A Trade between Similar but Nonequivalent Intrasubunit and Intersubunit Contacts in Cro Dimer Evolution^,

Abstract

The homodimeric lambda Cro protein has a "ball-and-socket" interface that includes insertion of an aromatic side chain, Phe 58, from each subunit into a cavity in the hydrophobic core of the other subunit. This overlap between the subunit core and dimer interface hypothetically explains the strong dimerization and weak monomer stability of lambda Cro in comparison to homologues. According to a model developed here and in a previous study [LeFevre, K. R., and Cordes, M. H. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2345-2350], the socket cavity evolved in part by replacement of a buried tryptophan in an ancestral stable monomer with a smaller side chain (Ala 33 in lambda Cro). The resulting core defect was in effect repaired by insertion of a different side chain (Phe 58) from a second subunit, generating the ball and socket. Consistent with such an evolutionary trade between intrasubunit and intersubunit interactions, we showed in the previous study that restoration of the ancestral Trp 33 in lambda Cro stabilized the monomer and reduced the extent of dimerization. Here, we report the solution structure of a stable lambda Cro monomer containing the Ala33Trp mutation, which confirms that the restored tryptophan fulfills its ancestral role as a core side chain, filling part of the socket cavity occupied by Phe 58 in the wild-type dimer. The structure also reveals, however, that the cavity is not completely filled by Trp 33, suggesting that its formation could have involved multiple mutations that reduced side chain volume. We offer suggestive evidence of a role of mutations at a second position.

Transitive homology-guided structural studies lead to discovery of Cro proteins with 40% sequence identity but different folds

“…Still, Pfl 6 dimerizes less strongly than Cro. On the basis of site-directed mutagenesis, we proposed previously that the low micromolar dimerization of Cro did not result directly from its ␤-sheet fold but derived in part from specific side-chain mutations yielding a hydrophobic ''ball and socket'' at both ends of the interface (19,26). These previous studies implicated Ala 33 and Phe 58 as key players in dimer evolution (Right Inset, Fig.…”

A role for indels in the evolution of Cro protein folds

“…For Xfaso 1, all indels were introduced into the wild‐type sequence background, whereas for Pfl 6 the indels were constructed an I58D background, with the exception of the VPAER and RARGR inserts, which were constructed in a wild‐type background. Wild‐type Pfl 6 and I58D have essentially identical thermal stabilities and circular dichroism spectra at 25 μ M protein concentration, but wild‐type Pfl 6 dimerizes with a K d of ∼1 m M , whereas I58D remains monomeric even at high concentrations because of disruption of a hydrophobic dimer interface . The reduced dimerization of I58D facilitates NMR investigations .…”

Two Structures of a λ Cro Variant Highlight Dimer Flexibility but Disfavor Major Dimer Distortions upon Specific Binding of Cognate DNA

“…The Cro protein from bacteriophage λ is a prototypical model system for gene regulatory systems, 1 protein-DNA interactions involving the helixturn-helix motif, 2-4 protein stability, 5-7 protein oligomerization, [8][9][10][11] and protein design and engineering. 7,12,13 A fascinating and important aspect of Cro is its dimerization.…”