Mechanism of actin polymerization with yeast formin Bni1p

Formin proteins are actin nucleators and elongators which can be
found in most eukaryotic cells. In this work, structure-function
relationships between yeast formin Bni1p and actin polymerization
were studied. In the first part of this work, it was attempted to
clone and express formin constructs derived from yeast Bni1p
(Saccharomyces cerevisiae), including the key FH2 domain and a
modified FH1 domain. Biomathematical models involving both
diffusion and concentration-limited actin recruitment kinetics
could be tested with such proteins. Cloning was mostly successful,
but only the FH2 domain alone was expressed. In the second part of
this work, a salt effect on FH2 mediated actin nucleation was
discovered by means of pyrene assays and epifluorescence
microscopy. Potassium chloride (KCl) is a downregulator of FH2
nucleation activity: a higher KCl concentration leads to a
significantly lower actin polymerization speed (kp, m), to a bigger
lag time (tlag) and to a bigger t1/2, with the respective actin
filament length distributions. The salt effect was shown to be
significant in a KCl concentration range from 10 mM to 90 mM at two
different FH2 concentration, but not in absence of FH2. The
critical KCl concentration is lowered in the presence of FH2. Some
initial experiments with sodium chloride point to a non-specific
nature of this salt. This is in agreement with the electrostatic
nature of the salt effect, which was studied further by
computational means: A decrease of the KCl concentration leads to
lower binding free energies of the protein-protein interactions in
the crystallographically characterized actin-FH2 complex 1Y64. This
is especially the case for the electrostatic Coulomb interaction of
a specific area ("lasso" site). ANCHOR calculation results of
solvent accessible surface areas (SASAs) corroborate the importance
of this site. The experimentally found downregulation of FH2
mediated actin nucleation by KCl can therefore be explained by
reduced actin recruitment by the FH2 dimer: KCl diminishes the
surface charge of FH2 and actin and thus weakens electrostatic
Coulomb interactions. In future, this newly discovered salt effect
should be considered in experiments on formins, for example when
performing in vitro screens for FH2 inhibitors. The relevance of
this new salt effect in vivo remains to be demonstrated.