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FIG. 1. Free-energy diagram of aLP folding with and without its pro region at 4°C. In the absence its pro region (P), unfolded aLP (U) spontaneously folds to a molten globule-like intermediate (I), which proceeds at an extremely slow rate to N through a high-energy folding TS. The addition of pro region provides a catalyzed folding pathway (denoted by dashed lines) that lowers the high folding barrier and results in a thermodynamically stable inhibition complex N-P. * indicates measurement at 25°C. (Modified from ref. 6.)

minimum free energy. In fact, because of the marginal stability of I, the N state is actually significantly less thermodynamically stable than either the I or U states.

To surmount the high barrier to folding and the extraordinary thermodynamic instability of the native state, aLP has coevolved the pro region, which can assist the folding of aLP when supplied in cis or in trans. Addition of Pro to I results in rapid folding to the N state (0.037 s–1) and recovery of functional protease (Fig. 1; ref. 6). Pro acts as a foldase, facilitating aLP folding by binding tightly to the folding transition state of the protease, lowering the barrier by 18.2 kcal/mol. In this manner, Pro serves as a potent catalyst, increasing the rate of aLP folding by 3×109. In addition, Pro is the tightest binding inhibitor known for the native protease (Ki=3×10–10 M; refs. 8 and 9), making Pro a single-turnover catalyst. This tight binding serves a critical function in aLP folding by shifting the thermodynamic equilibrium in favor of folded aLP (Pro·N is 3.4 kcal/mol more stable than Pro-I; Fig. 1).

The product of the folding reaction is not active aLP but the inhibitory complex. Release of active aLP requires that the Pro region be removed by proteolysis. Once Pro is degraded, the active protease becomes kinetically trapped in the metastable N state, with the high barrier preventing unfolding to the more thermodynamically favored unfolded states. In this way, promediated folding provides the only efficient means of folding aLP to its metastable native conformation.

Structures of Pro and Pro·aLP Complex. Recently determined crystal structures of Pro and the Pro·N complex illuminate Pro·aLP interactions (10). Alone, Pro adopts a novel C shaped a/ß-fold, consisting of an N-terminal helix, two compact globular domains (N domain, C domain) connected by a nearly rigid hinge region, and a C-terminal tail (Fig. 2a). Each globular domain contributes a three-stranded ß-sheet to the concave surface of the molecule and at least one a-helix that packs against these ß-sheets to form the convex surface. The N-terminal helix appears highly flexible, changing orientations in different crystal environments. Two of the three Pro molecules in the crystallographic asymmetric unit show different conformations for the N-terminal helix, whereas the third molecule reveals the helix to be disordered. Similarly, the

FIG. 2. (a) Topology of Pro as described in the text. A disordered loop in the Pro C domain is shown in red. (b) Schematic of primary sequence alignments of pro regions from nine bacterial serine proteases. Alignments were determined by using the aLP Pro structure as a guide. Regions of sequence homology correspond to specific secondary structures in the Pro structure, with the Pro C-terminal domain being the most conserved region. N-terminal sequences lacking homology are depicted by thin black lines. aLP, Lysobacter enzymogenes aLP (17); SGPC, Streptomyces griseus protease C (18); RPI, Rarobacter faecitabitus protease I (19); SGPD, S.griseus protease D (20); SGPE, S.griseus protease E (21); TFPA, Themomonaspora fusca serine protease (22); SAL, Streptomyces lividans protease (23); SGPA, S.griseus protease A (24); SGPB, S.griseus protease B (24).



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