MD Simulations of the dsRBP DGCR8 Reveal Correlated Motions that may Facilitate pri-miRNA Binding.
DGCR8, a protein containing two dsRNA binding domains in tandem, is vital for nuclear maturation of primary miRNAs (pri-miRNAs) in connection with the RNase III enzyme Drosha. The crystal structure of the DGCR8 “Core” (493-720) shows a unique well ordered structure of the linker region between the two dsRBDs, which differs from the flexible linker connecting the two dsRBDs in the antiviral response protein, PKR. To better understand the interfacial interactions between the two dsRBDs, we have run extensive MD simulations of the isolated dsRBDs (505-583 and 614-691) and the Core. The simulations reveal correlated reorientations of the two domains relative to one another, with the well ordered linker and C-terminus serving as a pivot. The results demonstrate that motions at the domain interface dynamically impact the conformation of the RNA binding surface and may provide an adaptive separation distance necessary to interact with a variety of different pri-miRNAs with heterogeneous structures.
Figure 1.
A ribbon diagram representing the crystal structure of DGCR8 Core (PDB 2YT4, residues 505-701) shows a well structured linker (H3 and H4) and a C-terminal helix (H5, black). The arrows indicate the proposed RNA binding sites. The portion of DGCR8 studied here can be seen in the schematic.
The interfacial interactions of DGCR8 were studied using MD simulations of three different constructs derived from the crystal structure of the RNA-free Core (PDB 2YT4): DGCR8-dsRBD1 (505-583), DGCR8-dsRBD2 (614-691), and DGCR-Core (505-701). Two loops are absent in the crystal structure due to low electron density and were therefore modeled back into the structure before running the simulations (see methods). Analysis proceeded following the calculation of 250 ns isothermal-isobaric (NPT) trajectories of each construct.
Protein stability was checked by analyzing the backbone RMSD from the starting crystal structure over the course of the trajectory. The high RMSD seen in the Core simulation is sufficient to cause concern in a single globular domain that the structure is unstable. However, DGCR8 is not a single global domain and the somewhat large RMSD is reasonable for a multiple domain protein if it can be attributed to the two domains reorienting themselves relative to each other, while still retaining their overall structure. Mathematically, this would tend to inflate the RMSD because no single reference structure would exist that serves well for the RMSD calculation over the entire time course.
Figure 2. RMSD traces show the overall stability of the dsRBDs during the MD simulations and highlight the rearrangement of the domains relative to each other in the Core simulation. Ribbon bundles show that the large Core RMSD is not due to local unfolding of the linker, but rather to rigid body reorientation of the two dsRBDs. Both bundles are created by taking structures from the simulation every 50 ns. For full details, please see our publication (referenced below).
The bundles shown in Figure 2, generated by superimposing only RBD1 (D) or only RBD2 (E), rule out local unfolding of the secondary elements in the linker, as they are clearly retained. Additionally, from the ribbon bundles we find that the reason that dsRBD2 has a higher RMSD than dsRBD1 is because loop 1′ of dsRBD2 fluctuates more than loop 1 of dsRBD1 when superimposed. Thus from the ribbon bundle structures, we have shown that the high RMSD from the Core is not from the structure being unstable, but the rigid body movement of the domains relative to each other. These motions are intriguing, because they have the potential to dynamically adjust the separation distance and orientation of the two RNA binding surfaces that must work together during miRNA binding
Correlated binding domain motions are demonstrated in Figure 3, based on anharmonic normal mode (ANM) calculations (left) and isotropically distributed ensemble (IDE) analysis of the Core MD trajectory (right). The largest amplitude eigenmode of the ANM calculation features correlated hinging motion in the dsRBDs, reminiscent of a butterfly flapping its wings. The motions of the domains are centered on the α-helix formed by the C-terminal residues that packs between the dsRBDs. Keeping with the butterfly analogy, the two wings (formed by the two dsRBDs) “flap” in an anti-correlated movement that changes the distance between the two proposed RNA binding surfaces, rather than in a correlated twisting motion that would leave the distance between the binding surfaces unchanged. The largest amplitude internal eigenmode of the IDE analysis confirms the overall features of the ANM analysis. These two methods differ fundamentally in both the method of calculation and force field employed, strongly supporting the physical significance of the results.
Figure 3. Correlated motion of the two dsRBDs as predicted by ANM analysis of the starting structure (left) and IDE analysis of the trajectory (right) indicates that collective dynamics may play an important role in miRNA binding by DGCR8.
Our work demonstrates that motions at the domain interface dynamically impact the conformation of the RNA binding surface and may provide an adaptive separation distance necessary to interact optimally with a variety of different pri-miRNAs with heterogeneous structures.
For experimental details and full resolution images, please see:
Wostenberg, C. & Showalter, S.A. (2010) “MD Simulations of the dsRBP DGCR8 Reveal Correlated Motions that may Facilitate pri-miRNA Binding.” Biophys. J., 99, 248-256.