Capping protein (CP) regulates actin dynamics by binding the barbed ends of actin filaments. Removal of CP may be one means to harness actin polymerization for processes such as cell movement and endocytosis. Here we structurally and biochemically investigated a CP interaction (CPI) motif present in the otherwise unrelated proteins CARMIL and CD2AP. The CPI motif wraps around the stalk of the mushroom-shaped CP at a site distant from the actin-binding interface, which lies on the top of the mushroom cap. We propose that the CPI motif may act as an allosteric modulator, restricting CP to a low-affinity, filament-binding conformation. Structure-based sequence alignments extend the CPI motif–containing family to include CIN85, CKIP-1, CapZIP and a relatively uncharacterized protein, WASHCAP (FAM21). Peptides comprising these CPI motifs are able to inhibit CP and to uncap CP-bound actin filaments.

Gelsolin consists of six homologous domains (G1-G6), each containing a conserved Ca-binding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1-G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1-G3/actin structure, whereas it is vacant in the equine version. In-depth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca 2؉ and responsible for opening the G2-G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Ca-binding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1-G3/actin reveals that the Ca 2؉ locks the G2-G3 interface. Thermal denaturation studies of G2-G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed.actin ͉ calcium activated ͉ calcium dependent ͉ TIRF

Two Zn2+ binding sites were found in the Aeromonas hydrophila AE036 metallo-beta-lactamase. The affinity of the first binding site for Zn2+ ions is so high that the dissociation constant could not be determined, but it is significantly lower than 20 nM. The mono-Zn2+ form of the enzyme exhibits a maximum activity against its carbapenem substrates. The presence of a Zn2+ ion in the second lower affinity binding site results in a loss of enzymatic activity with a Ki value of 46 microM at pH 6.5. The kinetic analysis is in agreement with a noncompetitive inhibition mechanism. The Zn content of the A. hydrophila enzyme is also strongly pH-dependent. With an external Zn2+ ion concentration of 0.4 microM, occupancy of the higher affinity site by metal ions is lower than 10% at pH 5 and 10. The affinity for the second binding site seems to increase from pH 6 to 7.5. Fluorescence emission and circular dichroism spectra revealed slight conformational changes upon titration of the apoenzyme by Zn2+ ions, resulting in the successive saturation of the first and second binding sites. Differential scanning calorimetry transitions and intrinsic fluorescence emission spectra in the presence of increasing concentrations of urea demonstrate that the catalytic zinc strongly stabilizes the conformation of the enzyme whereas the di-Zn enzyme is even more resistant to thermal and urea denaturation than the mono-Zn enzyme. The Zn2+ dependency of the activity of this metallo-beta-lactamase thus appears to be very different from that of the homologous Bacteroides fragilis enzyme for which the presence of two Zn2+ ions per molecule of protein appears to result in maximum activity.