@article{walters_pop_scott_drag_swartz_mattos_salvesen_clark_2009, title={A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis}, volume={424}, ISSN={["1470-8728"]}, DOI={10.1042/bj20090825}, abstractNote={The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 Å (1 Å = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.}, journal={BIOCHEMICAL JOURNAL}, author={Walters, Jad and Pop, Cristina and Scott, Fiona L. and Drag, Marcin and Swartz, Paul and Mattos, Carla and Salvesen, Guy S. and Clark, A. Clay}, year={2009}, month={Dec}, pages={335–345} }
 @article{feeney_pop_swartz_mattos_clark_2006, title={Role of loop bundle hydrogen bonds in the maturation and activity of (pro) caspase-3}, volume={45}, ISSN={["0006-2960"]}, DOI={10.1021/bi0611964}, abstractNote={During maturation, procaspase-3 is cleaved at D175, which resides in a linker that connects the large and small subunits. The intersubunit linker also connects two active site loops that rearrange following cleavage and, in part, form the so-called loop bundle. As a result of chain cleavage, new hydrogen bonds and van der Waals contacts form among three active site loops. The new interactions are predicted to stabilize the active site. One unresolved issue is the extent to which the loop bundle residues also stabilize the procaspase active site. We examined the effects of replacing four loop bundle residues (E167, D169, E173, and Y203) on the biochemical and structural properties of the (pro)caspase. We show that replacing the residues affects the activity of the procaspase as well as the mature caspase, with D169A and E167A replacements having the largest effects. Replacement of D169 prevents caspase-3 autoactivation, and its cleavage at D175 no longer leads to an active enzyme. In addition, the E173A mutation, when coupled to a second mutation in the procaspase, D175A, may alter the substrate specificity of the procaspase. The mutations affected the active site environment as assessed by changes in fluorescence emission, accessibility to quencher, and cleavage by either trypsin or V8 proteases. High-resolution X-ray crystallographic structures of E167A, D173A, and Y203F caspases show that changes in the active site environment may be due to the increased flexibility of several residues in the N-terminus of the small subunit. Overall, the results show that these residues are important for stabilizing the procaspase active site as well as that of the mature caspase.}, number={44}, journal={BIOCHEMISTRY}, author={Feeney, Brett and Pop, Cristina and Swartz, Paul and Mattos, Carla and Clark, A. Clay}, year={2006}, month={Nov}, pages={13249–13263} }
 @article{feeney_pop_tripathy_clark_2004, title={Ionic interactions near the loop L4 are important for maintaining the active-site environment and the dimer stability of (pro)caspase 3}, volume={384}, number={Dec 15 2004}, journal={Biochemical Journal (London, England : 1984)}, author={Feeney, B. and Pop, C. and Tripathy, A. and Clark, A. C.}, year={2004}, pages={515–525} }
 @article{bose_pop_feeney_clark_2003, title={An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi034998x}, abstractNote={We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH.}, number={42}, journal={BIOCHEMISTRY}, author={Bose, K and Pop, C and Feeney, B and Clark, AC}, year={2003}, month={Oct}, pages={12298–12310} }
 @article{pop_feeney_tripathy_clark_2003, title={Mutations in the procaspase-3 dimer interface affect the activity of the zymogen}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi034999p}, abstractNote={The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of approximately 60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 A from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation.}, number={42}, journal={BIOCHEMISTRY}, author={Pop, C and Feeney, B and Tripathy, A and Clark, AC}, year={2003}, month={Oct}, pages={12311–12320} }