Key result
DNase I and thymosin beta4 bind to actin independently without overlapping binding sites, forming a ternary complex with identical KD values as pure actin.
DNase I and Tbeta4 bind to actin independently without overlapping binding sites, allowing for potential X-ray analysis of the ternary complex.
May enable X-ray analysis of the actin ternary complex; leaves open its functional roles in vivo.
We have recently described a method for identifying contact sites between actin and thymosin beta4 (Tbeta4) by following spectrophotometrically the extent and kinetics of distinct, thiol-specific crosslinking reactions between appropriate derivatives of the two proteins [Reichert et a]. (1996) J. Biol. Chem. 271, 1301-1308]. In the present study this method was used to show that such crosslinking, which is indicative of complex formation, occurs to the same extent with the actin-DNase I complex as with pure actin, although at a somewhat lower rate. Further evidence for the formation of the ternary complex was given by gel electrophoresis. From fluorescence spectroscopy the KD value of Tbeta4 from the actin-DNase I complex was found to be identical to that from pure actin. In line with these data, the capacity of actin for inhibiting DNase I was not affected by the addition of Tbeta4. In conclusion, DNase I and Tbeta4 are independent of each other in their interaction with actin, suggesting that the binding sites of thymosin beta4 and DNase I on actin do not overlap. A ternary complex of DNase I, actin and Tbeta4, if obtained in crystalline form, could thus provide an approach for studying the interface of Tbeta4 and actin by X-ray analysis.
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Reichert et al. (1996) studied this question. Thymosin beta4 and DNase I vs. Pure actin was evaluated on Complex formation and KD value. DNase I and thymosin beta4 bind to actin independently without overlapping binding sites, forming a ternary complex with identical KD values as pure actin.
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