A monocysteine approach for probing the structure and interactions of the UmuD protein.

AUTOR(ES)

Lee, M H

RESUMO

UmuD participates in a variety of protein-protein interactions that appear to be essential for its role in UV mutagenesis. To learn about these interactions, we have initiated an approach based on the construction of a series of monocysteine derivatives of UmuD and have carried out experiments exploring the chemistry of the unique thiol group in each derivative. In vivo and in vitro characterizations indicate that these proteins have an essentially native structure. In proposing a model for the interactions of UmuD in the homodimer, we have made the following assumptions: (i) the conformations of the mutant proteins are similar to that of the wild type, and (ii) the differences in reactivity of the mutant proteins are predominantly due to the positional effects of the single cysteine substitutions. The model proposes the following. The region including the Cys-24-Gly-25 cleavage site, Val-34, and Leu-44 are closer to the interface than the other positions tested as suggested by the relative ease of dimer cross-linking of the monocysteine derivatives at these positions by oxidation with iodine (I2) and by reaction with bis-maleimidohexane. The mutant with a Ser-to-Cys change at position 60 (SC60) is similar in iodoacetate reactivity to the preceding derivatives but cross-links less efficiently by I2 oxidation. This suggests that Ser-60, the site of the putative nucleophile in the cleavage reaction, is located further from the dimer interface or in a cleft region. Both Ser-19, located in the N-terminal fragment of UmuD that is removed by RecA-mediated cleavage, and Ser-67 are probably not as close to the dimer interface, since they are cross-linked more easily with bis-maleimidohexane than with I2. The SC67 mutant phenotype also suggests that this position is less important in RecA-mediated cleavage but more important in a subsequent role for UmuD in mutagenesis. Ala-89, Gln-100, and Asp-126 are probably not particularly solvent accessible and may play important roles in protein architecture.

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