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 structure of LacI member, PurR, bound to DNA: minor groove binding by alpha helices.

The three-dimensional structure of a ternary complex of the purine repressor, PurR, bound to both its corepressor, hypoxanthine, and the 16-base pair purF operator site has been solved at 2.7 A resolution by x-ray crystallography. The bipartite structure of PurR consists of an amino-terminal DNA-binding domain and a larger carboxyl-terminal corepressor binding and dimerization domain that is similar to that of the bacterial periplasmic binding proteins. The DNA-binding domain contains a helix-turn-helix motif that makes base-specific contacts in the major groove of the DNA. Base contacts are also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. Critical to hinge helix-minor groove binding is the intercalation of the side chains of Leu54 and its symmetry-related mate, Leu54', into the central CpG-base pair step. These residues thereby act as "leucine levers" to pry open the minor groove and kink the purF operator by 45 degrees.

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.

Crystal structure of the lactose operon repressor and its complexes with DNA and inducer.

The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-beta-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

Structural comparison of the free and DNA-bound forms of the purine repressor DNA-binding domain.

BACKGROUND: The purine repressor (PurR) regulates genes that encode enzymes for purine biosynthesis. PurR has a two domain structure with an N-terminal DNA-binding domain (DBD) and a C-terminal corepressor-binding domain (CBD). The three dimensional structure of a ternary complex of PurR bound to both corepressor and a specific DNA sequence has recently been determined by X-ray crystallography. RESULTS: We have determined the solution structure of the PurR DBD by NMR. It contains three helices, with the first and second helices forming a helix-turn-helix motif. The tertiary structure of the three helices is very similar to that of the corresponding region in the ternary complex. The structure of the hinge helical region, however, which makes specific base contacts in the minor groove of DNA, is disordered in the DNA-free form. CONCLUSION: The stable formation of PurR hinge helices requires PurR dimerization, which brings the hinge regions proximal to each other. The dimerization of the hinge helices is likely to be controlled by the CBD dimerization interface, but is induced by specific-DNA binding.

Crystallization and preliminary X-ray analysis of an Escherichia coli purine repressor-hypoxanthine-DNA complex.

The purine repressor (PurR) is a DNA-binding protein, which together with a purine corepressor serves to regulate de novo purine and pyrimidine biosynthesis in Escherichia coli. PurR belongs to the structurally homologous lac repressor family of transcription regulators. A PurR-hypoxanthine-DNA complex has been crystallized, with DNA encompassing the high affinity purF operator site and which is 16 base-pairs long with 5'-deoxynucleoside overhangs on each complementary strand. The crystals diffract to better than 2.6 A and take the orthorhombic space group C222(1), with unit cell dimensions a = 175.9 A, b = 94.8 A and c = 81.8 A. The structure determination of this PurR-hypoxanthine-DNA complex will provide the first high resolution view of a Lacl member-DNA complex.

Crystallization and preliminary X-ray studies on the co-repressor binding domain of the Escherichia coli purine repressor.

The purine repressor is a putative helix-turn-helix DNA-binding protein that regulates several genetic loci important in purine and pyrimidine metabolism in Escherichia coli. The protein is composed of two domains, an N-terminal DNA-binding domain and a C-terminal core that binds the purine co-repressors, guanine and hypoxanthine. The co-repressor binding domain (residues 53 to 341) has been crystallized from polyethylene glycol 600-MgCl2 solutions. They are of the monoclinic form, space group P2(1), with a = 38.2 A, b = 125.7 A, c = 61.8 A and beta = 100.2 degrees. They diffract to a resolution of at least 2.2 A and contain two monomers per asymmetric unit. The importance of the structural determination of this domain is underscored by the high degree of sequence homology displayed within the effector binding sites among a sub-class of helix-turn-helix proteins, of which LacI and GalR are members. The structure of the PurR co-repressor binding domain will provide a high resolution view of one such domain and could serve as a possible model for future effector site structural determinations. Perhaps more important will be this structure's contribution to the further understanding of how protein-DNA interactions are modulated.

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 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.

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.

The stretch-inactivated channel, a vanilloid receptor variant, is expressed in small-diameter sensory neurons in the rat.

Exposure to hypertonic conditions is known to produce pain and activate small-diameter sensory neurons. Recently, the vanilloid receptor variant and stretch-inactivated ion channel (SIC) was cloned and shown to mediate an inward current in response to cell shrinkage. Since other vanilloid receptors have been previously shown to mediate nociception, we investigated whether SIC is expressed in sensory neurons. Using reverse transcription-polymerase chain reaction and in situ hybridization techniques, we identified SIC in the neurons of dorsal root and trigeminal ganglia. Furthermore, SIC was found to be present almost exclusively in the small-diameter sensory neurons, which includes the nociceptive population. Since SIC is activated by cell shrinkage, it may participate in the mediation of pain produced by hypertonic stimuli.

Mechanisms of whole blood-induced cerebral arterial contraction.

Using rabbit cerebral arteries in an in vitro chamber, we examined the cerebral arterial contraction initiated by clotting whole blood. By using methysergide maleate and a novel thromboxane synthetase inhibitor, 1-carboxyheptylimidazole (1-CHI), we studied the contributions of both serotonin and the prostaglandin metabolite thromboxane A2. Nontreated platelet-rich plasma (PRP) in the presence of methysergide produced a reliable contraction, whereas platelet-poor plasma did not. PRP from a rabbit pretreated with 1-CHI (50 mg/kg) compared to nontreated PRP caused a significantly smaller contraction. Blockade of this cerebral arterial contraction occurred without the disruption of platelet aggregation. Whole blood (1 ml) plus thrombin produced a consistent contraction over the 1 hour that was monitored. Whole blood drawn from a rabbit pretreated with 1-CHI (50 mg/kg) produced a smaller contraction, which began to dissipate in 5 minutes. When nontreated whole blood was added to the chamber in the presence of methysergide maleate (1.3 X 10(-5) g/ml), a contraction less than control was produced, and it persisted at 30 minutes. When whole blood pretreated with 1-CHI (50 mg/kg) was added to the chamber containing methysergide, there was a transient contraction that dissipated to nearly zero at 30 minutes. From our results, it is apparent that the thromboxane synthetase inhibitor has a profound effect on the later phase of blood-induced vasoconstriction. In contrast, the serotonin antagonist affected primarily the initial vasoconstriction and left the later phase largely unaltered. The role of thrombin, used to initiate coagulation, was also examined, and it was found to have a minimal direct constrictive effect when in a plasma solution.

The anticancer antibiotic mithramycin-A inhibits TRPV1 expression in dorsal root ganglion neurons.

Activation of peripheral nociceptors by products of inflammation has been shown to be dependent on specific sensory transducing elements such as the capsaicin receptor, TRPV1. The development of high-affinity antagonists to TRPV1 as well as to other receptors capable of detecting noxious stimuli has now become a major focus in analgesic development. Another critical feature of nociception is the relative abundance of a particular pain transducing receptor under normal or pathophysiologic conditions. Increases in expression and/or changes in distribution of nociceptive receptors such as TRPV1 have been correlated with progression of tissue injury and persistence of pain behaviors. Although some details are emerging as to what regulates nociceptor-specific gene expression, compounds that could potentially be used to block or reverse over-expression of nociceptive gene expression are essentially absent. In our efforts to better understand the transcriptional regulation of TRPV1 in sensory neurons, we identified an anticancer agent, mithramycin-A, that decreased TRPV1 expression in primary rat dorsal root ganglion (DRG) neurons. Mithramycin-A dose-dependently (10-50 nM) decreased endogenous TRPV1 mRNA content and appeared to decrease TRPV1-like protein expression in DRG neurons. We also observed that mithramycin-A directed a decrease in the number of capsaicin-responsive DRG neurons without a significant change in the capsaicin-response magnitudes. Interestingly, mithramycin-A also reduced the mRNA encoding Sp1 and Sp4 in DRG neurons, transcription factors previously found to positively regulate TRPV1 expression in sensory neurons. Taken together, we propose that mithramycin-A directs an inhibitory effect on a subpopulation of capsaicin-responsive DRG neurons that utilize Sp1-like factors for TRPV1 expression. Given the therapeutic correlate of mithramycin-A effectiveness in the treatment of certain cancers, small molecule transcriptional inhibitors such as mithramycin-A may serve as useful tools of discovery in pain transduction and possibly future analgesic development.

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.

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.

Structural analysis of the purine repressor, an Escherichia coli DNA-binding protein.

The purine repressor protein, PurR, is a member of the lac repressor, LacI, family of Escherichia coli DNA-binding proteins that bind DNA via a highly conserved N-terminal helix-turn-helix motif. Additionally, the members of this family display strong sequence homologies between their larger C-terminal effector binding/oligomerization domains. Analysis of the PurR primary and secondary structures reveals that its C-terminal corepressor binding domain is highly homologous to another group of E. coli-binding proteins, the periplasmic binding proteins, particularly to the ribose-binding protein (RBP). The high resolution x-ray structure of RBP allows this protein to serve as a template with which to model the predicted secondary structure of the corepressor binding domain of PurR. Similarly, the N-terminal DNA binding domain of PurR can be modeled using the NMR-determined structure of the corresponding region (residues 1-56) from LacI as a template. Combining the two, results in a description of the likely secondary structure topology of PurR and implicates residues important for corepressor binding and dimerization. CD spectroscopic studies on PurR, its corepressor binding domain and RBP result in secondary structure estimates nearly identical with those obtained by sequence analyses, thereby providing further corroborating physical evidence for this topological assignment.

Allosteric transition intermediates modelled by crosslinked haemoglobins.

The structural end-points of haemoglobin's transition from its low-oxygen-affinity (T) to high-oxygen-affinity (R) state, have been well established by X-ray crystallography, but short-lived intermediates have proved less amenable to X-ray studies. Here we use chemical crosslinking to fix these intermediates for structural characterization. We describe the X-ray structures of three haemoglobins, alpha 2 beta 1S82 beta, alpha 2 beta 1Tm82 beta and alpha 2 beta 1,82Tm82 beta, which were crosslinked between the amino groups of residues beta Val1 and beta Lys82 by 3,3'-stilbenedicarboxylic acid (S) or trimesic acid (Tm) while in the deoxy state, and saturated with carbon monoxide before crystallization. alpha 2 beta 1S82 beta, which has almost normal oxygen affinity, is completely in the R-state conformation; however, alpha 2 beta 1Tm82 beta and alpha 2 beta 1,82Tm82 beta, both of which have low oxygen affinity, have been prevented from completing their transition into the R state and display many features of a transitional intermediate. These haemoglobins therefore represent a snapshot of the nascent R state.

Mechanism of corepressor-mediated specific DNA binding by the purine repressor.

The modulation of the affinity of DNA-binding proteins by small molecule effectors for cognate DNA sites is common to both prokaryotes and eukaryotes. However, the mechanisms by which effector binding to one domain affects DNA binding by a distal domain are poorly understood structurally. In initial studies to provide insight into the mechanism of effector-modulated DNA binding of the lactose repressor family, we determined the crystal structure of the purine repressor bound to a corepressor and purF operator. To extend our understanding, we have determined the structure of the corepressor-free corepressor-binding domain of the purine repressor at 2.2 A resolution. In the unliganded state, structural changes in the corepressor-binding pocket cause each subunit to rotate open by as much as 23 degrees, the consequences of which are the disengagement of the minor groove-binding hinge helices and repressor-DNA dissociation.

The X-ray structure of the PurR-guanine-purF operator complex reveals the contributions of complementary electrostatic surfaces and a water-mediated hydrogen bond to corepressor specificity and binding affinity.

The purine repressor, PurR, is the master regulatory protein of de novo purine nucleotide biosynthesis in Escherichia coli. This dimeric transcription factor is activated to bind to cognate DNA operator sites by initially binding either of its physiologically relevant, high affinity corepressors, hypoxanthine (Kd = 9.3 microM) or guanine (Kd = 1.5 microM). Here, we report the 2.5-A crystal structure of the PurR-guanine-purF operator ternary complex and complete the atomic description of 6-oxopurine-induced repression by PurR. As anticipated, the structure of the PurR-guanine-purF operator complex is isomorphous to the PurR-hypoxanthine-purF operator complex, and their protein-DNA and protein-corepressor interactions are nearly identical. The former finding confirms the use of an identical allosteric DNA-binding mechanism whereby corepressor binding 40 A from the DNA-binding domain juxtaposes the hinge regions of each monomer, thus favoring the formation and insertion of the critical minor groove-binding hinge helices. Strikingly, the higher binding affinity of guanine for PurR and the ability of PurR to discriminate against 2-oxopurines do not result from direct protein-ligand interactions, but rather from a water-mediated contact with the exocyclic N-2 of guanine, which dictates the presence of a donor group on the corepressor, and the better electrostatic complementarity of the guanine base and the corepressor-binding pocket.

Crystal structures of Toxoplasma gondii HGXPRTase reveal the catalytic role of a long flexible loop.

Crystal structures of substrate-free and XMP-soaked hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) of the opportunistic pathogen Toxoplasma gondii have been determined to 2.4 and 2.9 A resolution, respectively. HGXPRTase displays the conserved PRTase fold. In the structure of the enzyme bound to its product, a long flexible loop (residues 115-126) is located away from the active site. Comparison to the substrate-free structure reveals a striking relocation of the loop, which is poised to cover the catalytic pocket, thus providing a mechanism by which the HG(X)PRTases shield their oxocarbonium transition states from nucleophilic attack by the bulk solvent. The conserved Ser 117-Tyr 118 dipeptide within the loop is brought to the active site, completing the ensemble of catalytic residues.