HPPK-HP-MgAMPCPP mimics most closely the natural ternary complex of HPPK and provides details of protein-substrate interactions. The coordination of the two Mg(2+) ions helps create the correct geometry for the one-step reaction of pyrophosphoryl transfer, for which we suggest an in-line single displacement mechanism with some associative character in the transition state. The rigidity of the adenine-binding pocket and hydrogen bonds are responsible for adenosine specificity. The nonconserved residues that interact with the substrate might be responsible for the species-dependent properties of an isozyme.

The formation of protein complexes between phosphorylated R-Smads and Smad4 is a central event in the TGF-beta signaling pathway. We have determined the crystal structure of two R-Smad/Smad4 complexes, Smad3/Smad4 to 2.5 angstroms, and Smad2/Smad4 to 2.7 angstroms. Both complexes are heterotrimers, comprising two phosphorylated R-Smad subunits and one Smad4 subunit, a finding that was corroborated by isothermal titration calorimetry and mutational studies. Preferential formation of the R-Smad/Smad4 heterotrimer over the R-Smad homotrimer is largely enthalpy driven, contributed by the unique presence of strong electrostatic interactions within the heterotrimeric interfaces. The study supports a common mechanism of Smad protein assembly in TGF-beta superfamily signaling.

High-mobility group A2 (HMGA2) is commonly overexpressed in large leiomyomas. HMGA2 is an important regulator of cell growth, differentiation, apoptosis, and transformation. As a predicted target of Let-7 microRNAs (Let-7s), HMGA2 can be repressed by Let-7s in vitro. MicroRNA profiling analysis revealed that Let-7s were significantly dysregulated in uterine leiomyomas: high in small leiomyomas and lower in large leiomyomas. To evaluate whether Let-7 repression of HMGA2 plays a major role in leiomyomas, we analyzed the molecular relationship of HMGA2 and Let-7s, both in vitro and in vivo. We first characterized that exogenous Let-7 microRNAs could directly repress the dominant transcript of HMGA2, HMGA2a. This repression was also identified for two cryptic HMGA2 transcripts in primary leiomyoma cultures. Second, we found that the endogenous Let-7s were biologically active and played a major role in the regulation of HMGA2. Then, we illustrated that Let-7 repression of HMGA2 inhibited cellular proliferation. Finally, we examined the expression levels of Let-7c and HMGA2 in a large cohort of leiomyomas (n = 120), and we found high levels of Let-7 and low levels of HMGA2 in small leiomyomas, and low levels of Let-7 and high levels of HMGA2 in large leiomyomas. Our findings suggest that the Let-7 -mediated repression of HMGA2 mechanism can be an important molecular event in

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP). Because HPPK is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent. Thermodynamic analysis shows that Mg(2+) is important not only for the binding of nucleotides but also for the binding of HMDP. Transient kinetic analysis shows that a step or steps after the chemical transformation are rate-limiting in the reaction catalyzed by HPPK. The pre-steady-state kinetics is composed of a burst phase and a steady-state phase. The rate constant for the burst phase is approximately 50 times larger than that for the steady-state phase. The latter is very similar to the k(cat) value measured by steady-state kinetics. A set of rate constants for the individual steps of the HPPK-catalyzed reaction has been determined by a combination of stopped-flow and quench-flow analyses. These results form a thermodynamic and kinetic framework for dissecting the roles of active site residues in the substrate binding and catalysis by HPPK.