D. H. Ohlendorf

Proposed α-helical super-secondary structure associated with protein-DNA recognition

Abstract 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.

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

SummaryAmino 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.

Comparison of the structures of Cro and λ repressor proteins from bacteriophage λ

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of λ repressor protein, both from bacteriophage λ, are compared. The second and third α-helices, α2 and α3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and λ repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first α-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the α1 and α2 helices of λ repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar α2-α3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the α2-α3 units of the respective proteins aligning on the DNA in exactly the same way.

High resolution structural studies of cro repressor protein and implications for dna recognition

Cro repressor is a small dimeric protein that binds to specific sites on the DNA of bacteriophage lambda. 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 alpha-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 alpha-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, lambda 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.

How does cro repressor recognize its DNA target sites

Abstract The structure of the cro repressor from bacteriophage λ suggests that a pair of α-helices of this gene-regulatory protein bind to its sequence-specific sites on the DNA. These protein α-helices lie within successive major grooves of the DNA, and amino acid side chains make multiple hydrogen bonds with the accessible parts of the DNA base pairs. Each α-helix is part of a common two-helical DNA-binding fold that apparently occurs in many proteins that regulate gene expression.

Crystallographic data for complexes of the Cro repressor with DNA.

Complexes of the bacteriophage lambda Cro repressor with two DNA duplexes have been crystallized. The DNA sequences are equimolar mixtures of ApTpCpApCpC and its complementary strand and ApCpCpGpCpApApGpG and its complementary strand, which are both parts of the lambda OR3 operator. The space group of both co-crystals is C2221 with cell dimensions a = 81.1 A, b = 89.2 A, and c = 80.0 A. Analysis of dissolved crystals shows that they respectively contain approximately two hexamers per Cro dimer and one nonamer per dimer. The co-crystals diffract to about 3 A resolution and appear suitable for structural studies.