Prediction of 3-D Structure of the Cro Protein from Phage 434

Abstract

A comparative model building process has been utilized to predict (he 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.     

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.     

Thermodynamic analysis of the structural stability of phage 434 Cro protein

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) M(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) K(-)(1) for DeltaC(p)()(,U), the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent DeltaC(p)()(,U) of 0.95 +/- 0.30 kcal mol(-)(1) K(-)(1) or a temperature-dependent value of 1.00 +/- 0.10 kcal mol(-)(1) K(-)(1) at 25 degrees C. DeltaC(p)()(,U) and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 degrees C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6)(-)(85)), but is close to the value reported for the putative monomeric lambda Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.     

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.     

Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein.

The phage 434 Cro protein, the N-terminal domain of its repressor (R1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. The intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in R1-69 in 7 M urea has been proposed as a folding initiation site. To understand the early events in the folding of 434 Cro, and for comparison with R1-69 and lambda6-85, we examined the conformational behavior of five peptides covering the entire 434 Cro sequence in water, 40% (by volume) TFE/water, and 7 M urea solutions using CD and NMR. Each peptide corresponds to a helix and adjacent residues as identified in the native 434 Cro NMR and crystal structures. All are soluble and monomeric in the solution conditions examined except for the peptide corresponding to the 434 Cro helix 4, which has low water solubility. Helix formation is observed for the 434 Cro helix 1 and helix 2 peptides in water, for all the peptides in 40% TFE and for none in 7 M urea. NMR data indicate that the helix limits in the peptides are similar to those in the native protein helices. The number of side-chain NOEs in water and TFE correlates with the helix content, and essentially none are observed in 7 M urea for any peptide, except that for helix 5, where a hydrophobic cluster may be present. The low intrinsic folding propensities of the five helices could account for the observed stability and folding behavior of 434 Cro and is, at least qualitatively, in accord with the results of the recently described diffusion-collision model incorporating intrinsic helix propensities.     

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.     

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.     

Secondary structure switching in Cro protein evolution

We report the solution structure of the Cro protein from bacteriophage P22. Comparisons of its sequence and structure to those of lambda Cro strongly suggest an alpha-to-beta secondary structure switching event during Cro evolution. The folds of P22 Cro and lambda Cro share a three alpha helix fragment comprising the N-terminal half of the domain. However, P22 Cro's C terminus folds as two helices, while lambda Cro's folds as a beta hairpin. The all-alpha fold found for P22 Cro appears to be ancestral, since it also occurs in cI proteins, which are anciently duplicated paralogues of Cro. PSI-BLAST and transitive homology analyses strongly suggest that the sequences of P22 Cro and lambda Cro are globally homologous despite encoding different folds. The alpha+beta fold of lambda Cro therefore likely evolved from its all-alpha ancestor by homologous secondary structure switching, rather than by nonhomologous replacement of both sequence and structure.     

Primary structure of an EcoRI fragment of lambda imm434 DNA containing regions cI-cro of phage 434 and cII-o of phage lambda.

Digestion of phage lambda imm434 DNA with restriction endonuclease EcoRI yields 7 fragments. The shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage DNA portion proximal to it. The complete primary structure of this fragment has been determined using the chemical method of DNA sequencing. Hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cII gene of phage lambda, as well as NH2-terminal amino-acid sequences of the cI protein of phage 434 and the O protein of phage lambda, have been deduced solely on the basis of the DNA sequence. The fragment studied contains also the pR and probably prm promoters and the oR operator of phage 434. The sequence coding for them differs from the respective DNA sequence of phage lambda.     

New 434-specific DNA binding protein copurified with the 434 tof protein from lambda imm434cI dv carrier cells of Escherichia coli

A tof-like protein that has 434-specific DNA binding activity has been copurified with the 434 tof protein from lambda imm434cI dv carrier cells. The apparent molecular weight of the new 434-specific DNA binding protein is 9,000 to 9,500, a little higher than that of the 434 tof protein, as estimated by SDS gel electrophoresis. Amino acid analysis revealed the protein to be an arginine-rich component whereas the 434 tof protein is a lysine-rich component. The specific binding reaction of the new protein to lambda imm434dv DNA is distinct from that of the 434 tof protein in respect to the sigmoid shape of the binding curve and to the temperature dependency. This suggests that the specific binding to lambda imm434dv DNA observed with the new protein is due not to a trace of the 434 tof protein contaminating the new protein preparation but rather to the new protein itself. The NH2-terminal 11 residues of the new 434-specific DNA binding protein were sequenced by manual Edman degradation. This technique revealed that the new protein is not a fragment of the 434 tof, cII, or O protein or an NH2-terminal fragment of the cI repressor. The origin and the physiological roles of the new 434-specific DNA binding protein remain unknown.     

Prediction of protein structure from ideal forms

For many years it has been accepted that the sequence of a protein can specify its three-dimensional structure. However, there has been limited progress in explaining how the sequence dictates its fold and no attempt to do this computationally without the use of specific structural data has ever succeeded for any protein larger than 100 residues. We describe a method that can predict complex folds up to almost 200 residues using only basic principles that do not include any elements of sequence homology. The method does not simulate the folding chain but generates many thousands of models based on an idealized representation of structure. Each rough model is scored and the best are refined. On a set of five proteins, the correct fold score well and when tested on a set of larger proteins, the correct fold was ranked highest for some proteins more than 150 residues, with others being close topological variants. All other methods that approach this level of success rely on the use of templates or fragments of known structures. Our method is unique in using a database of ideal models based on general packing rules that, in spirit, is closer to an ab initio approach.     

Prediction of protein function from protein sequence and structure

The sequence of a genome contains the plans of the possible life of an organism, but implementation of genetic information depends on the functions of the proteins and nucleic acids that it encodes. Many individual proteins of known sequence and structure present challenges to the understanding of their function. In particular, a number of genes responsible for diseases have been identified but their specific functions are unknown. Whole-genome sequencing projects are a major source of proteins of unknown function. Annotation of a genome involves assignment of functions to gene products, in most cases on the basis of amino-acid sequence alone. 3D structure can aid the assignment of function, motivating the challenge of structural genomics projects to make structural information available for novel uncharacterized proteins. Structure-based identification of homologues often succeeds where sequence-alone-based methods fail, because in many cases evolution retains the folding pattern long after sequence similarity becomes undetectable. Nevertheless, prediction of protein function from sequence and structure is a difficult problem, because homologous proteins often have different functions. Many methods of function prediction rely on identifying similarity in sequence and/or structure between a protein of unknown function and one or more well-understood proteins. Alternative methods include inferring conservation patterns in members of a functionally uncharacterized family for which many sequences and structures are known. However, these inferences are tenuous. Such methods provide reasonable guesses at function, but are far from foolproof. It is therefore fortunate that the development of whole-organism approaches and comparative genomics permits other approaches to function prediction when the data are available. These include the use of protein-protein interaction patterns, and correlations between occurrences of related proteins in different organisms, as indicators of functional properties. Even if it is possible to ascribe a particular function to a gene product, the protein may have multiple functions. A fundamental problem is that function is in many cases an ill-defined concept. In this article we review the state of the art in function prediction and describe some of the underlying difficulties and successes.     

Prediction of protein secondary structure from amino acid sequence

The conformational parametersP _k for each amino acid species (j=1–20) of sequential peptides in proteins are presented as the product ofP _i,k , wherei is the number of the sequential residues in thekth conformational state (k=-helix,-sheet,-turn, or unordered structure). Since the average parameter for ann-residue segment is related to the average probability of finding the segment in the kth state, it becomes a geometric mean of (P _k )_av=(P _i,k ) ^1/n with amino acid residuei increasing from 1 ton. We then used ln(P_k)_av to convert a multiplicative process to a summation, i.e., ln(P _k ) _av =(1/n)P _i,k (i=1 ton) for ease of operation. However, this is unlike the popular Chou-Fasman algorithm, which has the flaw of using the arithmetic mean for relative probabilities. The Chou-Fasman algorithm happens to be close to our calculations in many cases mainly because the difference between theirP _k and our InP _k is nearly constant for about one-half of the 20 amino acids. When stronger conformation formers and breakers exist, the difference become larger and the prediction at the N- and C-terminal-helix or-sheet could differ. If the average conformational parameters of the overlapping segments of any two states are too close for a unique solution, our calculations could lead to a different prediction.     

Crystal structure of the receptor-binding protein head domain from Lactococcus lactis phage bIL170

Lactococcus lactis, a gram-positive bacterium widely used by the dairy industry, is subject to lytic phage infections. In the first step of infection, phages recognize the host saccharidic receptor using their receptor binding protein (RBP). Here, we report the 2.30-A-resolution crystal structure of the RBP head domain from phage bIL170. The structure of the head monomer is remarkably close to those of other lactococcal phages, p2 and TP901-1, despite any sequence identity with them. The knowledge of the three-dimensional structures of three RBPs gives a better insight into the module exchanges which have occurred among phages.     

The structure of a repressor: crystallographic data for the Cro regulatory protein of bacteriophage lambda.

Crystals of the bacteriophage λ Cro repressor protein that are suitable for X-ray diffraction studies have been obtained. Preliminary crystallographic analysis reveals that the space group is R32, the cell dimensions in the hexagonal system are $\text{a = b = 91·9}\text{A}\text{?}\text{, c = 268·9}\text{A}\text{?}$, and there are three dimers per asymmetric unit.     

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.     

QSE: A new 3-D solvent exposure measure for the analysis of protein structure

Solvent exposure of amino acids measures how deep residues are buried in tertiary structure of proteins, and hence it provides important information for analyzing and predicting protein structure and functions. Existing methods of calculating solvent exposure such as accessible surface area, relative accessible surface area, residue depth, contact number, and half-sphere exposure still have some limitations. In this article, we propose a novel solvent exposure measure named quadrant-sphere exposure (QSE) based on eight quadrants derived from spherical neighborhood. The proposed measure forms a microenvironment around Cα atom as a sphere with a radius of 13??, and subdivides it into eight quadrants according to a rectangular coordinate system constructed based on geometric relationships of backbone atoms. The number of neighboring Cα atoms whose labels are the same is given as the QSE value of the center Cα atom at hand. As evidenced by histograms that show very different distributions for different structure configurations, the proposed measure captures local properties that are characteristic for a residue's eight-directional neighborhood within a sphere. Compared with other measures, QSE provides a different view of solvent exposure, and provides information that is specific for different tertiary structure. As the experimental results show, QSE measure can potentially be used in protein structure analysis and predictions.