Thermodynamic, enzymatic and structural effects of removing a salt bridge at the base of loop 4 in (pro)caspase-3.
Interactions between loops 2, 2' and 4, known as the loop bundle, stabilize the active site of caspase-3. Loop 4 (L4) is of particular interest due to its location between the active site and the dimer interface. We have disrupted a salt bridge between K242 and E246 at the base of L4 to determine its role in overall conformational stability and in maintaining the active site environment. Stability measurements show that only the K242A single mutant decreases stability of the dimer, whereas both single mutants and the double mutant demonstrate much lower activity compared to wild-type caspase-3. Structural studies of the caspase-3 variants show the involvement of K242 in hydrophobic interactions that stabilize helix 5, near the dimer interface, and the role of E246 appears to be to neutralize the positive charge of K242 within the hydrophobic cluster. Overall, the results suggest E246 and K242 are important in procaspase-3 for their interaction with neighboring residues, not with one another. Conversely, formation of the K242-E246 salt bridge in caspase-3 is needed for an accurate, stable conformation of loop L4 and proper active site formation in the mature enzyme.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695-7622, USA.
Jad
J
Walters
Paul
P
Swartz
Carla
C
Mattos
A Clay
AC
Clark
D053148:Caspase 3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D053148:Caspase 3
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