D-Glu residue at position 2 in the direction of the main chain in a conventional tripeptide, is compensated by the formation of a γ-linkage to the amino group of diaminopimelic acid, mimicking the peptide bond between residues 2 and 3 of a conventional tripeptide.

The structure and interactions of SpoIISA and SpoIISB, a toxin-antitoxin system in Bacillus subtilis.

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σ(F). Inappropriate spoIISA expression causes lysis of vegetatively growing B. subtilis cells and Escherichia coli cells when expressed heterologously, effects that are countered by co-expression of spoIISB, identifying SpoIISA-SpoIISB as a toxin-antitoxin system. SpoIISA has three putative membrane-spanning segments and a cytoplasmic domain. Here, the crystal structure of a cytoplasmic fragment of SpoIISA (CSpoIISA) in complex with SpoIISB has been determined by selenomethionine-multiwavelength anomalous dispersion phasing to 2.5 Å spacing, revealing a CSpoIISA(2)·SpoIISB(2) heterotetramer. CSpoIISA has a single domain α/ß structure resembling a GAF domain with an extended α-helix at its N terminus. The two CSpoIISA protomers form extensive interactions through an intermolecular four-helix bundle. Each SpoIISB chain is highly extended and lacking tertiary structure. The SpoIISB chains wrap around the CSpoIISA dimer, forming extensive interactions with both CSpoIISA protomers. CD spectroscopy experiments indicate that SpoIISB is a natively disordered protein that adopts structure only in the presence of CSpoIISA, whereas surface plasmon resonance experiments revealed that the CSpoIISA·SpoIISB complex is stable with a dissociation constant in the nanomolar range. The results are interpreted in relation to sequence conservation and mutational data, and possible mechanisms of cell killing by SpoIISA are discussed.

Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

The structure of Rph, an exoribonuclease from Bacillus anthracis, at 1.7 A resolution.

Maturation of tRNA precursors into functional tRNA molecules requires trimming of the primary transcript at both the 5' and 3' ends. Cleavage of nucleotides from the 3' stem of tRNA precursors, releasing nucleotide diphosphates, is accomplished in Bacillus by a phosphate-dependent exoribonuclease, Rph. The crystal structure of this enzyme from B. anthracis has been solved by molecular replacement to a resolution of 1.7 A and refined to an R factor of 19.3%. There is one molecule in the asymmetric unit; the crystal packing reveals the assembly of the protein into a hexamer arranged as a trimer of dimers. The structure shows two sulfate ions bound in the active-site pocket, probably mimicking the phosphate substrate and the phosphate of the 3'-terminal nucleotide of the tRNA precursor. Three other bound sulfate ions point to likely RNA-binding sites.

Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027.

Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.

A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases.

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an α-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site.

The structure of the oligopeptide-binding protein, AppA, from Bacillus subtilis in complex with a nonapeptide.

Besides their role as a source of amino acids for Bacillus subtilis, exogenous peptides play important roles in the signalling pathways leading to the development of competence and sporulation. B.subtilis has three peptide transport systems all belonging to the ATP-binding cassette family, a dipeptide permease (Dpp) and two oligopeptide permeases (Opp and App) with overlapping specificity. These comprise a membrane-spanning channel through which the peptide passes, a pair of ATPases which couple ATP hydrolysis to peptide translocation and a lipid-modified, membrane-anchored extracellular "binding-protein" that serves as the receptor for the system. Here, we present the crystal structure of a soluble form of the peptide-binding protein AppA, which has been solved to 1.6 A spacing by anomalous scattering and molecular replacement methods. The structure reveals a protein made of two distinct lobes with a topology similar to those of DppA from Escherichia coli and OppA from Salmonella typhimurium. Examination of the interlobe region reveals an enlarged pocket, containing electron density defining a nonapeptide ligand. The main-chain of the peptide is well defined and makes a series of polar contacts with the protein including salt-bridges at both its termini. The side-chain density is ambiguous in places, consistent with the interpretation that a population of peptides is bound, whose average electron density resembles the amino acid sequence N-VDSKNTSSW-C.

The crystal structure of YloQ, a circularly permuted GTPase essential for Bacillus subtilis viability.

yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell. It encodes a polypeptide of 298 residues with motifs characteristic of GTPases. As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.6 A spacing, revealing a three-domain organisation. At the heart of the molecule is the putative GTPase domain, which exhibits a classical alpha/beta nucleotide-binding fold with a topology very similar to that of Ras and Era. However, as anticipated from the order in which the conserved G protein motifs appear in the sequence, the GTPase domain fold in YloQ is circularly permuted with respect to the classical GTPases. The nucleotide-binding pocket in YloQ is unoccupied, and analysis of the phosphate-binding (P) loop indicates that conformational changes in this region would be needed to accommodate GTP. The GTPase domain is flanked at its N terminus by a beta-barrel domain with an oligonucleotide/oligosaccharide-binding (OB) fold, and at its C terminus by an alpha-helical domain containing a coordinated zinc ion. This combination of protein modules is unique to YloQ and its orthologues. Sequence comparisons reveal a clustering of conserved basic and aromatic residues on one face of the OB domain, perhaps pointing to a role for YloQ in nucleic acid binding. The zinc ion in the alpha-helical domain is coordinated by three cysteine residues and a histidine residue in a novel ligand organisation. The juxtaposition of the switch I and switch II regions of the G domain and the OB and zinc-binding domains suggests that chemical events at the GTPase active site may be transduced into relative movements of these domains. The pattern of conserved residues and electrostatic surface potential calculations suggest that the OB and/or Zn-binding domains participate in nucleic acid binding consistent with a possible role for YloQ at some stage during mRNA translation.

Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis.

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway. The crystal structure of purine nucleoside phosphorylase (DeoD) from B. anthracis has been solved by molecular replacement at 2.24 A resolution and refined to an R factor of 18.4%. This is the first report of a DeoD structure from a Gram-positive bacterium.

Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centre.

The BA4499 and BA5696 genes of Bacillus anthracis encode proteins homologous to manganese superoxide dismutase, suggesting that this organism has an expanded repertoire of antioxidant proteins. Differences in metal specificity and quaternary structure between the dismutases of prokaryotes and higher eukaryotes may be exploited in the development of therapeutic antibacterial compounds. Here, the crystal structure of two Mn superoxide dismutases from B. anthracis solved to high resolution are reported. Comparison of their structures reveals that a highly conserved residue near the active centre is substituted in one of the proteins and that this is a characteristic feature of superoxide dismutases from the B. cereus/B. anthracis/B. thuringiensis group of organisms.

Structure of the phosphatase domain of the cell fate determinant SpoIIE from Bacillus subtilis.

Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA polymerase sigma factor σ(F) from an inhibitory complex with the anti-sigma factor SpoIIAB. SpoIIE therefore couples morphological development to differential gene expression. Here, we determined the crystal structure of the phosphatase domain of SpoIIE to 2.6 Å spacing, revealing a domain-swapped dimer. SEC-MALLS (size-exclusion chromatography with multi-angle laser light scattering) analysis however suggested a monomer as the principal form in solution. A model for the monomer was derived from the domain-swapped dimer in which 2 five-stranded ß-sheets are packed against one another and flanked by α-helices in an αßßα arrangement reminiscent of other PP2C-type phosphatases. A flap region that controls access of substrates to the active site in other PP2C phosphatases is diminished in SpoIIE, and this observation correlates with the presence of a single manganese ion in the active site of SpoIIE in contrast to the two or three metal ions present in other PP2C enzymes. Mapping of a catalogue of mutational data onto the structure shows a clustering of sites whose point mutation interferes with the proper coupling of asymmetric septum formation to sigma factor activation and identifies a surface involved in intramolecular signaling.

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis.

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI. Each forms a pair of α-helices that hook together to form an intermolecular four-helix bundle. Here, we are interested in the assembly of the SinR tetramer and its binding to DNA. Size-exclusion chromatography with multi-angle laser light scattering and crystallographic analysis reveal that a DNA-binding fragment of SinR (residues 1-69) is a monomer, while a SinI-binding fragment (residues 74-111) is a tetramer arranged as a dimer of dimers. The SinR(74-111) chain forms two α-helices with the organisation of the dimer similar to that observed in the SinR-SinI complex. The tetramer is formed through interactions of residues at the C-termini of the four chains. A model of the intact SinR tetramer in which the DNA binding domains surround the tetramerisation core was built. Fluorescence anisotropy and surface plasmon resonance experiments showed that SinR binds to an oligonucleotide duplex, 5'-TTTGTTCTCTAAAGAGAACTTA-3', containing a pair of SinR consensus sequences in inverted orientation with a K(d) of 300 nM. The implications of these data for promoter binding and the curious quaternary structural transitions of SinR upon binding to (i) SinI and (ii) the SinR-like protein SlrR, which "repurposes" SinR as a repressor of autolysin and motility genes, are discussed.

Structure of the catalytic domain of the human mitochondrial Lon protease: proposed relation of oligomer formation and activity.

ATP-dependent proteases are crucial for cellular homeostasis. By degrading short-lived regulatory proteins, they play an important role in the control of many cellular pathways and, through the degradation of abnormally misfolded proteins, protect the cell from a buildup of aggregates. Disruption or disregulation of mammalian mitochondrial Lon protease leads to severe changes in the cell, linked with carcinogenesis, apoptosis, and necrosis. Here we present the structure of the proteolytic domain of human mitochondrial Lon at 2 A resolution. The fold resembles those of the three previously determined Lon proteolytic domains from Escherichia coli, Methanococcus jannaschii, and Archaeoglobus fulgidus. There are six protomers in the asymmetric unit, four arranged as two dimers. The intersubunit interactions within the two dimers are similar to those between adjacent subunits of the hexameric ring of E. coli Lon, suggesting that the human Lon proteolytic domain also forms hexamers. The active site contains a 3(10) helix attached to the N-terminal end of alpha-helix 2, which leads to the insertion of Asp852 into the active site, as seen in M. jannaschii. Structural considerations make it likely that this conformation is proteolytically inactive. When comparing the intersubunit interactions of human with those of E. coli Lon taken with biochemical data leads us to propose a mechanism relating the formation of Lon oligomers with a conformational shift in the active site region coupled to a movement of a loop in the oligomer interface, converting the proteolytically inactive form seen here to the active one in the E. coli hexamer.

Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen.

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity.

Structural rearrangement accompanying ligand binding in the GAF domain of CodY from Bacillus subtilis.

The GAF domain is a simple module widespread in proteins of diverse function, including cell signalling proteins and transcription factors. Its structure, typically spanning 150 residues, has three tiers: a basal layer of two or more alpha-helices, a middle layer of beta-pleated sheet and a top layer formed by segments of the polypeptide that connect strands of the beta-sheet. In structures of GAF domains in complex with their effectors, these polypeptide segments envelop the ligand, enclosing it in a cavity whose base is formed by the beta-sheet, such that ligand binding and release must be accompanied by conformational rearrangements of the distal portion of the structure. Descriptions of binding are presently limited by the absence of a GAF domain for which both liganded and unliganded structures are known. Earlier, we solved the crystal structure of the GAF domain of CodY, a branched-chain amino acid and GTP-responsive regulator of the transcription of stationary-phase and virulence genes in Bacillus, in complexes with isoleucine and valine. Here, we report the structure of this domain in its unliganded form, allowing definition of the structural changes accompanying ligand binding. The core of the protein and its dimerisation interface are essentially unchanged, in agreement with circular dichroism spectroscopy experiments that show that the secondary structure composition is unperturbed by ligand binding. There is however extensive refolding of the binding site loops, with up to 15-A movements of the coiled segment linking beta3 and beta4, such that the binding pocket is not formed in the absence of the ligand. The implications of these structural rearrangements for ligand affinity and specificity are discussed. Finally, saturation-transfer-difference NMR spectroscopy showed binding of isoleucine but not that of GTP to the GAF domain, suggesting that the two cofactors do not have a common binding site.