Bacteriophage Lambda Cro-Operator Complex

Caroline Hitsman '25 and Ada Wigfield '25

Contents:

I. Introduction

The bacteriophage λ Cro protein is a sequence-specific DNA-binding protein. It's part of the phages gene regulation system, in which Cro helps determine whether the phage undergoes lysis. Cro binds to specific operator DNA sequences, and it recognizes these sequences with its helix-turn-helix motif. Earlier structures showed some general ideas of how Cro interacts with DNA, but lower resolution made it hard to see the fine details. The higher-resolution structures shows the exact Cro conformational changes once it binds DNA and how it makes direct hydrogen-bonding and van der Waals contacts with bases in the DNA major groove. Cro relies heavily on direct readout of bases for specificity, and the way it differentiates between different operators comes from the changes between these interactions. Shown in this depiction is one half of the functioning Cro homodimer.

II. General Structure

Cro is a protein made of 66 amino acids per monomer and it functions as a dimer when it is bound to DNA. Each monomer has a compacted fold containing three and three . The second and third helices form a motif that sits in the of the operator DNA. The β-strands form a that helps stabilize the structure and also contributes to the dimer interface on the DNA. The Cro dimer interface is part of the β3 strand (residues 54–56) and sticks out toward the other subunit. It ends in a hook that includes Phe58 and Pro59. Phe58 from one monomer fits into a hydrophobic pocket in the other monomer’s core, which contributes to the tightly packed hydrophobic . This flexibility is important because when Cro binds DNA both the protein and the DNA undergo significant conformational changes. In the bound state, the recognition helices insert into the major groove symmetrically. The DNA bends about 40°, narrowing the center of the . Only the HTH regions of Cro actually the DNA bases directly, while other parts of the protein help stabilize the interaction by contacting the backbone.

III. Recognition Helix & Operator Contact

In total, only of the 17 base-pairs of the operator make contact with each Cro dimer (14 contacts per homodimer). Each operator sequence makes majority of contact with Cro through the sequence 5'-GGTG-3'. makes two hydrogen bonds with Ade(+2).
The side chain hydroxyl of forms a bifurcated hydrogen bond to both the O6 and N7 of G(-4). The NH2 side-chain of donates one hydrogen bond to a phosphate group of the DNA backbone. The side-chain donates hydrogen bonds to both N7 and O6 of G(-6), in addition to N7 of G(-7). The three bases contacted by each half of the Cro dimer are primarily recognized through or hydrophobic interactions, specifically with the methyl groups of thymine bases. occur between Gln16 and the methyl group of Thy(+1). Van der Waals contacts happen between Ser28, Ala29, and the methyl group of Thy(-5).Thr17 makes van der Waals interactions with the methyl group of T(+1), as well as the methyl group of T(+3).

IV. Cro Conformational Changes

The binding of the recognition helices α2 and α3 to the operator regions causes the HTH to rotate 53°. This rotation is caused by multiple ‘torsion-angle’ changes along the β ribbon. The most notable occurs at Glu53 and Glu54, where the β2β3-ribbon transitions into the exposed Residues 16 through 33 of the HTH turn about 1 angstrom away from the central β3-strand near Additionally, hydrogen bonding between the amide groups of results in the recognition helix pulling away from the aromatic ring in Phe58. This movement corresponds to the opening of a channel, which accommodates the DNA backbone. These conformational changes do not require the alteration of any hydrogen bonds.

VI. References

Brennan RG, Roderick SL, Takeda Y, Matthews BW. Protein-DNA conformational changes in the crystal structure of a lambda Cro-operator complex Catabolite Gene Activator Protein. Proc Natl Acad Sci U S A. 1990 Oct;87(20):8165-9. doi: 10.1073/pnas.87.20.8165. PMID: 2146682; PMCID: PMC54913.

Albright RA, Matthews BW. Crystal structure of lambda-Cro bound to a consensus operator at 3.0 A resolution. J Mol Biol. 1998 Jul 3;280(1):137-51. doi: 10.1006/jmbi.1998.1848. PMID: 9653037.

A. Mondragón, S.C. Harrison. The phage 434 CroOR1 complex at 2.5 Å resolution. Journal of Molecular Biology. 1991 May 20;219(2): 321-334. doi: 10.1016/0022-2836(91)90568-Q. PMID: 2038059

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