The Ca²⁺ Influence on Calmodulin Unfolding Pathway: A Steered Molecular Dynamics Simulation Study

<div><p>The force-induced unfolding of calmodulin (CaM) was investigated at atomistic details with steered molecular dynamics. The two isolated CaM domains as well as the full-length CaM were simulated in N-C-terminal pulling scheme, and the isolated N-lobe of CaM was studied specially in two other pulling schemes to test the effect of pulling direction and compare with relevant experiments. Both Ca²⁺-loaded CaM and Ca²⁺-free CaM were considered in order to define the Ca²⁺ influence to the CaM unfolding. The results reveal that the Ca²⁺ significantly affects the stability and unfolding behaviors of both the isolated CaM domains and the full-length CaM. In Ca²⁺-loaded CaM, N-terminal domain unfolds in priori to the C-terminal domain. But in Ca²⁺-free CaM, the unfolding order changes, and C-terminal domain unfolds first. The force-extension curves of CaM unfolding indicate that the major unfolding barrier comes from conquering the interaction of two EF-hand motifs in both N- and C- terminal domains. Our results provide the atomistic-level insights in the force-induced CaM unfolding and explain the observation in recent AFM experiments.</p> </div

The CaM structures and the simulation system setup.

<p>(A) the structure of the Ca2+-loaded full-length CaM, including two domains: N-lobe and C-lobe, and four EF-hand motifs: EF1, EF2, EF3, and EF4. EFβ-scaffold is a short β-sheet coupled two EF-hand motifs. (B) The sketch map of SMD simulation for Ca²⁺-loaded full-length CaM. Two red balls represent the pulled and constrained atoms, respectively. Black arrow represents the direction of the pulling force. The blue dots represent the water solvent. The green balls in both panels represent the Ca²⁺ ions.</p

MD simulation results of force-induced unfolding of isolated Ca²⁺-loaded and Ca²⁺-free N-lobe unfolding with non-N-C-terminal pulling directions using 4 ns snapshot of equilibration as the initial structure and ν = 5 Å/ns.

<p>External forces were applied on the N-lobe residue 17 (A, B) or residue 38(C, D) to unfold the sequence between the constrained C-terminal and the residue where force is applied. Left: (I) Force-extension curve; (II) Contact area between EF1 and EF2 of N-lobe; and (III) Backbone hydrogen bonds of EFβ-scaffold coupled EF1 and EF2. Right: snapshots of the unfolding trajectories. The external force was applied at the atoms presented with black solid circle (residues 17 or 38), and the C-terminal residue 74 is constrained.</p

MD simulation results of full-length CaM using 4 ns snapshot of equilibration as the initial structure and ν = 5 Å/ns: A) Ca²⁺-loaded state, and B) Ca²⁺-free state.

<p>Above: (I) the force-extension curve; (II) the contact area of two EF-hand motifs in either domain; (III) gyration radius of two domains; and the backbone hydrogen bonds of EFβ-scaffold in N-lobe(IV) and C-lobe(V). Bottom: Five structural snapshots correspond to the special force points labeled in the force-extension curves of Ca²⁺-loaded CaM and seven structural snapshots correspond to the special force points labeled in the force-extension curves of Ca²⁺-free CaM. In all structural snapshots, the N-terminal is at the left side and the C-terminal is at the right side. The forces are applied at the atoms presented with the black solid circle.</p

The influence of cell membrane and SNAP25 linker loop on the dynamics and unzipping of SNARE complex

<div><p>The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is composed of three neuronal proteins VAMP2, Syntaxin and SNAP25, which plays a core role during the process of membrane fusion. The zipping assembly of the SNARE complex releases energies and drives the vesicle and cell membrane into close proximity. In this study, we use all-atom molecular dynamics simulations to probe the dynamics of SNARE and its unzipping process in the context of membrane at the atomistic details. Our results indicated that the NTD of SNARE core domain is relatively more stable than CTD, which is in agreement with previous experiments. More importantly, possible interactions between the linker loop (LL) region of SNAP25 and VAMP2 are observed, suggests that the LL region may facilitate VAMP2 binding and SNARE initiation. The forced unzipping of SNARE in the presence of membrane and LL of SNAP25 reveals the possible pathway for energy generation of SNARE zipping, provides information to understand how force may regulate the cooperativity between the membrane and the SNARE complex.</p></div

Summary of MD simulations performed.

<p>Summary of MD simulations performed.</p

LL region of SNAP25 forms interactions with VAMP2.

<p>(A)snapshot of the free MD simulation. The interactions on the N-terminal residues of NTD region are highlighted, with VAMP2 residues 25 to 41 shown in gray and SNAP25 residues 125–130 shown in purple. A specific salt bridge between VAMP2 E41 and SNAP25 R124 is also indicated. (B)The time-course of the distance VAMP2 E41 and SNAP25 R124 for Free2, a distance below 3.2Å can be viewed as salt bridge fromed. (C)Number of contacts between of SNAP25 with VAMP2 residues 24 to 41.</p

The dynamics of each SNARE layer.

<p>(A)Averaged root mean squared fluctuation of each layer in the simulations. (B) Time-course of the numbers of hydrogen bond between the layer residues with their surroundings for each layers. (C)Average number of Hydrogen bond between the layer residues with their surroundings for the last 20 ns of the two simulation (80–100 ns).</p

The free molecular dynamics simulations.

<p>(A)The RMSD of three different simulations, resutls for SNARE core domain and LL region are shown. (B)The change of the angle between SNARE core domain and the membrane. (C)Change of the angle between the transmembrane domain of Syntaxin and the membrane.</p

SNARE complex and the simulation setup.

<p>(A)Schematic of ternary SNARE four helix bundle with Syntaxin(yellow), VAMP2(red), SNAP25(green). The notation of SNARE layers is indicated. The sequence of linker loop of SNAP25 is shown, 4 palmitoylated cysteines anchored to the membrane were shown in red. (B)Assembly of SNARE complex with the membrane, with the palmitoylated cysteines on SNAP25 highlighted. (C)The initial simulaiton system. The SNARE core domain is placed ~ 40° reletive to the membrane.</p