Role of loop bundle hydrogen bonds in the maturation and activity of (Pro)caspase-3.
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.
Feeney
Brett
B
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622, USA.
Pop
Cristina
C
Swartz
Paul
P
Mattos
Carla
C
Clark
A Clay
AC
D000595:Amino Acid Sequence; D001483:Base Sequence; D053148:Caspase 3 / Q000737:chemistry / Q000378:metabolism*; D003461:Crystallography; D017931:DNA Primers; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D017931:DNA Primers; D053148:Caspase 3