Prediction of 3-D structure of the Cro protein from phage 434

A comparative model building process has been utilized to predict the three-dimensional structure of the bacteriophage 434 Cro protein. Amino acid sequence similarities between the 434 Cro protein and other bacteriophage repressor and Cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific DNA binding proteins, to derive the model. From this model the interactions between the 434 Cro protein and its operator DNA have been deduced. These proposed interactions are consistent with the known properties of the bacteriophage 434 Cro protein.     

High Resolution Structural Studies of Cro Repressor Protein and Implications for DNA Recognition

Abstract

Cro repressor is a small dimeric protein that binds to specific sites on the DNA of bacteriophage λ. The structure of Cro has been determined and suggests that the protein binds to its sequence-specific sites with a pair of two-fold related α-helices of the protein located within successive major grooves of the DNA.

From the known three-dimensional structure of the repressor, model building and energy refinement have been used to develop a detailed model for the presumed complex between Cro and DNA. Recognition of specific DNA binding sites appears to occur via multiple hydrogen bonds between amino acid side chains of the protein and base pair atoms exposed within the major groove of DNA. The Cro:DNA model is consistent with the calculated electrostatic potential energy surface of the protein.

From a series of amino acid sequence and gene sequence comparisons, it appears that a number of other DNA-binding proteins have an α-helical DNA-binding region similar to that seen in Cro. The apparent sequence homology includes not only DNA-binding proteins from different bacteriophages, but also gene-regulatory proteins from bacteria and yeast. It has also been found that the conformations of part of the presumed DNA-binding regions of Cro repressor, λ repressor and CAP gene activator proteins are strikingly similar. Taken together, these results strongly suggest that a two-helical structural unit occurs in the DNA-binding region of many proteins that regulate gene expression. However, the results to date do not suggest that there is a simple one-to-one recognition code between amino acids and bases.

Crystals have been obtained of complexes of Cro with six-base-pair and nine-basepair DNA oligomers, and X-ray analysis of these co-crystals is in progress.     

Proposed alpha-helical super-secondary structure associated with protein-dna recognition

Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.     

A comparative study of dynamic structures between phage 434 Cro and repressor proteins by normal mode analysis

Two DNA binding proteins, Cro and the amino-terminal domain of the repressor of bacteriophage 434 (434 Cro and 434 repressor) that regulate gene expression and contain a helix-turn-helix (HTH) motif responsible for their site-specific DNA recognition adopt very similar three-dimensional structures when compared to each other. To reveal structural differences between these two similar proteins, their dynamic structures, as examined by normal mode analysis, are compared in this paper. Two kinds of structural data, one for the monomer and the other for a complex with DNA, for each protein, are used in the analyses. From a comparison between the monomers it is found that the interactions of Ala-24 in 434 Cro or Val-24 in 434 repressor, both located in the HTH motif, with residues 44, 47, 48, and 51 located in the domain facing the motif, and the interactions between residues 17, 18, 28, and 32, located in the HTH motif, cause significant differences in the correlative motions of these residues. From the comparison between the monomer and the complex with DNA for each protein, it was found that the first helix in the HTH motif is distorted in the complex form. While the residues in the HTH motif in 434 Cro have relatively larger positive correlation coefficients of motions with other residues within the HTH motif, such correlations are not large in the HTH motif of 434 repressor. It is suggestive to their specificity because the 434 repressor is less specific than 434 Cro. Although a structural comparison of proteins has been performed mainly from a static or geometrical point of view, this study demonstrates that the comparison from a dynamic point of view, using the normal mode analysis, is useful and convenient to explore a difference that is difficult to find only from a geometrical point of view, especially for proteins very similar in structure. © 1996 Wiley-Liss, Inc.     

A hybrid sequence approach to the paracelsus challenge

Inspired by the Paracelsus Challenge of Rose and Creamer (Proteins 19:1–3, 1994), we have designed a protein sequence that is 50% identical to an all-helical protein but is intended to fold into a largely β-sheet structure. Rather than attempt a de novo design, our strategy was to construct a hybrid sequence based on a helical “parent” protein (434 Cro) and a “target” protein with the desired fold (the B1 domain of protein G). The hybrid sequence (Crotein-G) is 50% identical to 434 Cro but is also 62% identical to the B1 domain of protein G. We also created a variant of Crotein-G (ZCrotein-G) that contains a potential His₃Cys₁ zinc binding site. At low protein concentrations and in the presence of 20% 2,2,2-trifluoroethanol (TFE) (v/v), the circular dichroism spectra of the designed proteins are distinct from that of 434 Cro and similar to that of the B1 domain of protein G. However, the proteins fail to denature in a cooperative manner. Furthermore, aggregation occurs at moderate protein concentrations or in the absence of TFE. Addition of zinc to ZCrotein-G does not promote structure formation. In summary, 434 Cro has been altered to something that may resemble the B1 domain of protein G, but the protein does not adopt a native structure. Proteins 30:136–143, 1998. © 1998 Wiley-Liss, Inc.     

Combinatorial redesign of the DNA binding specificity of a prokaryotic helix-turn-helix repressor

Redesign of the bacteriophage 434 Cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized DNA sequences. Site-directed, combinatorial mutagenesis of the 434 Cro helix-turn-helix (HTH) motif generated libraries of new variants which were screened for binding to new target sequences. Multiple mutations of 434 Cro that functionally converted wild-type (wt) 434 Cro DNA binding-sequence specificity to that of a lambda bacteriophage-specific repressor were identified. The libraries contained variations within the HTH sequence at only three positions. In vivo and in vitro analysis of several of the identified 434 Cro variants showed that the relatively few changes in the recognition helix of the HTH motif of 434 Cro resulted in specific and tight binding of the target DNA sequences. For the best 434 Cro variant identified, an apparent K(d) for lambda O(R)3 of 1 nM was observed. In competition experiments, this Cro variant was observed to be highly selective. We conclude that functional 434 Cro repressor variants with new DNA binding specificities can be generated from wt 434 Cro by mutating just the recognition helix. Important characteristics of the screening system responsible for the successful identifications are discussed. Application of the techniques presented here may allow the identification of DNA binding protein variants that functionally affect DNA regulatory sequences important in disease and industrial and biotechnological processes.     

Computer modeling studies of the structure of a repressor

Due to advances in molecular biology the DNA sequences of structural genes coding for proteins are often known before a protein is characterized or even isolated. The function of a protein whose amino acid sequence has been deduced from a DNA sequence may not even be known. This has created greater interest in the development of methods to predict the tertiary structures of proteins. The a priori prediction of a protein's structure from its amino acid sequence is not yet possible. However, since proteins with similar amino acid sequences are observed to have similar three-dimensional structures, it is possible to use an analogy with a protein of known structure to draw some conclusions about the structure and properties of an uncharacterized protein. The process of predicting the tertiary structure of a protein relies very much upon computer modeling and analysis of the structure. The prediction of the structure of the bacteriophage 434 cro repressor is used as an example illustrating current procedures.     

Crystallization and X-ray diffraction studies of a 434 Cro-DNA complex

Crystals have been obtained of the bacteriophage 434 Cro protein bound to a synthetic DNA operator. An analysis of the packing shows that the complexes stack end-to-end along crystallographic axis, forming long rods with non-crystallographic 11(3) screw symmetry. The average number of DNA base-pairs per turn is 10.27, which is somewhat more overwound as compared with the 434 repressor-DNA crystals of Anderson et al. Diffraction extends to 3 A along the rod direction and to 5 A in perpendicular directions.     

The phage 434 Cro/OR1 complex at 2.5 A resolution

The crystal structure of phage 434 Cro protein in complex with a 20 base-pair DNA fragment has been determined to 2.5 A resolution. The DNA fragment contains the sequence of the OR1 operator site. The structure shows a bent conformation for the DNA, straighter at the center and more bent at the ends. The central base-pairs adopt conformations with significant deviations from coplanarity. The two molecules interact extensively along their common interface, both through hydrogen bonds and van der Waals interactions. The significance of these interactions for operator binding and recognition is discussed.     

Folding kinetics of phage 434 Cro protein

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At <2 M urea, decreased folding amplitudes and nonlinear urea dependencies that are apparent at pH 6 indicate deviation from two-state behavior and suggest the formation of an early folding intermediate. The folding behavior of 434 Cro and why it folds 2 orders of magnitude slower than lambda(6-85) are rationalized in terms of the lower intrinsic helix stabilities and putative charge interactions in 434 Cro.     

Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.

The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator.     

Substituting an alpha-helix switches the sequence-specific DNA interactions of a repressor

It has been suggested that many DNA-binding proteins use an alpha-helix for specific sequence recognition. We have used amino acid sequence homologies to identify the presumptive DNA-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. The 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, OR, and they make different contacts in each binding site, as revealed by the chemical probe dimethyl sulfate. We substituted the putative recognition alpha-helix of 434 repressor with the putative recognition alpha-helix of 434 cro protein to create a hybrid protein named repressor*. The specific DNA contacts made by repressor* are like those of 434 cro protein.     

Changes in the Functional Activity of Phi11 Cro Protein is Mediated by Various Ions

Phi11, a temperate bacteriophage of Staphylococcus aureus, has been found to harbor a cro repressor gene which facilitates Phi11 to adopt the lytic mode of development. The Cro protein has been found to bind very specifically to a 15-bp operator DNA, located in the Phi11 cI–cro intergenic region [1]. To investigate the effects exerted by different ions upon the interaction between Cro and its cognate operator DNA, we have employed gel shift assays as well as circular dichroism spectral analysis. In this communication, we have shown that NH₄ ⁺ and acetate^? ions better facilitated the binding of Cro with its cognate operator as compared to Na⁺, K⁺ and Li⁺. Interestingly, Mg²⁺, carbonate^2? and Citrate^3? have an inhibitory effect upon the binding. The effect of the said ions upon the structure of Cro was also investigated by circular dichroism and it was found that other than Citrate^3? ions, none of the other ions destabilised the protein. On the other hand, Mg²⁺ and carbonate^2? ions maintained the structure of the protein but severely hampered its functional activity. Citrate^3? ions severely unfolded Cro and also inhibited its function. Considering all the data, NH₄ ⁺ and acetate^? ions appeared to be more suitable in maintaining the biological activity of Cro.     

100ps Molecular Dynamic Simulation of d(TATCACC)2

Abstract

A heptanucleotide sequence d(TATCACC)2 from OR3 region of bacteriophage X is considered sufficient for the recognition of Cro protein. We present here results on molecular dynamic simulations on this sequence for 100 ps in 0.02 ps interval. The simulations are done using computer program GROMOS. The conformational results are averaged over each ps. The IUPAC torsional parameters for 100 conformations are illustrated using a wheal and a dial systems. Several other stereochemical parameters such as H-bonding lengths and angles, sugar puckers, helix twist and roll angles as also distances between opposite strand phosphorus are depicted graphically. We find that there is rupture of terminal H-bonds. The bases are tilted and shifted away from the helix axis giving rise to bifurcated H-bonds. H- bonds are seen even in between different base pairs. The role of these dynamic structural changes in the recognition of OR3 operator by Cro protein is discussed in the paper.     

Structure of phage 434 Cro protein at 2.35 A resolution

The crystal structure of phage 434 Cro protein has been determined and refined against 2.35 A data to an R-factor of 19.5%. The protein comprises five alpha-helices and shows the helix-turn-helix motif found in other repressor proteins.     

Design of lambda Cro fold: solution structure of a monomeric variant of the de novo protein

One of the classical DNA-binding proteins, bacteriophage lambda Cro, forms a homodimer with a unique fold of alpha-helices and beta-sheets. We have computationally designed an artificial sequence of 60 amino acid residues to stabilize the backbone tertiary structure of the lambda Cro dimer by simulated annealing using knowledge-based structure-sequence compatibility functions. The designed amino acid sequence has 25% identity with that of natural lambda Cro and preserves Phe58, which is important for formation of the stably folded structure of lambda Cro. The designed dimer protein and its monomeric variant, which was redesigned by the insertion of a beta-hairpin sequence at the C-terminal region to prevent dimerization, were synthesized and biochemically characterized to be well folded. The designed protein was monomeric under a wide range of protein concentrations and its solution structure was determined by NMR spectroscopy. The solved structure is similar to that of a monomeric variant of natural lambda Cro with a root-mean-square deviation of the polypeptide backbones at 2.1A and has a well-packed protein core. Thus, our knowledge-based functions provide approximate but essential relationships between amino acid sequences and protein structures, and are useful for finding novel sequences that are foldable into a given target structure.     

Many gene-regulatory proteins appear to have a similar α-helical fold that binds DNA and evolved from a common precursor

Summary Amino acid and DNA sequence comparisons suggest that many sequence-specific DNA-binding proteins have in common and homologous region of about 22 amino acids. This region corresponds to two consecutive α-helices that occur in bot Cro and cI repressor proteins of bacteriophage λ and in catabolite gene activator protein ofEscherichia coli and are presumed to interact with DNA. The results obtained here suggest that this α-helical DNA-binding fold occurs in many proteins that regulate gene expression. It also appears that this DNA-binding unit evolved from a common evolutionary precursor.     

A Model for the Complex Between the Helix Destabilizing Protein GP32 of Bacteriophage T4 and Single-Stranded DNA

Abstract

A model for the structure of the complex between the helix-destabilizing protein of bacteriophage T4, GP32, and single-stranded DNA is proposed. In this model the bases are arranged in a helix, that is characterized by a relatively large distance between successive bases, a substantial base tilt, in combination with a small rotation per base. This helix is further organized into a tertiary structure, possibly a superhelix, of which the corresponding protein shell corresponds to the relatively rigid and rod-like structure that is observed in hydrodynamic experiments. It is proposed that similar structural features apply to other single-stranded DNA binding proteins in complex with polynucleotides.     

Identification by computer sequence analysis of transcriptional regulator proteins in Dictyostelium discoideum and Serratia marcescens

We have performed computer searches in the database of known protein sequences for proteins similar in sequence to bacteriophage regulatory proteins of known 3-D structure. The searches are more selective than other methods due to the use of a length-dependent threshold in sequence similarity, above which structural homology is implied with high certainty. Two probable DNA binding proteins were identified which are predicted to have a three-dimensional structure very similar to bacteriophage cro and repressor proteins. Approximate three-dimensional model coordinates are available from the authors. Both proteins contain the helix-turn-helix sequence motif typical of a wide class of DNA binding proteins and their function is deduced by analogy to sequence-similar proteins of known function. We predict that the Y.Smal protein in the restriction-modification enzyme gene locus of the enterobacterium serratia marcescens is a regulator of endonuclease expression; and, that the vegetative specific gene VSH7 of the slime mold dictyostelium discoideum codes for a regulator of gene expression specific for the slime mold growth phase before the onset of the developmental program. Point mutations that would have a strong effect on growth regulation phenotype are suggested. The VSH7 protein would be the first eukaryotic representative of the cro/phage repressor class.