Supplemental Material

This article contains the following supporting material:

• Sec14p Conformational Dynamics Video
• Simulation Of Sec14p Conformational Dynamics Video
• Simulation Of Sec14p Conformational Dynamics B Video
• Figure S1 - Helix A₁₀/T₄ moves as a rigid body toward helix A₉ and away from helix A₁₂. (A) Distances between the Cα-atoms of helix A₁₀/T₄ residue K₂₂₉ and helix A₁₂ residue E₂₅₀ are plotted as a function of MD simulation time (black). This plot reports the distances between the C-terminal end of helix A₁₀/T₄ and helix A₁₂. A similar plot between the Cα-atoms of helix A₁₀/T₄ residue F₂₃₁ and helix A₉ residue R₁₉₅ as a function of MD simulation time is also shown (gray). The R₁₉₅-F₂₃₁ distances report ‘status-of-closure’ for the A₁₀/T₄/A₁₁ helical gate. A backbone rendition of Sec14p viewed from the posterior highlights residues K₂₂₉ (green sphere), E₂₅₀ (red sphere), and G₂₆₆ (blue sphere), as indicated. (B) Helices A₉ and A₁₂ remain relatively fixed during the simulation. Distances between the Cα-atoms of helix A₉ residue R₁₉₅ and helix A₁₂ residue E₂₅₀ are plotted as a function of MD simulation time. This plot reports the distances between the helices A₉ and A₁₂. An en face backbone rendition of Sec14p highlights residues R₁₉₅ (purple sphere) and E₂₅₀ (red sphere), as indicated. (C) Distances between the Cα-atoms of helix A₁₀/T₄ residue K₂₂₉ and helix A₁₂ residue E₂₅₀ (black) are plotted as a function of MD simulation time for Sec14p^G266D. As in (A) for Sec14p, this plot reports the distances between the C-terminal end of helix A₁₀/T₄ and helix A₁₂ in Sec14p^G266D. The R₁₉₅-F₂₃₁ ‘status-of-closure’distances as a function of MD simulation time are also shown (gray). A backbone rendition of Sec14p viewed from the posterior highlights residues K₂₂₉ (green sphere), E₂₅₀ (red sphere), and G₂₆₆ (blue sphere), as indicated.
• Figure S2 - The N-terminus of helix A₁₀/T₄ is relatively fixed. The indicated distance monitoring plots of Cα-backbone residues for Sec14p are in black and the corresponding Sec14p^G266D plots are in gray. (A) Helices A₁₀/T₄ and β-strand B₄ remain fixed during the simulation. Distances between the Cα-atoms of helix A₁₀/T₄ residue F₂₂₁ and β-strand B₄ residue I₂₁₄ are plotted as a function of MD simulation time. This plot reports the distances between the helix A₁₀/T₄ and β-strand B₄. The distances remain relatively constant throughout the simulation. An en face backbone rendition of Sec14p highlights residues I₂₁₄ (gold sphere) and F₂₂₁ (green sphere), as indicated. (B) The termini of helices A₁₀/T₄ and A₉ remain fixed during the Sec14p simulation but slide towards each other in the Sec14p^G266D simulation. Distances between the Cα-atoms of helix A₁₀/T₄ residue F₂₂₁ and helix A₉ residue A₁₈₇ are plotted as a function of MD simulation time. This plot reports the distances between the N-termini of helices A₁₀/T₄ and A₉. The distances remain relatively constant for Sec14p throughout the simulation (black) but decrease in the Sec14p^G266D simulation. An en face backbone rendition of Sec14p highlights residues A₁₈₇ (purple sphere) and F₂₂₁ (green sphere), as indicated.
• Figure S3 - Superimposition representing average domain displacement of helix A₁₀/T₄ in Sec14p and Sec14p^G266D simulations over the given time intervals: Sec14p ‘open’ conformation (11.1-13.2 ns) (green), Sec14p ‘partially closed’ conformer (25.3-25.6 ns) (purple), Sec14p^G266D ‘closed’ conformer (6.4-14.4 ns) (gold), as indicated.
• Figure S4 - Correlation of (φ, ψ) angles with respect to the opening and closing of the gate. (A) Plot shows correlation between changes in φ and ψ angles (torsion angles along the protein backbone) for each residue with position of the gate as measured by the distance between the Cα atoms of helix A₉ residue R₁₉₅ and helix A₁₀/T₄ residue F₂₃₁ that reports ‘status-of-closure’ of the A₁₀/T₄/A₁₁ helical gate. The correlation of backbone conformation with gate position identifies residues which have “hinge-like” behavior during the simulation. Positions with different “hinge-like” behaviors in the Sec14p and Sec14p^G266D simulations are noted, and the correlation heat map (red corresponds to greatest correlation, blue to least) is shown. The correlation lists of φ and ψ angles for each residue are available from the authors by request. (B) Locations of positions with variations in “hinge-like” behavior are noted. Secondary structural elements: β-strands β₁ (gold), β₄ (green), β₅ (purple), and 3₁₀-helix T₅ (red) are indicated. Residue G₂₆₆ is shown as a blue sphere.
• Figure S5 - Anatomy of the hinge unit. (A) Representation of the hinge FY component that is supported by π-stacking of B₄ residue Y₂₁₃ and B₅ residue F₂₄₁ (gold spheres in the backbone rendition of the Sec14p posterior view at left; G₂₆₆ is depicted as a blue sphere for reference). These Y₂₁₃ and F₂₄₁ residues are identified and rendered in space-fill mode (purple) in the ribbon diagram at right, as indicated. (B) Hinge residue I₂₄₂ of the KII component fits into a cleft formed by the A₁₀/T₄ residues F₂₂₁ and S₂₂₂. F₂₂₁ and S₂₂₂ are rendered as green spheres and I₂₄₂ as a gold sphere in the backbone rendition of the Sec14p en face view at left. Residues F₂₂₁, S₂₂₂ and I₂₄₂ are identified and rendered in space-fill mode (gray, purple and gold, respectively) in the ribbon diagram at right, as indicated.
• Figure S6 - Distance monitoring of backbone hydrogen bond interactions within the T₅ helical component. Sec14p distances are in black while the corresponding Sec14p^G266D distances are rendered gray. (A) P₂₆₁-F₂₆₄ hydrogen bond interactions are maintained in both Sec14p and Sec14p^G266D. (B) G₂₆₅-V₂₆₂ hydrogen bond interactions maintained in Sec14p are lost in Sec14p^G266D. (C) V₂₆₂-S₂₆₈ hydrogen bond interactions are intermittent in Sec14p but are lost in Sec14p^G266D. (D) S₂₆₈-L₂₆₀ hydrogen bond interactions maintained in Sec14p are lost in Sec14p^G266D. (E) The N₂₅₉-P₂₅₆ hydrogen bond is stably maintained throughout the Sec14p simulation. (F) The K₂₆₇-A₂₅₇ hydrogen bond is stably maintained throughout both the Sec14p and Sec14p^G266D simulations. (G) Residue A₂₅₇ engages in a unique and intermittent interaction with D₂₆₆ in Sec14p^G266D that is irretrievably broken at 14 ns.
• Figure S7 - Distance monitoring of the hydrogen bond interactions examined between the A₁₂LT₅ and B₁LB₂ substructures. (A) A main-chain to side-chain hydrogen bond between residues T₁₁₄ and N₂₅₉ is maintained throughout the Sec14p (black) simulation but is erratic in the case of Sec14p^G266D (gray). (B) An intermittent side-chain to side-chain hydrogen-bonding interaction is maintained between D₁₁₅OD1 and Q₂₅₄NE2 in the Sec14p simulation (black), but is lost in the Sec14p^G266D simulation (gray). (C) An intermittent side-chain to side-chain hydrogen-bonding interaction is maintained between D₁₁₅OD2 and Q₂₅₄NE2 in the Sec14p simulation (black), but is lost in the Sec14p^G266D simulation (gray). (D) A main-chain hydrogen bond between the K₁₁₆ amide and the Q₂₅₄ carbonyl is maintained in Sec14p (black) but is broken in Sec14p^G266D (gray).