Conformational prediction and circular dichroism studies on the lac repressor

Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.     

Orientation and oligomerization specificity of the Bcr coiled-coil oligomerization domain

The Bcr oligomerization domain, from the Bcr-Abl oncoprotein, is an attractive therapeutic target for treating leukemias because it is required for cellular transformation. The domain homodimerizes via an antiparallel coiled coil with an adjacent short, helical swap domain. Inspection of the coiled-coil sequence does not reveal obvious determinants of helix-orientation specificity, raising the possibility that the antiparallel orientation preference and/or the dimeric oligomerization state are due to interactions of the swap domains. To better understand how structural specificity is encoded in Bcr, coiled-coil constructs containing either an N- or C-terminal cysteine were synthesized without the swap domain. When cross-linked to adopt exclusively parallel or antiparallel orientations, these showed similar circular dichroism spectra. Both constructs formed coiled-coil dimers, but the antiparallel construct was approximately 16 degrees C more stable than the parallel to thermal denaturation. Equilibrium disulfide-exchange studies confirmed that the isolated coiled-coil homodimer shows a very strong preference for the antiparallel orientation. We conclude that the orientation and oligomerization preferences of Bcr are not caused by the presence of the swap domains, but rather are directly encoded in the coiled-coil sequence. We further explored possible determinants of structural specificity by mutating residues in the d position of the coiled-coil core. Some of the mutations caused a change in orientation specificity, and all of the mutations led to the formation of higher-order oligomers. In the absence of the swap domain, these residues play an important role in disfavoring alternate states and are especially important for encoding dimeric oligomerization specificity.     

Genetic studies of the lac repressor. XII. Amino acid replacements in the DNA binding domain of the Escherichia coli lac repressor

Out of the first 62 residues of the lac repressor, 38 positions have been substituted by at least one amino acid exchange. The total number of replacements in this region is 131. Data from several studies are considered.     

The lac repressor

Few proteins have had such a strong impact on a field as the lac repressor has had in Molecular Biology. Over 40 years ago, Jacob and Monod [Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3 (1961) 318] proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. It is a cogent depiction of how a set of 'structural' genes may be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. In bacteria, the genes required for lactose utilization are negatively regulated when a repressor molecule binds to an upstream cis activated operator. The repressor and its operator together form a genetic switch, the lac operon. The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite, the repressor undergoes a conformational change that reduces its affinity for the operator. The structures of the lac repressor and its complexes with operator DNA and effector molecules have provided a physical platform for visualizing at the molecular level the different conformations the repressor and the molecular basis for the switch. The structures of lac repressor, bound to its operator and inducer, have also been invaluable for interpreting a plethora of biochemical and genetic data.     

The tetramerization domain of the Mnt repressor consists of two right-handed coiled coils.

The tetrameric Mnt repressor is involved in the genetic switch between the lysogenic and lytic growth of Salmonella bacteriophage P22. The solution structure of its C-terminal tetramerization domain, which holds together the two dimeric DNA-binding domains, has been determined by NMR spectroscopy. This structure reveals an assembly of four alpha-helical subunits, consisting of a dimer of two antiparallel coiled coils with a unique right-handed twist. The superhelical winding is considerably stronger and the interhelical separation closer than those found in the well-known left-handed coiled coils in fibrous proteins and leucine zippers. An unusual asymmetry arises between the two monomers that comprise one right-handed coiled coil. A difference in the packing to the adjacent monomer of the other coiled coil occurs with an offset of two helical turns. The two asymmetric monomers within each coiled coil interconvert on a time scale of seconds. Both with respect to symmetry and handedness of helical packing, the C2 symmetric four-helix bundle of Mnt differs from other oligomerization domains that assemble DNA-binding modules, such as that in the tumor suppressor p53 and the E. coli lac repressor.     

Effects of dominant-negative lac repressor mutations on operator specificity and protein stability

The specific binding of dominant-negative (I-d) lactose (lac) repressors to wild-type (wt) as well as mutant (Oc) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target. Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322. Twelve of these lacI-d missense mutations were transferred from F'lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13-lacI phages [Mott et al., Nucl. Acids Res. 12 (1984) 4139-4152]. The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-beta-D-thiogalactoside (IPTG), as well as binding to wt operator. The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against repressor expressed from a chromosomal lacIq gene. Six of the I-d repressors were partially degraded in vivo. All repressors bound IPTG with approximately the affinity of wt repressor. Repressors having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17-25) were examined in vivo for their affinities for a series of single site Oc operators. Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator [Ebright, Proc. Natl. Acad. Sci. USA 83 (1986) 303-307], the His-18 repressor did not affect specificity. This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions.     

Local mobility of the lac repressor molecule

The rotational mobility of lac repressor from Escherichia coli was investigated by nanosecond fluorescence depolarization spectroscopy. A single rotational correlation time (φ) of the repressor was observed by monitoring the emission anisotropic decay of the intrinsic tryptophan fluorescence. The small value of φ (9·5 ns) suggests that one or both of the two tryptophan residues in the repressor are located in a flexible segment of the protein molecule. This segmental flexibility is enhanced by binding of inducer (isopropyl-β-d-thiogalactoside) to the repressor while it is restrained by binding of anti-inducer (glucose) or small DNA fragments, as indicated by the changes in φ. Further time-dependent emission anisotropy studies with an extrinsic fluorescent probe, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonate, covalently attached to the repressor yielded two rotational correlation times. The shorter φ_S (6·7 ns) also corresponds to a segmental flexibility whereas the longer φ_L (118 ns) represents the rotational motion of the entire repressor molecule. Both the values of φ_S and φ_L vary by addition of inducer or anti-inducer in a manner similar to that observed for the intrinsic tryptophan fluorescence but they are insensitive to addition of DNA fragments. The changes in local mobility of the lac repressor molecule observed in these studies may provide some insight into how inducer (or anti-inducer) destabilizes (or stabilizes) the repressor-operator complex.     

A model for the binding of lac repressor to the lac operator

A model is suggested for the lac repressor binding to the lac operator in which the repressor polypeptide chain sequences from Gly 14 to Ala 32 and from Ala 53 to Leu 71 are involved in specific interaction with operator DNA. A correspondence between the protein and DNA sequences is found which explains specificity of the repressor binding to the lac operator. The model can be extended to describe specific binding of other regulatory proteins to DNA.     

NMR study of the interaction between the lac repressor and the lac operator

Binding of the lac repressor headpiece, the N-terminal region of the lac repressor, to the lac operator of Escherichia coli was studied by 1H-NMR spectroscopy. Two DNA fragments, of 51 base pairs and 62 base pairs, containing the lac operator region, were investigated. The signals of their hydrogen-bonded imino protons were well resolved in the 500-MHz NMR spectra. The spectra of the free lac operator DNA are similar to those obtained from ring-current-shift calculations for a B-DNA structure. Complex formation with the headpiece led to small but nevertheless characteristic changes in the spectra. The fact that very few imino resonances shifted upon addition of headpiece, as well as the variety in direction and size of these chemical shifts, indicate the formation of a specific complex between the lac repressor and the lac operator. The observed changes in the resonance positions exclude the intercalation of tyrosine residues of the headpiece between adjacent base pairs of the lac operator as well as the formation of a cruciform structure. They rather reflect a small conformational transition in the DNA itself, caused for example by an alteration in the tilt of a few base pairs or a shift of the keto-enol tautomeric equilibrium of the bases towards the enolic form.     

Site-specific DNA-affinity chromatography of the lac repressor.

To test the feasibility of site-specific DNA-affinity chromatography, E. coli lac repressor was bound to an operator-containing DNA column, and in parallel to a non-operator DNA column. Salt gradient elution shows: 1) elution from non-operator DNA was near 250mM KCl or NaCl; interpretation of this result suggests the usefulness of the procedure for studying salt-dependence of DNA-protein affinities; 2) elution from operator-containing DNA was delayed (average elution = 1000mM salt), demonstrating a feasibility of site-specific DNA-affinity chromatography, if one provides a sufficiently favorable ratio of specific to non-specific DNA binding sites; 3) repressor eluted from operator-containing DNA over a very broad salt range, which may represent chromatography-generated repressor heterogeneity.     

The interaction of the recognition helix of lac repressor with lac operator.

We have constructed a system which allows systematic testing of repressor--operator interactions. The system consists of two plasmids. One of them carries a lac operon in which lac operator has been replaced by a unique restriction site into which synthetic operators can be cloned. The other plasmid carries the gene coding for the repressor, in our case a semisynthetic lacI gene of which parts can be exchanged in a cassette-like manner. A galE host allows us to select for mutants which express repressors with altered specificities. Here we report the change of specificity in the lac system by changing residues 1 and 2 of the recognition helix of lac repressor. The specificity changes are brought about cooperatively by the change of both residues. Exchanges of just one residue broaden the specificity. Our results hint that the recognition helix of lac repressor may possibly have the opposite orientation to those in Lambda cro protein or 434 CI repressor.