Helix unfolding/refolding characterizes the functional dynamics of Staphylococcus aureus Clp protease

Abstract

The ATP-dependent Clp protease (ClpP) plays an essential role not only in the control of protein quality but also in the regulation of bacterial pathogen virulence, making it an attractive target for antibacterial treatment. We have previously determined the crystal structures of Staphylococcus aureus ClpP (SaClpP) in two different states, extended and compressed. To investigate the dynamic switching of ClpP between these states, we performed a series of molecular dynamics simulations. During the structural transition, the long and straight helix E in the extended SaClpP monomer underwent an unfolding/refolding process, resulting in a kinked helix very similar to that in the compressed monomer. As a stable intermediate in the molecular dynamics simulation, the compact state was suggested and subsequently identified in x-ray crystallographic experiment. Our combined studies also determined that Ala(140) acted as a "hinge" during the transition between the extended and compressed states, and Glu(137) was essential for stabilizing the compressed state. Overall, this study provides molecular insights into the dynamics and mechanism of the functional conformation changes of SaClpP. Given the highly conserved sequences of ClpP proteins among different species, these findings potentially reflect a switching mechanism for the dynamic process shared in the whole ClpP family in general and thus aid in better understand the principles of Clp protease assembly and function.

Keywords: Bacterial Pathogenesis; Clp Protease; Computational Biology; Crystal Structure; Energy Landscape; Molecular Dynamics; Principle-Component Analysis; Protein Dynamics.

FIGURE 1.

Three states observed in ClpP. A, structure of SaClpP (Protein Data Bank code 3STA) showing the ClpP tetradecamer in the extended state. The axial N-terminal loops are shown in green, the head domain is shown in cyan, and the handle domain is shown in magenta. B, structure of SaClpP (Protein Data Bank code 4EMM) showing ClpP in the compact state. The color coding used is the same as that in A. The broken line in magenta represents the disordered part of the handle domain (bottom). C, structure of SaClpP (Protein Data Bank code 3ST9) showing the enzyme in the compressed state. D, alignment of three states. The extended, compact, and compressed monomers are shown in pink, purple, and magenta, respectively.

FIGURE 2.

Helical unfolding/refolding process of ClpP extended monomer. A, Cα RMSD values of the head domain and handle domain in ClpP^ex/mono versus simulation time. The RMSD values of the head domain and handle domain are shown in green and red, respectively. B, secondary structures as a function of time for ClpP^ex/mono in trajectory as calculated using DSSP. The structures were analyzed every 100 ps. C, snapshot structures of ClpP^ex/mono extracted from the trajectory in which the secondary structure was obtained using DSSP analysis.

FIGURE 3.

Conformational changes of oligomeric SaClpP. A, the catalytic triads and Asp¹⁷⁰-Arg¹⁷¹ network in extended SaClpP are shown along the axial inside proteolytic chamber. The three monomers in contact with each other are colored in green, cyan, and pink. B, Cα RMSD values of the head domain and handle domain of ClpP^ex (top) and ClpP^{ex/D170A/R171A} (bottom) versus simulation time, respectively. C, motions corresponding to PC1 of ClpP^ex/hepta. D, motions corresponding to PC1 of one monomer extracted from ClpP^ex/hepta. E, height of ClpP^ex/hepta (excluding the N-terminal loops) versus simulation time. The starting structure is shown in the left panel, whereas the structure of the 1500-ns snapshot is shown in the right panel. F, radius of ClpP^ex/hepta versus simulation time. The N-terminal loops define a pore surrounded by Asp¹⁹. The whole structure is surrounded by Asp¹⁶³. Top, radius of the whole structure. Bottom, radius of N-terminal pore. G, the surface presentation of the extended (left) and compressed SaClpP (right). The detailed protocol for estimating the radius is the same as in F.

FIGURE 4.

Intermediate compact state during the transition between the extended and compressed states. A, top, energy landscape for the conformational transition of ClpP^ex/mono. Reaction coordinates were defined according to PC1 and PC2 obtained from PCA. Bottom, typical conformations of local energy minima. B, structural superimposition of compact states from several organisms and the second energy minimum structure of the energy landscape. C, structural superimposition of compressed state and the third energy minimum structure of the energy landscape. D, hydrolysis of the fluorogenic peptide Suc-LY-AMC by wild-type SaClpP and two mutants (A140G and A140P). Kinetic constants were determined with the GraphPad Prism 5 software by plotting enzyme velocity against substrate concentration. Error bars (S.E.) are shown based on three independent repeats. RFU, relative fluorescence units.

FIGURE 5.

Role of E137 in the stabilization of the compressed state. A, conformational stabilization of handle helix E. The residues involved in anchoring helix E in a kinked conformation are shown as sticks, with the hydrogen bonds depicted as black dashed lines. B, overlay of all seven monomers of the compressed SaClpP. Glu¹³⁷ is shown in a stick representation. C, distance of atom OE1 in Glu¹³⁷ and OD1 in Asp³⁸ versus simulation time. D, overall superimposition of the two rings from the E137A mutant (slate) and wild-type extended SaClpP (pink) is presented in cylindrical schematics. The superimposition was carried out using PyMOL and yielded a small RMSD (0.22 Å for the tetradecamer), indicating high similarity between these structures. E, secondary structures as a function of time for ClpP^cpr/mono and ClpP^cpr/E137A in trajectories as calculated using DSSP. Top, profile of secondary structure transformation of ClpP^cpr/mono. Bottom, profile of secondary structure transformation of ClpP^cpr/E137A.

FIGURE 6.

Postdegradation and recovery in the functional cycle of ClpP. Structures of the three SaClpP states showing four monomers are presented as schematic illustrations (top). Surface traces are also shown. The N-terminal loops are in green, the head domain is in cyan, and the handle domain is in magenta. The Asp¹⁷⁰-Arg¹⁷¹ network is shown as a closed lock, and its destruction is shown as an open lock. Glu¹³⁷ is shown as an orange hook. Residues interacting with Glu¹³⁷ are shown as purple circles. The structures of the three SaClpP states in the monomer are shown as schematic traces (bottom) and colored as described above.