IQ motif selectivity in human IQGAP1: binding of myosin essential light chain and S100B.

IQGAPs are cytoskeletal scaffolding proteins which link signalling pathways to the reorganisation of actin and microtubules. Human IQGAP1 has four IQ motifs each of which binds to calmodulin. The same region has been implicated in binding to two calmodulin-like proteins, the myosin essential light chain Mlc1sa and the calcium and zinc ion binding protein S100B. Using synthetic peptides corresponding to the four IQ motifs of human IQGAP1, we showed by native gel electrophoresis that only the first IQ motif interacts with Mlc1sa. This IQ motif, and also the fourth, interacts with the budding yeast myosin essential light chain Mlc1p. The first and second IQ motifs interact with S100B in the presence of calcium ions. This clearly establishes that S100B can interact with its targets through IQ motifs in addition to interacting via previously reported sequences. These results are discussed in terms of the function of IQGAP1 and IQ motif recognition.

Divergence of chemical function in the alkaline phosphatase superfamily: Structure and mechanism of the P-C bond cleaving enzyme phosphonoacetate hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and P-32-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

A hydrogel based zinc(ii) sensor for use in fluorescent multi-well plate analysis

A polymeric hydrogel containing a photoinduced electron transfer (PET) based probe for Zn(ii) has been formulated into the wells of a 96-well plate. Upon addition of Zn(ii) ions to selected wells, the fluorescence of the gel was observed to increase in a concentration dependent manner in the 0.25-1.75 mM range. The millimolar binding constant observed for this probe is higher than that reported for other Zn(ii) probes in the literature and offers the possibility to determine the concentration of this ion in environments where the Zn(ii) concentration is high. The combination of the multi-well plate set-up with fluorescence detection offers the possibility of high-throughput screening using low sample volumes in a timely manner. To the best of our knowledge, this is the first reported example of a polymeric hydrogel sensor for zinc with capability for use in fluorescence multi-well plate assay.