Cofilin dissociates Arp2/3 complex and branches from actin filaments.

BACKGROUND: Actin-based cellular motility requires spatially and temporally coordinated remodeling of a network of branched actin filaments. This study investigates how cofilin and Arp2/3 complex, two main players in the dendritic nucleation model, interact to produce sharp spatial transitions between densely branched filaments and long, unbranched filaments. RESULTS: We found that cofilin binding reduces both the affinity of actin filaments for Arp2/3 complex and the stability of branches. We used fluorescence spectroscopy to measure the kinetics of cofilin association with filaments and the resulting dissociation of Arp2/3 complex and TIRF microscopy to visualize filament severing and the loss of actin filament branches. Cofilin severs filaments optimally when few actin subunits are occupied but dissociates branches rapidly only at higher occupancies. Effective debranching is nevertheless achieved, as a result of cooperative binding and reduced affinity of Arp2/3 complex for the filament, at cofilin concentrations below those required for direct competition. CONCLUSIONS: Cofilin rapidly dissociates Arp2/3 complex and branches by direct competition for binding sites on the actin filament and by propagation of structural changes in the actin filament that reduce affinity for Arp2/3 complex.

DNA polymerase catalysis in the absence of Watson-Crick hydrogen bonds: analysis by single-turnover kinetics.

We report the first pre-steady-state kinetic studies of DNA replication in the absence of hydrogen bonds. We have used nonpolar nucleotide analogues that mimic the shape of a Watson-Crick base pair to investigate the kinetic consequences of a lack of hydrogen bonds in the polymerase reaction catalyzed by the Klenow fragment of DNA polymerase I from Escherichia coli. With a thymine isostere lacking hydrogen-bonding ability in the nascent pair, the efficiency (k(pol)/Kd) of the polymerase reaction is decreased by 30-fold, affecting the ground state (Kd) and transition state (k(pol)) approximately equally. When both thymine and adenine analogues in the nascent pair lack hydrogen-bonding ability, the efficiency of the polymerase reaction is decreased by about 1000-fold, with most of the decrease attributable to the transition state. Reactions using nonpolar analogues at the primer-terminal base pair demonstrated the requirement for a hydrogen bond between the polymerase and the minor groove of the primer-terminal base. The R668A mutation of Klenow fragment abolished this requirement, identifying R668 as the probable hydrogen-bond donor. Detailed examination of the kinetic data suggested that Klenow fragment has an extremely low tolerance of even minor deviations of the analogue base pairs from ideal Watson-Crick geometry. Consistent with this idea, some analogue pairings were better tolerated by Klenow fragment mutants having more spacious active sites. In contrast, the Y-family polymerase Dbh was much less sensitive to changes in base pair dimensions and more dependent upon hydrogen bonding between base-paired partners.

Generation of an active monomer of rabbit muscle creatine kinase by site-directed mutagenesis: the effect of quaternary structure on catalysis and stability.

Cytosolic creatine kinase exists in native form as a dimer; however, the reasons for this quaternary structure are unclear, given that there is no evidence of active site communication and more primitive guanidino kinases are monomers. Three fully conserved residues found in one-half of the dimer interface of the rabbit muscle creatine kinase (rmCK) were selectively changed to alanine by site-directed mutagenesis. Four mutants were prepared, overexpressed, and purified: R147A, R151A, D209A, and R147A/R151A. Both the R147A and R147A/R151A were confirmed by size-exclusion chromatography and analytical ultracentrifugation to be monomers, whereas R151A was dimeric and D209A appeared to be an equilibrium mixture of dimers and monomers. Kinetic analysis showed that the monomeric mutants, R147A and R147A/R151A, showed substantial enzymatic activity. Substrate binding affinity by R147A/R151A was reduced approximately 10-fold, although k(cat) was 60% of the wild-type enzyme. Unlike the R147A/R151A, the kinetic data for the R147A mutant could not be fit to a random-order rapid-equilibrium mechanism characteristic of the wild-type, but could only be fit to an ordered mechanism with creatine binding first. Substrate binding affinities were also significantly lower for the R147A mutant, but k(cat) was 11% that of the native enzyme. Fluorescence measurements using 1-anilinonaphthalene-8-sufonate showed that increased amounts of hydrophobic surface area are exposed in all of the mutants, with the monomeric mutants having the greatest amounts of unfolding. Thermal inactivation profiles demonstrated that protein stability is significantly decreased in the monomeric mutants compared to wild-type. Denaturation experiments measuring lambda(max) of the intrinsic fluorescence as a function of guanidine hydrochloride concentration helped confirm the quaternary structures and indicated that the general unfolding pathway of all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is not a prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.