Structural basis for distinct operational modes and protease activation in AAA+ protease Lon

Abstract

Substrate-bound structures of AAA+ protein translocases reveal a conserved asymmetric spiral staircase architecture wherein a sequential ATP hydrolysis cycle drives hand-over-hand substrate translocation. However, this configuration is unlikely to represent the full conformational landscape of these enzymes, as biochemical studies suggest distinct conformational states depending on the presence or absence of substrate. Here, we used cryo–electron microscopy to determine structures of the Yersinia pestis Lon AAA+ protease in the absence and presence of substrate, uncovering the mechanistic basis for two distinct operational modes. In the absence of substrate, Lon adopts a left-handed, “open” spiral organization with autoinhibited proteolytic active sites. Upon the addition of substrate, Lon undergoes a reorganization to assemble an enzymatically active, right-handed “closed” conformer with active protease sites. These findings define the mechanistic principles underlying the operational plasticity required for processing diverse protein substrates.

Modular and coordinated activity of AAA+ active sites in the double-ring ClpA unfoldase of the ClpAP protease

“…A competing model posits that probabilistic ATP hydrolysis at many positions within a AAA+ ring can drive a power stroke, thereby eliminating the requirement for adjacent subunits to fire in a strictly sequential pattern ( 15 , 16 ). This model is also consistent with observed states in recent cryo-EM structures ( 42 – 44 ), mixed-ring experiments showing that ClpX and HslU can function with only a subset of ATPase active AAA+ modules ( 15 , 16 ), and single-molecule experiments that demonstrate ClpXP translocation occurs with different step sizes in a stochastic pattern ( 45 , 46 ). Our ClpA experiments lend strong support that ClpA and ClpAP function via a nonstrictly sequential model for both ATP hydrolysis and work by the D1 and D2 rings.…”

Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

“…This findings indicates that the two threonine residues are likely to play an important role in the threading process, possibly via specific interactions with residues in Rca's pore-loop 1 and 2 (20,21). We also further note that the observed 2 amino acid step interval would be consistent with successive zipper-like interactions that have been described to be utilized for substrate engagement and threading by the central pore in multiple other AAA+ proteins (38)(39)(40)(41)(42).In RbcL, the hydroxl side chain of Y20 forms a hydrogen bond to E60, a key catalytic residue that interacts with K334 that is positioned at the apex of Loop 6, and thought to orient the CO 2 molecule for gas addition (43). We hypothesized that this interaction could act to disrupt the active site when the N-terminus is displaced by Rca-threading.…”

Structures of the Human LONP1 Protease Reveal Regulatory Steps Involved in Protease Activation

“…1 and Supplementary Table 1). The organization is reminiscent of the left-handed, open lockwasher configurations previously observed for substrate-free bacterial Lon 26,28 , although human LONP1 appears to oligomerize with a slightly smaller helical pitch than the bacterial forms (Fig. 1c,d and Supplementary Fig.…”

“…Interestingly, the identified interactions between the pore loop 2 aromatics of ATP1 and ATP2 and the translocating substrate extend the overall interactions between human LONP1 and substrate deeper into the central chamber. The presence of a methionine, which cannot establish intercalating interactions with substrate, at the equivalent residue in Y. pestis Lon (M432), is likely responsible for a lack of observable pore loop 2 interactions in its structure 28 ( Fig. 2b and Supplementary Fig.…”

“…The LONP1 protease consists of an N-terminal domain involved in substrate recognition and oligomerization, a AAA+ (ATPases associated with diverse cellular activities) domain that that powers substrate translocation, and a C-terminal serine protease domain involved in substrate proteolysis 1,4,18 . Numerous biophysical approaches have been employed to gain insight into the macromolecular structure and function of bacterial Lon [19][20][21][22][23][24][25][26][27] , and recent cryo-electron microscopy (cryo-EM) studies of bacterial Lon in both substrate-free and substrate-bound conformations revealed the structural basis for substrate processing and protease activation 28 . However, few structural studies have been directed towards understanding the molecular mechanism of protease activation and substrate processing by human LONP1.…”