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.

Fig. 1. Architectures of the substrate-bound Lon^ENZ and substrate-free, Lon^OFF configurations.

(A) Cutaway view of the substrate-bound Y. pestis Lon^ENZ atomic model (center) flanked by orthogonal exterior views of the ATPase (left) and protease (right) domain rings. Each subunit of the homohexamer is assigned a distinct color depending on its position in the spiral staircase, and the cryo-EM density of the substrate is shown as a solid isosurface colored orange. Nucleotides are depicted using a sphere representation. (B) The different orientations of individual protomers relative to the protease domain, produced by orienting all the protease domains to a common view. Subunits are colored as in (A). The descending and ascending movements of the NTD^3H and ATPase domains relative to their proteases are accentuated by dashed lines shown above the NTD^3H. Dihedral angle measurements between ATPase and protease domains demonstrate a gradual expansion of descending subunits in the spiral staircase and compression in the two seam subunits. (C) Cutaway view of the substrate-free Y. pestis Lon^OFF atomic model (center) flanked by orthogonal exterior views of the ATPase (left) and protease (right) domain rings. Subunits are colored to correspond to their position in the Lon^ENZ staircase architecture after transitioning. Nucleotides are depicted using a sphere representation. (D) The similar orientations of the individual protomers of the substrate-free structure are produced by orienting all the protease domains to a common view without changing axial position. The descending movement of the subunits is accentuated by dashed lines shown above the NTD^3H. Dihedral angle measurements between ATPase and protease domains demonstrate subtle changes in the expansion and compression of the six descending subunits.

Fig. 2. Substrate translocation is mediated by residues in pore loop 1 and the NTD^3H.

(A) Top: Cutaway view of the Lon^OFF cryo-EM density with the NTD^3H colored light blue, ATPase domains colored light gray, and protease domains colored dark gray. Bottom: Cutaway view of the substrate-bound Lon cryo-EM density with NTD^3H colored light blue, ATPase domains colored light gray, protease domains colored dark gray, and substrate colored orange. Sphere representations of spiraling methionine (M284) side chains in the NTD^3H and tyrosine residues of pore loop 1 are colored dark blue and hot pink, respectively. (B) The NTD^3H pores of substrate-free (top) and substrate-bound Lon (bottom) shown as a molecular surface representation and colored by subunit as in Fig. 1A. During substrate translocation, NTD^3H forms a pore above the central channel. (C) A seven-residue polyalanine chain is modeled into the substrate density found in the closed Lon structure shown in a transparent orange surface representation. Y398 and I399 from pore loop 1 are shown using stick representations with associated cryo-EM density zoned around these residues in gray. While Y398 and I399 show intercalating, zipper-like interactions with substrate in ATP1 to ATP3, Y398, and I399 of pore loop 1 in ATP′ are positioned further away from the central channel. Pore loop 1 residues in the seam subunits (ADP1 and ADP2) are detached from the substrate.

Fig. 3. Lon proteolytic active site forms cleft for substrates in Lon^ENZ and is autoinhibited in Lon^OFF.

(A) An axial view of the chamber of the sixfold symmetric protease ring from the substrate-bound Lon^ENZ structure is shown as a molecular surface with subunits are colored as in Fig. 1, with the six catalytic serine-containing loops (673 to 677) emphasized using a more saturated color. These loops organize into a ring-like assembly, generating a series of substrate-binding grooves. (B) A cutaway side view of the substrate-bound structure shown as a molecular surface. A close-up view of the cryo-EM density of the proteolytic active site is shown to the right as a gray mesh, with the atomic model showing the catalytic dyad (magenta) and serine-containing loop (purple stick representation). The serine-containing loop is likely stabilized in this extended conformation, in part, by hydrogen bonding between D676 (pink) and the peptide backbone of a nearby loop (light purple). (C) An axial view of the chamber of the protease ring from the Lon^OFF structure is shown and colored as in (A). The serine-containing loops are no longer interacting because of the separation of the protease domains in this conformation. (D) A cutaway side view of the substrate-free structure showing the location of the protease active sites in the open, exposed proteolytic chamber. A close-up view of the cryo-EM density of the proteolytic active site is shown as in (B), showing that in Lon^OFF, the catalytic serine-containing loop adopts a 3₁₀ helix that sterically occludes the proteolytic active site. D676 and K722 are within hydrogen-bonding distance, further limiting cleavage by the catalytic dyad. (E) A five–amino acid polyalanine peptide (orange) was modeled into the substrate-binding groove of Lon^ENZ (represented using a transparent space-filling representation of the atomic model), based on the position of bortezomib covalently bound to S679 (PDB: 4YPM) (57). This demonstrates how an unfolded peptide substrate putatively docks into the active site for proteolytic cleavage by Lon protease. (F) The rearrangement of the serine-containing loop during the transition from Lon^OFF (dark gray/purple) to Lon^ENZ (light gray/purple). This rearrangement is also shown in movie S2.

Fig. 4. The PS1βH motif of Lon connects pore loop 1 to adjacent nucleotide-binding pockets.

(A) Individual units of the AAA+ domains from the Lon^ENZ structure are aligned and shown overlaid using a ribbon representation. The two units are (i) the small ATPase subdomains (left) and (ii) the NTD^3H and large subdomain. Each subunit is colored according to the same color scheme assigned in Fig. 1. Superimposing these subdomains shows an average Cα RMSD of 0.81, indicating that these regions individually move as rigid units throughout the hydrolysis cycle. (B) ATP1 and ATP2 subunits are highlighted using a coil representation in the context of Lon structures (smoothed surface representation, ATPase domains are white, and protease domains are gray). The PS1βHs are colored purple and pink in ATP1 and ATP2, respectively. Sensor-1 at the C-terminal base of the PS1βH is denoted in ATP2. Close-ups of the regions enclosed by the boxes are shown in (C) and (D). (C) Y456, at turn of the PS1βH, stabilizes the NTD^3H through proximal interactions with conserved aromatic residues Y294 and W297. (D) Pore loop 1 interactions with substrate are stabilized through interactions between E458 in the PS1βH and two tandem arginine residues in pore loop 1. (E) Remodeling cis and trans subunit interactions in the nucleotide-binding pocket drives sequential ATP hydrolysis cycle and stepwise substrate translocation. In the nucleotide-binding pocket of two adjacent ATP-bound subunits (e.g., ATP1 and ATP2; left), nucleotide is stabilized by interactions with the arginine finger (R484) in trans and sensor-2 (R542) in cis. Furthermore, a bridging glutamate residue (E447) at the N-terminal base of the PS1βH motif engages a cluster of basic residues within the nucleotide-binding pocket, stabilizing the intersubunit interface. In the ATP′ nucleotide-binding pocket (middle), this organization is disrupted upon E447 being retracted from the nucleotide-binding pocket upon nucleotide hydrolysis, causing the subunit to compress and bringing sensor-2 and other motifs involved in ATP hydrolysis (i.e., Walker A and Walker B motifs) in the proximity of the bound nucleotide. The ADP1 (right) and ADP2 (not shown) nucleotide-binding pockets reveal a similar organization to that observed in ATP′, indicating that nucleotide exchange is necessary to “reset” the hydrolysis cycle.

Fig. 5. Summary of the mechanism of substrate translocation in Lon protease.

(A) In the absence of substrate, Lon that is fully ADP-bound adopts a left-handed, open lock washer configuration with autoinhibited protease active sites. Simultaneous ATP and substrate binding to the uppermost protomer initiates the reorganization toward the proteolytically active Lon^ENZ conformer. Subunits then progressively exchange ADP for ATP in a counterclockwise manner. The Lon^ENZ conformer is achieved upon binding a total of four ATP molecules, finalized with one hydrolysis event to close the ring. Hydrolysis occurs in the subunit that had previously been at the top of the spiral (green) when it reaches the lowest position of the ATPase spiral within the closed hexamer. The rearrangement results in a seven to eight-residue translocation of the substrate peptide, which is now threaded through the center of the central ATPase channel. (B) The Lon^ENZ conformation is competent for hand-over-hand translocation, and the protease domains form an enclosed sixfold symmetric ring and with active protease sites. Around-the-ring ATP hydrolysis drives substrate translocation, with the pore loops of the ATP-bound subunits engaging with substrate (substrate-interacting pore loops shown using space-filling representation). ATP binding and hydrolysis at opposite sides of the ring result in rigid-body movements of the three upper-most ATP-bound subunits (dotted outline), leading to two-residue translocation steps toward the protease. (C) Cutaway view showing progression of substrate along the central axis, being translocated two amino acids per hydrolysis event, and substrate binding within the active site for cleavage. As in (B), substrate-interacting pore loops shown using space-filling representation. Substrate is directed to the protease, where it can be positioned for cleavage. A summary of the Lon^ENZ mechanochemical cycle is shown in movie S1. (D) Inability of the pore loop 1 of an ADP-bound subunit to bind substrate either due to reaching the terminus of a substrate or encountering a tightly folded domain will trigger a return to the Lon^OFF conformation. The remaining ATP- and substrate-bound subunits will continue around-the-ring hydrolysis and translocation, but each will remain in the ADP-bound conformation after nucleotide hydrolysis. Remaining nonproteolyzed substrate peptides are released. A summary of the entire conformational cycle is shown in movie S3.