Improved model of a LexA repressor dimer bound to recA operator

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Delta(1-67) S119A, S119A, G85D and Delta(1-67) quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the 'cleavable' cleavage site region found in the crystal structures is actually the more relevant form of the region in wild-type LexA since it agrees with the value of the pre-exponential Arrhenius factor for its autocleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a 'conformational switch' inferred from the crystal structures is meaningless.     

Improved Model of a LexA Repressor Dimer Bound to recA Operator

Abstract

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Δ_1–67 S119A, S119A, G85D and Δ_1-67 quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the ‘cleavable’ cleavage site region found in the crystal structures is actually the more relevant form of the region in wildtype LexA since it agrees with the value of the pre-exponential Arrhenius factor for its auto- cleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a ‘conformational switch’ inferred from the crystal structures is meaningless.     

Model of a LexA repressor dimer bound to recA operator

A complete three dimensional model (RCSB000408; PDB code 1qaa) for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor is proposed. A model of interaction of the N-terminal operator binding domain 1-72 with the operator was available. We have modelled residues 106-202 of LexA on the basis of the crystal structure of a homologous protein, UmuD'. Residues 70-105 have been modelled by us, residues 70-77 comprising the real hinge, followed by a beta-strand and an alpha-helix, both interacting with the rest of the C-domain. The preexponential Arrhenius factor for the LexA autocleavage is shown to be approximately 10(9) s(-1) at 298K whereas the exponential factor is approximately 2 x 10(-12), demanding that the autocleavage site is quite close to the catalytic site but reaction is slow due to an activation energy barrier. We propose that in the operator bound form, Ala 84- Gly 85 is about 7-10A from the catalytic groups, but the reaction does not occur as the geometry is not suitable for a nucleophilic attack from Ser 119 Ogamma, since Pro 87 is held in the cis conformation. When pH is elevated or under the action of activated RecA, cleavage may occur following a cis --> trans isomerization at Pro 87 and/or a rotation of the region beta9-beta10 about beta7-beta8 following the disruption of two hydrogen bonds. We show that the C-C interaction comprises the approach of two negatively charged surfaces neutralized by sodium ions, the C-domains of the monomers making a new beta barrel at the interface burying 710A2 of total surface area of each monomer.     

Quaternary structure and function in phage lambda repressor: 1H-NMR studies of genetically altered proteins

The quaternary structure and dynamics of phage lambda repressor are investigated in solution by 1H-NMR methods. lambda repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. 1H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the "linker" polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84----Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.     

Large-scale purification, oligomerization equilibria, and specific interaction of the LexA repressor of Escherichia coli

A rapid large-scale procedure for the purification of the LexA repressor of Escherichia coli is described. This procedure allows one to get more than 100 mg of purified protein from 100 g of bacterial paste with a purity of at least 97%. This method is comparable to earlier, far more complicated purification procedures giving clearly smaller yields. It is shown that the LexA protein may be identified spectroscopically by a large A235/A280 ratio and very pronounced ripples in the absorption spectrum arising from a high amount of phenylalanine residues with respect to that of the other aromatic amino acids. Polyacrylamide gel electrophoresis has been used to study the specific interaction of LexA with a recA operator fragment. The quaternary structure of LexA has been studied by equilibrium ultracentrifugation and sedimentation velocity measurements. The sedimentation coefficient increases with increasing LexA concentration, indicating that LexA is involved in self-association. This finding has been confirmed by equilibrium ultracentrifugation. The results are best described by a monomer-dimer and a subsequent dimer-tetramer equilibrium, with an association constant of 2.1 X 10(4) M-1 for the dimer and 7.7 X 10(4) M-1 for the tetramer formation. These relatively small association constants determined under near-physiological pH and salt conditions suggest that in vivo LexA should be essentially in the monomeric state. The degree to which LexA decreases the electrophoretic mobility of a 175 base pair fragment harboring the recA operator suggests that the recA operator interacts nevertheless with a LexA dimer. However, our results may be also explained by the binding of a LexA monomer with a simultaneous bending of the DNA fragment.     

Digestion of the lambda cI repressor with various serine proteases and correlation with its three dimensional structure

Partial proteolysis of the lambda cI repressor has been carried out systematically with trypsin, chymotrypsin, elastase, endoproteinase Glu-C, kallikrein, and thrombin. The cleavage sites have been determined by (i) comparison of fragments produced and observed in SDS-polyacrylamide gel with known fragments and plots of distance migrated versus log (molecular weight of fragment), (ii) partial Edman sequencing of the stable C-terminal fragments to identify cleavage points, and (iii) electrospray mass spectrometry of fragments produced. Most cleavage points are found to occur in the region 86-137, saving some in the N-terminal domain observed for trypsin and Glu-C. Region 86-137 can be further subdivided into three regions 86-91, 114-121, and 128-137 prone to cleavage, with intermediate regions resistant to cleavage to all six proteases. These resistant regions show that much of the region 93-131 previously called a 'linker' is actually part of the C-domain as first proposed in all models from our laboratory. Region 92-114 includes the cleavage site Ala-Gly, which must be buried in the intact repressor. The observed cleavage points in region 114-137 can be used to judge the best among three previously proposed models since they differ from each other in the structure of region 93-131. Model 1j5g is adjudged to be better than model 1lwq (which is based on 1kca, a crystal structure) as susceptible residues are more exposed in the former and lack of cleavages at six sites is better explained. Likewise, the models 1j5g and 1lwq are compared with a recent crystal structure of fragment 101-229 in 2ho0 and another low resolution crystal structure in 3bdn.     

A model for the LexA repressor DNA complex

A structural model for the interaction of the LexA repressor DNA binding domain (DBD) with operator DNA is derived by means of Monte Carlo docking. Protein–DNA complexes were generated by docking the LexA repressor DBD NMR solution structure onto both rigid and bent B-DNA structures while giving energy bonuses for contacts in agreement with experimental data. In the resulting complexes, helix III of the LexA repressor DBD is located in the major groove of the DNA and residues Asn-41, Glu-44, and Glu-45 form specific hydrogen bonds with bases of the CTGT DNA sequence. Ser-39, Ala-42, and Asn-41 are involved in a hydrophobic interaction with the methyl group of the first thymine base. Residues in the loop region connecting the two β-sheet strands are involved in nonspecific contacts near the dyad axis of the operator. The contacts observed in the docked complexes cover the entire consensus CTGT half-site DNA operator, thus explaining the specificity of the LexA repressor for such sequences. In addition, a large number of nonspecific interactions between protein and DNA is observed. The agreement between the derived model for the LexA repressor DBD/DNA complex and experimental biochemical results is discussed. © 1995 Wiley-Liss, Inc.     

Tetramerization of the LexA repressor in solution: implications for gene regulation of the E.coli SOS system at acidic pH

Structural changes on LexA repressor promoted by acidic pH have been investigated. Intense protein aggregation occurred around pH 4.0 but was not detected at pH values lower than pH 3.5. The center of spectral mass of the Trp increased 400 cm(-1) at pH 2.5 relatively to pH 7.2, an indication that LexA has undergone structural reorganization but not denaturation. The Trp fluorescence polarization of LexA at pH 2.5 indicated that its hydrodynamic volume was larger than its dimer at pH 7.2. 4,4'-Dianilino-1,1'-binaphthyl-5,5'- disulfonic acid (bis-ANS) experiments suggested that the residues in the hydrophobic clefts already present at the LexA structure at neutral pH had higher affinity to it at pH 2.5. A 100 kDa band corresponding to a tetramer was obtained when LexA was subject to pore-limiting native polyacrylamide gel electrophoresis at this pH. The existence of this tetrameric state was also confirmed by small angle X-ray scattering (SAXS) analysis at pH 2.5. 1D 1H NMR experiments suggested that it was composed of a mixture of folded and unfolded regions. Although 14,000-fold less stable than the dimeric LexA, it showed a tetramer-monomer dissociation at pH 2.5 from the hydrostatic pressure and urea curves. Albeit with half of the affinity obtained at pH 7.2 (Kaff of 170 nM), tetrameric LexA remained capable of binding recA operator sequence at pH 2.5. Moreover, different from the absence of binding to the negative control polyGC at neutral pH, LexA bound to this sequence with a Kaff value of 1415 nM at pH 2.5. A binding stoichiometry experiment at both pH 7.2 and pH 2.5 showed a [monomeric LexA]/[recA operator] ratio of 2:1. These results are discussed in relation to the activation of the Escherichia coli SOS regulon in response to environmental conditions resulting in acidic intracellular pH. Furthermore, oligomerization of LexA is proposed to be a possible regulation mechanism of this regulon.     

Crystal Structures of a Populus tomentosa 4-Coumarate:CoA Ligase Shed Light on Its Enzymatic Mechanisms

4-Coumaric acid:CoA ligase (4CL) is the central enzyme of the plant-specific phenylpropanoid pathway. It catalyzes the synthesis of hydroxycinnamate-CoA thioesters, the precursors of lignin and other important phenylpropanoids, in two-step reactions involving the formation of hydroxycinnamate-AMP anhydride and then the nucleophilic substitution of AMP by CoA. In this study, we determined the crystal structures of Populus tomentosa 4CL1 in the unmodified (apo) form and in forms complexed with AMP and adenosine 5′-(3-(4-hydroxyphenyl)propyl)phosphate (APP), an intermediate analog, at 2.4, 2.5, and 1.9 Å resolution, respectively. 4CL1 consists of two globular domains connected by a flexible linker region. The larger N-domain contains a substrate binding pocket, while the C-domain contains catalytic residues. Upon binding of APP, the C-domain rotates 81° relative to the N-domain. The crystal structure of 4CL1-APP reveals its substrate binding pocket. We identified residues essential for catalytic activities (Lys-438, Gln-443, and Lys-523) and substrate binding (Tyr-236, Gly-306, Gly-331, Pro-337, and Val-338) based on their crystal structures and by means of mutagenesis and enzymatic activity studies. We also demonstrated that the size of the binding pocket is the most important factor in determining the substrate specificities of 4CL1. These findings shed light on the enzymatic mechanisms of 4CLs and provide a solid foundation for the bioengineering of these enzymes.     

Papain does not cleave operator-bound lambda repressor: structural characterization of the carboxy terminal domain and the hinge

The circular dichroism spectra of three different purified carboxy terminal fragments 93-236, 112-236 and 132-236 of the bacteriophage lambda cI repressor have been measured and compared with those of the intact repressor and the amino terminal fragment 1-92. All three carboxy terminal fragments contain mostly beta-strands and loops, a minor helix content increasing with the size of the fragment, showing that the 93-131 region previously called a hinge is structured. Fourier transformed infrared spectra also showed that fragment 93-236 contains alpha-helices, alpha-sheets and turns but fragment 132-236 contains no detectable alpha-helix, only beta-sheets and turns. Papain is known to cleave the lambda repressor, but it is shown here that it cannot cleave the operator-bound repressor dimer. For the 132-236 fragment, both the wt and the SN228 mutant previously shown to be dimerization defective in the intact, gave similar dimerization properties as investigated by HPLC at 2 to 100 microM protein concentration, with a KD of 13.2 microM and 19.1 microM respectively. The papain cleavage for wt and SN228 proceed at equal rates for the first cleavage at 92-93; however, the subsequent cleavages are faster for SN228. The three Cys residues in the 132-236 fragment were found to be unreactive upon incubation with DTNB, indicating the thiol sulfur atoms are buried in the repressor carboxy terminal domain. Denaturation of the 132-236 fragment studied by tryptophan fluorescence shows two transitions centered at 1.5 M and 4.5 M of urea.     

Quarternary Structure and Function in Phage λ Repressor: 1H-NMR Studies of Genetically Altered Proteins

Abstract

The quaternary structure and dynamics of phage λ repressor are investigated in solution by ¹H-NMR methods. λ repressor contains two domains separable by proteolysis: an N-terminal domain that mediates sequence-specific DNA-A binding, and a C-terminal domain that contains strong dimer and higher-order contacts. The active species in operator recognition is a dimer. Although the crystal structure of an N-terminal fragment has been determined, the intact protein has not been crystallized, and there is little evidence concerning its structure. ¹H-NMR data indicate that the N-terminal domain is only loosely tethered to the C-terminal domain, and that its tertiary structure is unperturbed by proteolysis of the “linker” polypeptide. It is further shown that in the intact repressor structure a quaternary interaction occurs between N-terminal domains. This domain-domain interaction is similar to the dimer contact observed in the crystal structure of the N-terminal fragment and involves the hydrophobic packing of symmetry-related helices (helix 5). In the intact structure this interaction is disrupted by the single amino-acid substitution, Ile84→Ser, which reduces operator affinity at least 100-fold. We conclude that quaternary interactions between N-terminal domains function to appropriately orient the DNA-binding surface with respect to successive major grooves of B-DNA.     

Repression of the E coli recA gene requires at least two LexA protein monomers

To analyze the DNA binding domain of E coli LexA repressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated. A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis. Mutants defective in DNA binding were identified and then examined for dominance to lexA+. A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype. Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator. The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator. A mixture of mutant LexA408 and LexA+ proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites. These results suggest that at least 1 LexA protein monomer interacts with each operator half site. We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.     

Structure and characterization of RNase H3 from Aquifex aeolicus

The crystal structure of ribonuclease?H3 from Aquifex?aeolicus (Aae-RNase?H3) was determined at 2.0?? resolution. Aae-RNase?H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase?H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus?stearothermophilus RNase?H3 (Bst-RNase?H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase?H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase?H3 is relatively long and flexible, while that of Aae-RNase?H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase?H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase?H3 is important for substrate binding, and uses the flat surface of the β-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase?H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.     

Crystal structure of the lambda repressor C-terminal domain octamer.

The three-dimensional structure of the lambda repressor C-terminal domain (CTD) has been determined at atomic resolution. In the crystal, the CTD forms a 2-fold symmetric tetramer that mediates cooperative binding of two repressor dimers to pairs of operator sites. Based upon this structure, a model was proposed for the structure of an octameric repressor that forms both in the presence and absence of DNA. Here, we have determined the structure of the lambda repressor CTD in three new crystal forms, under a wide variety of conditions. All crystals have essentially the same tetramer, confirming the results of the earlier study. One crystal form has two tetramers bound to form an octamer, which has the same overall architecture as the previously proposed model. An unexpected feature of the octamer in the crystal structure is a unique interaction at the tetramer-tetramer interface, formed by residues Gln209, Tyr210 and Pro211, which contact symmetry-equivalent residues from other subunits of the octamer. Interestingly, these residues are also located at the dimer-dimer interface, where the specific interactions are different. The structures thus indicate specific amino acid residues that, at least in principle, when altered could result in repressors that form tetramers but not octamers.     

Role of protein--protein interactions in the regulation of transcription by trp repressor investigated by fluorescence spectroscopy.

In the present work, we have characterized the protein--protein interactions in the trp repressor (TR) from Escherichia coli using fluorescence spectroscopy. The steady-state and time-resolved fluorescence anisotropy of repressor labeled with 5-(dimethylamino)naphthalene-1-sulfonamide (DNS) was used to monitor subunit equilibria in the absence and presence of corepressor. In the absence of tryptophan, the repressor is in equilibrium between tetramers and dimers in the concentration range studied (approximately 0.04-40 microM in dimer). Binding of corepressor resulted in a marked destabilization of the tetramer. The beginning of a dimer-monomer dissociation transition was observed by monitoring the decrease in the intrinsic tryptophan emission energy upon dilution below 0.1 microM in dimer, indicating an upper limit for the dimer-dissociation constant near 1 nM. DNA titrations with a 26 base pair sequence containing the trp EDCBA operator performed in the absence and presence of the corepressor are consistent with a 1:1 dimer/operator stoichiometry in the presence of tryptophan, while the aporepressor binds with TR dimer/DNA stoichiometries greater than one and which depend upon both the concentration of protein and that of the operator. Using the multiple observable parameters available in fluorescence, we have thus carried out a thorough investigation of the coupled equilibria in this bacterial repressor. Our results are consistent with a physiologically relevant thermodynamic role for tetramerization in the regulatory function of the trp repressor. The present results which have brought to light novel protein--protein interactions in the trp repressor system indicate that fluorescence spectroscopic methods could prove quite useful in the study of the role of protein--protein interactions in eukaryotic systems as well.     

LexA repressor induces operator-dependent DNA bending

LexA, the repressor of the SOS system in Escherichia coli induces a substantial DNA bending upon interaction with the operator of the caa gene, which codes for the bacterial toxin colicin A. Analysis by gel electrophoresis of a family of DNA fragments of identical length, but bearing the caa operator at different positions, shows that DNA bending occurs close to or within the operator sequence upon LexA binding. In contrast, the interaction of LexA with the recA operator induces no detectable bending on 5% polyacrylamide gels. This difference between the two operators is likely to be due to an intrinsic bendability of the caa operator related to thymine tracts located on both sides of the operator. Such tracts do not exist in the recA operator. The free DNA fragments harbouring the caa operator show a slight tendency to bend even in the absence of the LexA repressor. The centre of this intrinsic bend is located close to or within the caa operator.