Disabling the folding catalyst is the last critical step in alpha-lytic protease folding

Abstract

Alpha-Lytic protease (alphaLP) is an extracellular bacterial pro-protease marked by extraordinary conformational rigidity and a highly cooperative barrier to unfolding. Although these properties successfully limit its proteolytic destruction, thereby extending the functional lifetime of the protease, they come at the expense of foldability (t(1/2) = 1800 yr) and thermodynamic stability (native alphaLP is less stable than the unfolded species). Efficient folding has required the coevolution of a large N-terminal pro region (Pro) that rapidly catalyzes alphaLP folding (t(1/2) = 23 sec) and shifts the thermodynamic equilibrium in favor of folded protease through tight native-state binding. Release of active alphaLP from this stabilizing, but strongly inhibitory, complex requires the proteolytic destruction of Pro. alphaLP is capable of initiating Pro degradation via cleavage of a flexible loop within the Pro C-terminal domain. This single cleavage event abolishes Pro catalysis while maintaining strong native-state binding. Thus, the loop acts as an Achilles' heel by which the Pro foldase machinery can be safely dismantled, preventing Pro-catalyzed unfolding, without compromising alphaLP native-state stability. Once the loop is cleaved, Pro is rapidly degraded, releasing active alphaLP.

Figure 1.

Identification of Pro degradation fragments created by αLP proteolysis. Pro (20 μM) refolds Int (2 μM) to native αLP, which in turn degrades Pro through a series of fragments identified using MALDI mass spectrometry and N-terminal sequencing (Table 1). Initial cleavage occurs within a flexible loop located in the Pro C-terminal domain. Pro alone shows no degradation under the same conditions.

Figure 2.

A time course of Pro degradation by αLP. The relative amounts of Pro and Pro fragments created during the catalyzed folding of Int (2 μM) by Pro (20 μM) are shown plotted as a function of time. The cleavage of full-length Pro (circles) occurs at approximately the same rate as the appearance of the 12.6-kD (squares), 9.32-kD (triangles), and 7.37-kD (diamonds) degradation fragments, as determined by single exponential fits of the data (0.63 ± 0.046 min⁻¹, 0.81 ± 0.15 min⁻¹, 0.94 ± 0.26 min⁻¹, and 0.78 ± 0.16 min⁻¹, respectively). Direct addition of native αLP resulted in vastly increased cleavage rates; yet, the extent of Pro degradation was only dependent on the final concentration of native αLP present (data not shown).

Figure 3.

ProTEVloop inhibition of native αLP. Increasing amounts of ProTEVloop_uncut (filled circles) and ProTEVloop_cut (open circles) reduce the proteolytic activity of 6 nM and 0.25 nM native αLP, respectively. ProTEVloop_uncut displays tight-binding inhibition of αLP (K_i = 0.26 ± 0.071 nM) similar to that of wild-type Pro (Table 2). Loop cleavage results in an approximately fourfold loss in affinity for native αLP (K_i = 1.4 ± 0.097 nM), with ProTEVloop_cut acting as a simple competitive inhibitor instead of a tight-binding inhibitor.

Figure 4.

The effect of loop cleavage on the catalyzed folding of αLP. (A) A full enzymatic profile of ProTEVloop_uncut-catalyzed αLP folding, in which catalyzed folding rate constants are plotted as a function of ProTEVloop_uncut concentration and the data are fit to a variant of the Michaelis-Menten equation, gives kinetic constants (k_cat and K_M) within error of those for wild-type Pro (Table 2). (B) The fraction of Int (0.8 μM) refolded to native αLP, both in the absence of catalyst (filled squares) and in the presence of either 3.75 μM intact (filled circles) or TEV-cleaved (open circles) ProTEVloop, is plotted as a function of time. The resulting progress curves illustrate that although ProTEVloop_cut accelerates folding (k_obs = 4.2×10⁻⁵ ± 0.29×10⁻⁵ min⁻¹) over that of the uncatalyzed reaction (inset), this rate of refolding is substantially slower than the ProTEVloop_uncut-catalyzed folding reaction (k_obs = 0.37 ± 0.036 min⁻¹). (C) The addition of increasing amounts of ProTEVloop_cut to wild-type Pro-catalyzed refolding reactions retards initial refolding rates, as ProTEVloop_cut competes for Int binding (K_i = 73 ± 13 μM), but cannot catalyze refolding to native αLP (see Table 2).

Figure 5.

Modeling of ProTEVloop-catalyzed αLP refolding demonstrates multiple turnover. The fraction of Int (0.8 μM) refolded to native αLP by 24 nM ProTEVloop_uncut, alone (filled circles) and in the presence of 9 μM ProTEVloop_cut (open circles), is plotted as a function of time with data generated from kinetic simulations of modeled ProTEVloop-catalyzed folding reactions (solid lines) for both refolding scenarios. The kinetic model is consistent with the observed folding behaviors, including the increase in αLP folding seen on addition of excess, but catalytically inactive, ProTEVloop_cut. Competition for native αLP binding appears to allow ProTEVloop_uncut to catalyze multiple rounds of αLP folding.