The Role Of Packing Defects In The Stability And Function Of The Intramembrane Protease Glpg

Although proteins are efficiently packed, structural analysis has revealed that packing defects (i.e., voids, tunnels and pockets) are prevalent inside proteins despite their unfavorable contribution to protein stability. It has been speculated that those packing defects may be necessary for ligand binding, transport or conformational changes required for function. Despite the potential importance, the role of packing defects in the stability and function of proteins is not clearly understood especially for membrane proteins. Here, using the rhomboid intramembrane protease GlpG as a model, we test the hypotheses that 1) improving the packing through cavity-filling substitution can be a general way to stabilize a membrane protein, and 2) if packing defects are critical for function, it would be possible to lock the protein conformation into either inactive or constitutively active state through engineering of the cavities. We first identified packing defects in several rhomboid proteases from different origins by homology modeling, and mapped conserved cavities onto the structure of GlpG. Then we designed cavity-filling substitutions, and measured the stability and activity of those variants. Among the 18 “cavity-filling” substitutions tested, three were significantly destabilizing (ΔΔGoU u003e 1 kcal/mol), three were moderately stabilizing (ΔΔGoU =0.6∼1.0 kcal/mol), and the rest retained the stability to the level similar to wild-type. Among the three stabilizing substitutions, one was fully active while the other two were inactive. Those two inactivating substitutions did not involve the active-site residues but targeted the sites that belonged to the previously identified more flexible regions in GlpG. This result suggests that the packing defects may be required for the movement of structural elements of proteins, and implies that protein packing may have evolved for function through the delicate balance between stability and flexibility.