Structural mechanisms of QacR induction and multidrug recognition.

The Staphylococcus aureus multidrug binding protein QacR represses transcription of the qacA multidrug transporter gene and is induced by structurally diverse cationic lipophilic drugs. Here, we report the crystal structures of six QacR-drug complexes. Compared to the DNA bound structure, drug binding elicits a coil-to-helix transition that causes induction and creates an expansive multidrug-binding pocket, containing four glutamates and multiple aromatic and polar residues. These structures indicate the presence of separate but linked drug-binding sites within a single protein. This multisite drug-binding mechanism is consonant with studies on multidrug resistance transporters.

The staphylococcal QacR multidrug regulator binds a correctly spaced operator as a pair of dimers.

Expression of the Staphylococcus aureus plasmid-encoded QacA multidrug transporter is regulated by the divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of qacA expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of qacA expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers.

Crystal structure of MtaN, a global multidrug transporter gene activator.

MtaN (Multidrug Transporter Activation, N terminus) is a constitutive, transcriptionally active 109-residue truncation mutant, which contains only the N-terminal DNA-binding and dimerization domains of MerR family member Mta. The 2.75 A resolution crystal structure of apo-MtaN reveals a winged helix-turn-helix protein with a protruding 8-turn helix (alpha5) that is involved in dimerization by the formation of an antiparallel coiled-coil. The hydrophobic core and helices alpha1 through alpha4 are structurally homologous to MerR family member BmrR bound to DNA, whereas one wing (Wing 1) is shifted. Differences between the orientation of alpha5 with respect to the core and the revolution of the antiparallel coiled-coil lead to significantly altered conformations of MtaN and BmrR dimers. These shifts result in a conformation of MtaN that appears to be incompatible with the transcription activation mechanism of BmrR and suggest that additional DNA-induced structural changes are necessary.

The basic helix-loop-helix domain of the aryl hydrocarbon receptor nuclear transporter (ARNT) can oligomerize and bind E-box DNA specifically.

The aryl hydrocarbon receptor nuclear transporter (ARNT) is a basic helix-loop-helix (bHLH) protein that contains a Per-Arnt-Sim (PAS) domain. ARNT heterodimerizes in vivo with other bHLH PAS proteins to regulate a number of cellular activities, but a physiological role for ARNT homodimers has not yet been established. Moreover, no rigorous studies have been done to characterize the biochemical properties of the bHLH domain of ARNT that would address this issue. To begin this characterization, we chemically synthesized a 56-residue peptide encompassing the bHLH domain of ARNT (residues 90-145). In the absence of DNA, the ARNT-bHLH peptide can form homodimers in lower ionic strength, as evidenced by dynamic light scattering analysis, and can bind E-box DNA (CACGTG) with high specificity and affinity, as determined by fluorescence anisotropy. Dimers and tetramers of ARNT-bHLH are observed bound to DNA in equilibrium sedimentation and dynamic light scattering experiments. The homodimeric peptide also undergoes a coil-to-helix transition upon E-box DNA binding. Peptide oligomerization and DNA affinity are strongly influenced by ionic strength. These biochemical and biophysical studies on the ARNT-bHLH reveal its inherent ability to form homodimers at concentrations supporting a physiological function and underscore the significant biochemical differences among the bHLH superfamily.

Crystal structures of SarA, a pleiotropic regulator of virulence genes in S. aureus.

Staphylococcus aureus is a major human pathogen, the potency of which can be attributed to the regulated expression of an impressive array of virulence determinants. A key pleiotropic transcriptional regulator of these virulence factors is SarA, which is encoded by the sar (staphylococcal accessory regulator) locus. SarA was characterized initially as an activator of a second virulence regulatory locus, agr, through its interaction with a series of heptad repeats (AGTTAAG) within the agr promoter. Subsequent DNA-binding studies have revealed that SarA binds readily to multiple AT-rich sequences of variable lengths. Here we describe the crystal structure of SarA and a SarA-DNA complex at resolutions of 2.50 A and 2.95 A, respectively. SarA has a fold consisting of a four-helix core region and 'inducible regions' comprising a beta-hairpin and a carboxy-terminal loop. On binding DNA, the inducible regions undergo marked conformational changes, becoming part of extended and distorted alpha-helices, which encase the DNA. SarA recognizes an AT-rich site in which the DNA is highly overwound and adopts a D-DNA-like conformation by indirect readout. These structures thus provide insight into SarA-mediated transcription regulation.

Crystal structure of the transcription activator BmrR bound to DNA and a drug.

The efflux of chemically diverse drugs by multidrug transporters that span the membrane is one mechanism of multidrug resistance in bacteria. The concentrations of many of these transporters are controlled by transcription regulators, such as BmrR in Bacillus subtilis, EmrR in Escherichia coli and QacR in Staphylococcus aureus. These proteins promote transporter gene expression when they bind toxic compounds. BmrR activates transcription of the multidrug transporter gene, bmr, in response to cellular invasion by certain lipophilic cationic compounds (drugs). BmrR belongs to the MerR family, which regulates response to stress such as exposure to toxic compounds or oxygen radicals in bacteria. MerR proteins have homologous amino-terminal DNA-binding domains but different carboxy-terminal domains, which enable them to bind specific 'coactivator' molecules. When bound to coactivator, MerR proteins upregulate transcription by reconfiguring the 19-base-pair spacer found between the -35 and -10 promoter elements to allow productive interaction with RNA polymerase. Here we report the 3.0 A resolution structure of BmrR in complex with the drug tetraphenylphosphonium (TPP) and a 22-base-pair oligodeoxynucleotide encompassing the bmr promoter. The structure reveals an unexpected mechanism for transcription activation that involves localized base-pair breaking, and base sliding and realignment of the -35 and -10 operator elements.

Consensus and variant cAMP-regulated enhancers have distinct CREB-binding properties.

Recent determination of the cAMP response element-binding protein (CREB) basic leucine zipper (bZIP) consensus CRE crystal structure revealed key dimerization and DNA binding features that are conserved among members of the CREB/CREM/ATF-1 family of transcription factors. Dimerization appeared to be mediated by a Tyr(307)-Glu(312) interhelical hydrogen bond and a Glu(319)-Arg(314) electrostatic interaction. An unexpected hexahydrated Mg(2+) ion was centered above the CRE in the dimer cavity. In the present study, we related these features to CREB dimerization and DNA binding. A Y307F substitution reduced dimer stability and DNA binding affinity, whereas a Y307R mutation produced a stabilizing effect. Mutation of Glu(319) to Ala or Lys attenuated dimerization and DNA binding. Mg(2+) ions enhanced the binding affinity of wild-type CREB to the palindromic CRE by approximately 20-fold but did not do so for divergent CREs. Similarly, mutation of Lys(304), which mediates the CREB interaction with the hydrated Mg(2+), blocked CREB binding to the palindromic but not the variant CRE sequences. The distinct binding characteristics of the K304A mutants to the consensus and variant CRE sequences indicate that CREB binding to these elements is differentially regulated by Mg(2+) ions. We suggest that CREB binds the consensus and variant CRE sequences through fundamentally distinct mechanisms.

Characterization of the DNA- and metal-binding properties of Vibrio anguillarum fur reveals conservation of a structural Zn(2+) ion.

The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under iron-replete conditions. Here we report in vitro solution studies on Vibrio anguillarum Fur binding to the consensus 19-bp Escherichia coli iron box in the presence of several divalent metals. We found that V. anguillarum Fur binds the iron box in the presence of Mn(2+), Co(2+), Cd(2+), and to a lesser extent Ni(2+) but, unlike E. coli Fur, not in the presence of Zn(2+). We also found that V. anguillarum Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.

Crystallization and preliminary X-ray diffraction studies on the DNA-binding domain of the multidrug transporter activation protein (MtaN) from Bacillus subtilis.

The N-terminal DNA-binding domain of the multidrug transporter activation protein (MtaN) was crystallized by the hanging-drop vapour-diffusion method using lithium chloride as a precipitant. The crystals are orthorhombic and belong to the space group I2(1)2(1)2(1), with unit-cell parameters a = 49.4, b = 67.8, c = 115. 0 A. Diffraction data have been collected at 100 K to 2.75 A resolution at a synchrotron-radiation source.

Recognition of multiple drugs by a single protein: a trivial solution of an old paradox.

Multidrug-efflux transporters recognize scores of structurally dissimilar toxic compounds and expel them from cells. The broad chemical specificity of these transporters challenges some of the basic dogmas of biochemistry and remains unexplained. To understand, at least in principle, how a protein can recognize multiple compounds, we analysed the transcriptional regulator of the Bacillus subtilis multidrug transporter Bmr. This regulator, BmrR, binds multiple dissimilar hydrophobic cations and, by activating the expression of the Bmr transporter, causes their expulsion from the cell. Crystallographic analysis of the complexes of the inducer-binding domain of BmrR with some of its inducers revealed that ligands cause disordering of the surface alpha-helix and penetrate the hydrophobic core of the protein, where they form multiple van der Waals and stacking interactions with hydrophobic amino acids and an electrostatic bond with the buried glutamic residue. Mutational analysis of the binding site suggests that each ligand forms a unique set of atomic contacts with the protein: each tested mutation exerted disparate effects on the binding of different ligands. The example of BmrR demonstrates that a protein can bind multiple compounds with micromolar affinities by using only electrostatic and hydrophobic interactions. Its ligand specificity can be broadened by the flexibility of the binding site. It therefore seems that the commonly expressed fascination with the broad specificity of multidrug transporters is misdirected and originates from an almost exclusive familiarity with the more sophisticated processes of specific molecular recognition that predominate among existing proteins.

The structure of a CREB bZIP.somatostatin CRE complex reveals the basis for selective dimerization and divalent cation-enhanced DNA binding.

The cAMP responsive element-binding protein (CREB) is central to second messenger regulated transcription. To elucidate the structural mechanisms of DNA binding and selective dimerization of CREB, we determined to 3.0 A resolution, the structure of the CREB bZIP (residues 283-341) bound to a 21-base pair deoxynucleotide that encompasses the canonical 8-base pair somatostatin cAMP response element (SSCRE). The CREB dimer is stabilized in part by ionic interactions from Arg(314) to Glu(319') and Glu(328) to Lys(333') as well as a hydrogen bond network that links the carboxamide side chains of Gln(322')-Asn(321)-Asn(321')-Gln(322). Critical to family selective dimerization are intersubunit hydrogen bonds between basic region residue Tyr(307) and leucine zipper residue Glu(312), which are conserved in all CREB/CREM/ATF-1 family members. Strikingly, the structure reveals a hexahydrated Mg(2+) ion bound in the cavity between the basic region and SSCRE that makes a water-mediated DNA contact. DNA binding studies demonstrate that Mg(2+) ions enhance CREB bZIP:SSCRE binding by more than 25-fold and suggest a possible physiological role for this ion in somatostatin cAMP response element and potentially other CRE-mediated gene expression.

The structural basis of repertoire shift in an immune response to phosphocholine.

The immune response to phosphocholine (PC)-protein is characterized by a shift in antibody repertoire as the response progresses. This change in expressed gene combinations is accompanied by a shift in fine specificity toward the carrier, resulting in high affinity to PC-protein. The somatically mutated memory hybridoma, M3C65, possesses high affinity for PC-protein and the phenyl-hapten analogue, p-nitrophenyl phosphocholine (NPPC). Affinity measurements using related PC-phenyl analogues, including peptides of varying lengths, demonstrate that carrier determinants contribute to binding affinity and that somatic mutations alter this recognition. The crystal structure of an M3C65-NPPC complex at 2.35-A resolution allows evaluation of the three light chain mutations that confer high-affinity binding to NPPC. Only one of the mutations involves a contact residue, whereas the other two have indirect effects on the shape of the combining site. Comparison of the M3C65 structure to that of T15, an antibody dominating the primary response, provides clear structural evidence for the role of carrier determinants in promoting repertoire shift. These two antibodies express unrelated variable region heavy and light chain genes and represent a classic example of the effect of repertoire shift on maturation of the immune response.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding.

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.

Recruitment of CREB binding protein is sufficient for CREB-mediated gene activation.

Phosphorylation of the transcription factor CREB leads to the recruitment of the coactivator, CREB binding protein (CBP). Recent studies have suggested that CBP recruitment is not sufficient for CREB function, however. We have identified a conserved protein-protein interaction motif within the CBP-binding domains of CREB and another transcription factor, SREBP (sterol-responsive element binding protein). In contrast to CREB, SREBP interacts with CBP in the absence of phosphorylation. We have exploited the conservation of this interaction motif to test whether CBP recruitment to CREB is sufficient for transcriptional activation. Substitution of six nonconserved amino acids from SREBP into the activation domain of CREB confers high-affinity, phosphorylation-independent CBP binding. The mutated CREB molecule, CREB(DIEDML), activates transcription in F9 teratocarcinoma and PC12 cells even in the absence of protein kinase A (PKA). Addition of exogenous CBP augments the level of transcription mediated by CREB(DIEDML), and adenovirus 12S E1A blocks transcription, implicating CBP in the activation process. Thus, recruitment of CBP to CREB is sufficient for transcriptional activation. Addition of PKA stimulates transcription induced by CREB(DIEDML) further, suggesting that a phosphorylation event downstream from CBP recruitment augments CREB signaling.

A structure-based mechanism for drug binding by multidrug transporters.

Multidrug transporters bind chemically dissimilar, potentially cytotoxic compounds and remove them from the cell. How these transporters carry out either of these functions is unknown. On the basis of crystal structures of the multidrug-binding domain of the transcription activator BmrR and mutagenesis studies on the bacterial multidrug transporter MdfA, we propose a possible mechanism for the binding of cationic lipophilic drugs by multidrug transporters. The key element of this mechanism includes a conformational change in the transporter that exposes a buried charged residue in the substrate-binding pocket and allows access to this site by only those drugs that are its steric and electrostatic complements.

The role of lysine 55 in determining the specificity of the purine repressor for its operators through minor groove interactions.

The interaction of the dimeric Escherichia coli purine repressor (PurR) with its cognate sequences leads to a 45 degrees to 50 degrees kink at a central CpG base step towards the major groove, as dyad-related leucine side-chains interdigitate between these bases from the minor groove. The resulting broadening of the minor groove increases the accessibility of the six central base-pairs towards minor groove interactions with residues from PurR. It has been shown that lysine 55 of PurR makes a direct contact with the adenine base (Ade8) directly 5' to the central CpG base-pair step in the high-affinity purF operator sequence. We have investigated the importance of this interaction in the specificity and affinity of wild-type PurR (WT) for its operators and we have studied a mutant of PurR in which Lys55 is replaced with alanine (K55A). Complexes of WT and K55A with duplex DNA containing pur operator sequences varied at position 8 were investigated crystallographically, and binding studies were performed using fluorescence anisotropy. The structures of the protein-DNA complexes reveal a relatively unperturbed global conformation regardless of the identity of the base-pair at position 8 or residue 55. In all structures the combination of higher resolution and a palindromic purF operator site allowed several new PurR.DNA interactions to be observed, including contacts by Thr15, Thr16 and His20. The side-chain of Lys55 makes productive, though varying, interactions with the adenine, thymine or cytosine base at position 8 that result in equilibrium dissociation constants of 2.6 nM, 10 nM and 35 nM, respectively. However, the bulk of the lysine side-chain apparently blocks high-affinity binding of operators with guanine at position 8 (Kd620 nM). Also, the high-affinity binding conformation appears blocked, as crystals of WT bound to DNA with guanine at position 8 could not be grown. In complexes containing K55A, the alanine side-chain is too far removed to engage in van der Waals interactions with the operator, and, with the loss of the general electrostatic interaction between the phosphate backbone and the ammonium group of lysine, K55A binds each operator weakly. However, the mutation leads to a swap of specificity of PurR for the base at position 8, with K55A exhibiting a twofold preference for guanine over adenine. In addition to defining the role of Lys55 in PurR minor groove binding, these studies provide structural insight into the minor groove binding specificities of other LacI/GalR family members that have either alanine (e.g. LacI, GalR, CcpA) or a basic residue (e.g. RafR, ScrR, RbtR) at the comparable position.

Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani.

The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis.

Characterization of the SarA virulence gene regulator of Staphylococcus aureus.

Staphylococcus aureus is a potent human pathogen that expresses a large number of virulence factors in a temporally regulated fashion. Two pleiotropically acting regulatory loci were identified in previous mutational studies. The agr locus comprises two operons that express a quorum-sensing system from the P2 promoter and a regulatory RNA molecule from the P3 promoter. The sar locus encodes a DNA-binding protein that activates the expression of both agr operons. We have cloned the sarA gene, expressed SarA in Escherichia coli and purified the recombinant protein to apparent homogeneity. The purified protein was found to be dimeric in the presence and absence of DNA and to consist mostly of alpha-helices. DNase I footprinting of SarA on the putative regulatory region cis to the agr promoters revealed three high-affinity binding sites composed of two half-sites each. Quantitative electrophoretic mobility shift assays (EMSAs) were used to derive equilibrium binding constants (KD) for the interaction of SarA with these binding sites. An unusual ladder banding pattern was observed in EMSA with a large DNA fragment including all three binding sites. Our data indicate that SarA regulation of the agr operons involves binding to multiple half-sites and may involve other sites located downstream of the promoters.

Structural basis of multidrug recognition by BmrR, a transcription activator of a multidrug transporter.

Multidrug-efflux transporters demonstrate an unusual ability to recognize multiple structurally dissimilar toxins. A comparable ability to bind diverse hydrophobic cationic drugs is characteristic of the Bacillus subtilis transcription regulator BmrR, which upon drug binding activates expression of the multidrug transporter Bmr. Crystal structures of the multidrug-binding domain of BmrR (2.7 A resolution) and of its complex with the drug tetraphenylphosphonium (2.8 A resolution) revealed a drug-induced unfolding and relocation of an alpha helix, which exposes an internal drug-binding pocket. Tetraphenylphosphonium binding is mediated by stacking and van der Waals contacts with multiple hydrophobic residues of the pocket and by an electrostatic interaction between the positively charged drug and a buried glutamate residue, which is the key to cation selectivity. Similar binding principles may be used by other multidrug-binding proteins.