14 research outputs found
In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.
The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum) plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L.) plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family
The backbone RMSD of PSI monomers as a function of time.
<p>Backbone RMSD of the PSI monomer at pH 4.5 (blue line) and pH 7.4 (orange line) are presented. The PSI monomer maintains its overall tertiary structure at both pH 4.5 and pH 7.4 similar to that of the native dimer structure until an abrupt change in RMSD at 230 ns and 198 ns for pH 4.5 and pH 7.4, respectively. At these times, the PSI jackknifes closed and adopts saposin-like fold characteristic of all known SAPLIP members.</p
Contribution of the first twenty PCA eigenvectors to the overall closing motion of the PSI.
<p>The contribution of the first 20 PCA eigenvectors to the closing motion of the PSI at active (<b>A</b>) and inactive (<b>B</b>) pH are presented. The first eigenvectors contribute 78.8% and 74.2% of the overall motions for the pH 4.5 and pH 7.4 simulations, respectively, and correspond to the collapse of helices α1/α2 onto helices α3/α4.</p
The potential of mean force (PMF) as a function of the distance between the COMs of PSI monomers.
<p>The PMF as a function of the intra-peptide distance between the PSI dimer monomers at pH 3.0 (blue line) and pH 7.4 (orange line) revealed that dissociation of the dimer requires increased energy as pH is lowered from pH 7.4 (95.7 kJ mol<sup>−1</sup>) to pH 3.0 (108.8 kJ mol<sup>−1</sup>), possibly the result of charge neutralization of carboxylate groups at acidic pH thereby minimizing charge-charge repulsion.</p
Structures of the PSI and orthorhombic Sap C.
<p>The crystal structure of potato (<i>Solanum tuberosum</i>) PSI (PDB ID 3RFI, <b>A</b>), with the missing linker region modelled and orthorhombic Sap C (PDB ID 2QYP, <b>B</b>) are presented. Like its Sap C homologue, potato PSI was crystalized as an extended dimer. The hinge-bending capability of the PSI is made possible by the flexible helix-helix junctions formed between α1/α2 and α3/α4, indicated by dashed arrows.</p
Radius of gyration (R<sub>g</sub>) of the PSI over the time course of the simulations.
<p>R<sub>g</sub> of the PSI at pH 4.5 (blue line) and pH 7.4 (orange line) as a function of time. In either case, the PSI was free to move in the extended state. Upon adoption of a saposin-like fold, the collapse of the hydrophobic concave face of the PSI onto itself limits movement, thereby restricting water access to the hydrophobic core.</p
The backbone RMSD of the PSI dimer as a function of time.
<p>Backbone root-mean-square deviation (RMSD) of the PSI dimer at pH 3.0 (<b>A</b>) and pH 7.4 (<b>B</b>) indicated little deviation, evidenced by the low RMSD of the PSI backbone atoms for both peptides comprising the PSI homodimer. Colours identify the individual peptide chains within the dimer.</p
The C<sub>α</sub> root-mean-square fluctuations (RMSFs) of the PSI dimer as a function of time.
<p>The C<sub>α</sub> RMSFs for helices at pH 3.0 (<b>A</b>) and pH 7.4 (<b>B</b>) were consistent throughout the time course of the simulations, remaining below 5 Å for the helical regions. Fluctuations of up to 10 Å were noted for the flexible linker region, in agreement with the hypothesis that the linker region is intrinsically disordered, providing evidence of PSI dimer secondary structure stability regardless of pH. Colours identify the individual peptide chains within the dimer.</p
Comparison of folded potato PSI to other SAPLIPs.
<p>Structural comparison of the folded potato PSI at pH 4.5 (blue) and pH 7.4 (green), averaged over the last 200 ns of the simulation trajectories, to the crystal structure of barley PSI (PDB ID 1QDM, magenta) and the crystal structure Sap C (PDB ID 2GTG, red). Potato PSI simulated at pH 7.4, simulated with parameters closely resembling the experimental parameters used for both barley PSI and Sap C, exhibited a compact globular structure consisting of a distorted four-α-helix bundle characteristic of other SAPLIPs. Potato PSI simulated at pH 4.5 adopted a compact four-α-helix bundle structure not previously observed for any SAPLIP. The linker regions of potato PSI are omitted for clarity.</p
Two-dimensional projections of the first two eigenvectors of the PSI monomer.
<p>Projection of the first two eigenvectors of the unbiased PSI simulations at pH 4.5 (<b>A</b>) and pH 7.4 (<b>B</b>). Both simulations transited from the extended dimer-like structure to a saposin fold-like conformation over the course of the 500 ns trajectories. The inactive pH simulation transited through three distinct clusters whereas the active pH simulation transitioned through several microstates before becoming trapped in the last densely populated cluster. The differences in the essential subspace sampled by the two differing pH ranges may be due to unspecific (hydrophobic) interactions sampled in the pH 4.5 simulations where charge neutralization minimizes like-charge repulsions.</p