A genetic and physical study of the interdomain linker of E. Coli AraC protein--a trans-subunit communication pathway.

Genetic experiments with full length AraC and biophysical experiments with its dimerization domain plus linker suggest that arabinose binding to the dimerization domain changes the properties of the inter-domain linker which connects the dimerization domain to the DNA binding domain via interactions that do not depend on the DNA binding domain. Normal AraC function was found to tolerate considerable linker sequence alteration excepting proline substitutions. The proline substitutions partially activate transcription even in the absence of arabinose and hint that a structural shift between helix and coil may be involved. To permit fluorescence anisotropy measurements that could detect arabinose-dependent dynamic differences in the linkers, IAEDANS was conjugated to a cysteine residue substituted at the end of the linker of dimerization domain. Arabinose, but not other sugars, decreased the steady-state anisotropy, indicating either an increase in mobility and/or an increase in the fluorescence lifetime of the IAEDANS. Time-resolved fluorescence measurements showed that the arabinose-induced anisotropy decrease did not result from an increase in the excited-state lifetime. Hence arabinose-induced decreases in anisotropy appear to result from increased tumbling of the fluorophore. Arabinose did not decrease the anisotropy in mutants incapable of binding arabinose nor did it alter the anisotropy when IAEDANS was conjugated elsewhere in the dimerization domain. Experiments with heterodimers of the dimerization domain showed that the binding of arabinose to one subunit of the dimer decreases the fluorescence anisotropy of only a fluorophore on the linker of the other subunit.

Heterodimers reveal that two arabinose molecules are required for the normal arabinose response of AraC.

AraC protein, which regulates expression of the l-arabinose operon in Escherichia coli, is a dimer whose DNA binding affinity for pairs of DNA half-sites is controlled by arabinose. Here we have addressed the question of whether the arabinose response of AraC requires the binding of one or two molecules of arabinose. This was accomplished by measuring the DNA dissociation rates of wild-type AraC and heterodimeric AraC constructs in which one subunit is capable of binding arabinose and the other subunit does not bind arabinose. Solutions consisting entirely of heterodimers were formed by spontaneous subunit exchange between two different homodimers, with heterodimers being trapped by the formation of an intersubunit disulfide bond between cysteine residues strategically positioned within the dimerization interface. We found that the normal arabinose response of AraC requires the binding of two arabinose molecules. These results provide additional constraints on mechanistic models for the action of AraC.

Solution structure of the DNA binding domain of AraC protein.

We report the solution structure of the DNA binding domain of the Escherichia coli regulatory protein AraC determined in the absence of DNA. The 20 lowest energy structures, determined on the basis of 1507 unambiguous nuclear Overhauser restraints and 180 angle restraints, are well resolved with a pair wise backbone root mean square deviation of 0.7 A. The protein, free of DNA, is well folded in solution and contains seven helices arranged in two semi-independent sub domains, each containing one helix-turn-helix DNA binding motif, joined by a 19 residue central helix. This solution structure is discussed in the context of extensive biochemical and physiological data on AraC and with respect to the DNA-bound structures of the MarA and Rob homologs.

Opposite allosteric mechanisms in TetR and CAP.

Regulation of the DNA binding affinity of an oligomeric protein can be considered to consist of an intrinsic component, in which the affinity of an individual DNA-binding domain is modulated in response to effector binding, and an extrinsic component, in which the relative position of the protein's two DNA-binding domains are altered so that they can or cannot contact both half-site operators simultaneously. We demonstrated directly that the TetR repressor utilizes an extrinsic mechanism and CAP, the catabolite activator protein, utilizes an intrinsic mechanism.

Functional modes of the regulatory arm of AraC.

One of the two crystal structures of the arm-dimerization domain determined in the absence of arbinose fails to show the arm, whereas the other structure does show it. The two structures lead to different pictures for the regulatory behavior of the arms. Trypsin digestion, fluorescence anisotropy, and NMR experiments presented here were designed to resolve the issue and show that in arm-dimerization domain, the arms are structured, although differently, in the presence and absence of arabinose. The arms have also been shown to interact with the DNA binding domains of AraC by their requirement for the immobilization of the DNA binding domains that is necessary for DNA looping and repression. The binding of arabinose has been shown to release the DNA binding domains and looping ceases. The picture resulting from the new experiments and the crystal structures of the arm-dimerization domain is that in the absence of arabinose, the arm adopts one structure on the dimerization domain and that the DNA binding domain then binds to this complex. Upon binding arabinose, the arm restructures and as a result, no longer serves as a gasket between the DNA binding domain and dimerization domain. The DNA binding domain is then released, subject only to the constraints imposed by the flexible linker connecting it to dimerization domain, and the protein relocates on the DNA and activates transcription.

DNA tape measurements of AraC.

A new method for measuring distances between points in the AraC-DNA complex was developed and applied. It utilizes variable lengths of single-stranded DNA that connect double-stranded regions containing the two half-site binding sequences of AraC. These distances plus the protein interdomain linker distances are compatible with two classes of structure for the dimeric AraC gene regulatory protein. In one class, the N-terminal regulatory arm of one dimerization domain is capable of interacting with the DNA-binding domain on the same polypeptide chain for a cis interaction. In the other class, the possible arm-DNA-binding domain interaction is trans, where it adds to the dimerization interface.

Structure and properties of a truely apo form of AraC dimerization domain.

The arabinose-binding pockets of wild type AraC dimerization domains crystallized in the absence of arabinose are occupied with the side chains of Y31 from neighboring domains. This interaction leads to aggregation at high solution concentrations and prevents determination of the structure of truely apo AraC. In this work we found that the aggregation does not significantly occur at physiological concentrations of AraC. We also found that the Y31V mutation eliminates the self-association, but does not affect regulation properties of the protein. At the same time, the mutation allows crystallization of the dimerization domain of the protein with only solvent in the arabinose-binding pocket. Using a distance difference method suitable for detecting and displaying even minor structural variation among large groups of similar structures, we find that there is no significant structural change in the core of monomers of the AraC dimerization domain resulting from arabinose, fucose, or tyrosine occupancy of the ligand-binding pocket. A slight change is observed in the relative orientation of monomers in the dimeric form of the domain upon the binding of arabinose but its significance cannot yet be assessed.

Subdomain organization and catalytic residues of the F factor TraI relaxase domain.

TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.

Flavin thermodynamics explain the oxygen insensitivity of enteric nitroreductases.

Bacterial nitroreductases are NAD(P)H-dependent flavoenzymes which catalyze the oxygen-insensitive reduction of nitroaromatics, quinones, and riboflavin derivatives. Despite their broad substrate specificity, their reactivity is very specific for two-electron, not one-electron, chemistry. We now describe the thermodynamic properties of the flavin mononucleotide cofactor of Enterobacter cloacae nitroreductase (NR), determined under a variety of solution conditions. The two-electron redox midpoint potential of NR is -190 mV at pH 7.0, and both the pH dependence of the midpoint potential and the optical spectrum of the reduced enzyme indicate that the transition is from neutral oxidized flavin to anionic flavin hydroquinone. The one-electron-reduced semiquinone states of both the free enzyme and an NR-substrate analogue complex are strongly suppressed based on optical spectroscopy and electron paramagnetic resonance measurements. This can explain the oxygen insensitivity of NR and its homologues, as it makes the execution of one-electron chemistry thermodynamically unfavorable. Therefore, we have established a chemical basis for the recent finding that a nitroreductase is a member of the soxRS oxidative defense regulon in Escherichia coli [Liochev, S. I., Hausladen, A., Fridovich, I. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (7), 3537-3539]. We also report binding affinities for the FMN cofactor in all three oxidation states either determined fluorometrically or calculated using thermodynamic cycles. Thus, we provide a detailed picture of the thermodynamics underlying the unusual activity of NR.

TraY DNA recognition of its two F factor binding sites.

F factor TraY, a ribbon-helix-helix DNA-binding protein, performs two roles in bacterial conjugation. TraY binds the F origin of transfer (oriT) to promote nicking of plasmid DNA prior to conjugative transfer. TraY also binds the P(Y) promoter to up-regulate tra gene expression. The two plasmid regions bound by TraY share limited sequence identity, yet TraY binds them with similar affinities. TraY recognition of the two sites was first probed using in vitro footprinting methods. Hydroxyl radical footprinting at both oriT and P(Y) sites indicated that bound TraY protected the DNA backbone bordering three adjacent DNA subsites. Analytical ultracentrifugation results for TraY:oligonucleotide complexes were consistent with two of these subsites being bound cooperatively, and the third being occupied at higher TraY concentrations. Methylation protection and interference footprinting identified several guanine bases contacted by or proximal to bound TraY, most located within these subsites. TraY affinity for variant oriT sequences with base substitutions at or near these guanine bases suggested that two of the three subsites correspond to high-affinity, cooperatively bound imperfect inverted GA(G/T)A repeats. Altering the spacing or orientation of these sites reduced binding. TraY mutant R73A failed to protect two symmetry-related oriT guanine bases in these repeats from methylation, identifying possible direct TraY-DNA contacts. The third subsite appears to be oriented as an imperfect direct repeat with its adjacent subsite, although base substitutions at this subsite did not reduce binding. Although unusual for ribbon-helix-helix proteins, this binding site arrangement occurs at both F TraY sites, consistent with it being functionally relevant.

Subunit organization in cytoplasmic dynein subcomplexes.

Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain-binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo.