Role of Loop Bundle Hydrogen Bonds in the Maturation and Activity of (Pro)caspase-3

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• 作者：Brett Feeney ; Cristina Pop ; Paul Swartz ; Carla Mattos ; A. Clay Clark
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：November 7, 2006
• 年：2006
• 卷：45
• 期：44
• 页码：13249 - 13263
• 全文大小：761K
• 年卷期：v.45,no.44(November 7, 2006)
• ISSN：1520-4995

文摘

During maturation, procaspase-3 is cleaved at D175, which resides in a linker that connectsthe large and small subunits. The intersubunit linker also connects two active site loops that rearrangefollowing cleavage and, in part, form the so-called loop bundle. As a result of chain cleavage, new hydrogenbonds and van der Waals contacts form among three active site loops. The new interactions are predictedto stabilize the active site. One unresolved issue is the extent to which the loop bundle residues alsostabilize 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 thatreplacing the residues affects the activity of the procaspase as well as the mature caspase, with D169Aand 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, whencoupled 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-raycrystallographic structures of E167A, D173A, and Y203F caspases show that changes in the active siteenvironment may be due to the increased flexibility of several residues in the N-terminus of the smallsubunit. Overall, the results show that these residues are important for stabilizing the procaspase activesite as well as that of the mature caspase.