Combining multi-scale modelling methods to decipher molecular motions of a branching sucrase from glycoside-hydrolase family 70

Abstract

Among α-transglucosylases from Glycoside-Hydrolase family 70, the ΔN123-GB-CD2 enzyme derived from the bifunctional DSR-E from L. citreum NRRL B-1299 is particularly interesting as it was the first described engineered Branching Sucrase, not able to elongate glucan polymers from sucrose substrate. The previously reported overall structural organization of this multi-domain enzyme is an intricate U-shape fold conserved among GH70 enzymes which showed a certain conformational variability of the so-called domain V, assumed to play a role in the control of product structures, in available X-ray structures. Understanding the role of functional dynamics on enzyme reaction and substrate recognition is of utmost interest although it remains a challenge for biophysical methods. By combining long molecular dynamics simulation (1μs) and multiple analyses (NMA, PCA, Morelet Continuous Wavelet Transform and Cross Correlations Dynamics), we investigated here the dynamics of ΔN123-GB-CD2 alone and in interaction with sucrose substrate. Overall, our results provide the detailed picture at atomic level of the hierarchy of motions occurring along different timescales and how they are correlated, in agreement with experimental structural data. In particular, detailed analysis of the different structural domains revealed cooperative dynamic behaviors such as twisting, bending and wobbling through anti- and correlated motions, and also two structural hinge regions, of which one was unreported. Several highly flexible loops surrounding the catalytic pocket were also highlighted, suggesting a potential role in the acceptor promiscuity of ΔN123-GBD-CD2. Normal modes and essential dynamics underlined an interesting two-fold dynamic of the catalytic domain A, pivoting about an axis splitting the catalytic gorge in two parts. The comparison of the conformational free energy landscapes using principal component analysis of the enzyme in absence or in presence of sucrose, also revealed a more harmonic basin when sucrose is bound with a shift population of the bending mode, consistent with the substrate binding event.

Fig 1. B-factors and flexible structural motifs along MD simulation.

Panel (A) represents the average fluctuations of Cα atoms represented by B-factors per residue calculated along MD simulation of ΔN₁₂₃-GBD-CD2. The strips in the background of the B-factor lines highlight structural motifs surrounding the active site, represented using the same color code on the panel (B), which represents the superimposed views of ten frames taken along the MD simulation. The highlighted regions are loop 2127–2138, helix-loop-helix motif corresponding to region 2324–2368 and its adjacent loop 2592–2605, then the 2290–2300 and 2757–2780 β-hairpin motifs in green, light blue, red, magenta and forest green, respectively. The shown side chains represent the two catalytic residues: the nucleophile D2210 and the acid base E2248. The panel (C) represents a schematic view of the five domains of ΔN123-GBD-CD2; detailed (β/α)₈ barrel domain A with cyan color (helices represented as rectangles and β-sheets as arrows), the domains B, C, IV and V in green, magenta, yellow and red, respectively. The structural motifs surrounding the active site are represented with the same color code than panel (B).

Fig 2. RMSD per residue and wavelet analysis.

Residual RMSD (A) and wavelet (C) analysis for each amino acid residue as function of MD simulation time. The bottom legends show the color used to discriminate discrete RMSD values in Angström (panel A) or wavelet period in nanoseconds (panel B). The right-edge strip indicates the different structural domains that compose ΔN₁₂₃-GDB-CD2 using the same color code as in panel D, the hinge between domains IV-V and the block D are highlighted on the right side. The bottom plot (B) shows an enlarged view of residual RMSD of flexible motifs surrounding the active site: loop 2127–2138, helix-loop-helix motif corresponding to residues 2324–2368 and its adjacent loop (residues 2592–2605), then the β-hairpin motifs composed respectively of residues 2290–2300 and 2757–2780, and the β-hairpin 1832–1854. The loop identifiers are colored in green, light blue, red, magenta, forest green and black respectively. The panel (D) highlights the five structural domains of ΔN₁₂₃-GDB-CD2 with different colors: domains B, C, IV and V in green, magenta, yellow and red, respectively, the domain A is represented with two colors; blue for the block D and cyan for the rest of the domain. A schematic representation of ΔN₁₂₃-GDB-CD2 structure is shown with delimitation of the different domains forming the U-shape.

Fig 3. Dynamical cross correlation (DCC) analysis.

(A) DCC map of ΔN₁₂₃GBD-CD2 calculated from 1μs MD simulation. The color scale from orange to blue corresponds to discrete correlation coefficient values (DCC) from -1.0 to +1.0. The different structural domains of ΔN₁₂₃GBD-CD2 are highlighted by strips on top and left side and using the same color code as in panel (B). Regions marked by the rectangles a-g are discussed in the main text. The panel (B) highlights the five structural domains of ΔN₁₂₃-GDB-CD2 with different colors: domains B, C, IV and V in green, magenta, yellow and red respectively, the domain A is represented with two colors; navy blue for the block D and cyan for the rest of the domain A.

Fig 4. Comparison between computed and experimental B-factors.

B-factors were calculated from the three first modes of NMA (blue), and EDA (orange) and plotted against experimental B-factors derived from X-ray structures of ΔN₁₂₃-GBD-CD2 (black) as a function of amino acid residues. The top edge strip shows the different domains of ΔN₁₂₃-GBD-CD2 using the same color code as in Figs 2 and 3.

Fig 5. Average unsigned dihedral backbone angles.

The panels (A, B) illustrate the average unsigned (absolute) deviation (AUD) of Φ, Ψangles, respectively, resulting from the three first modes of NMA. The top edge strip shows the structural domains of ΔN₁₂₃-GBD-CD2 using the same color code as in Figs 2 and 3. The red stars indicate the hinge regions Q1956 and T2004 from N-ter to C-ter respectively, the red arrow show the N1985 hinge residue. The panel (C) represents the superimposition of IV and V domains of the GTF180-ΔN (pdb entry: 3KLK) in magenta and ΔN123-GBD-CD2 (pdb entry: 3TTQ) in green. The spheres show the N-ter and C-ter residues of each enzyme. The location of hinge regions discussed in this paper are indicated by the red arrow and stars. The black arrow indicates the hinge region (D794-E795) identified in GTF180-ΔN [18,28].

Fig 6. Dynamical cross correlation from theoretical methods against experimental data.

DCC map of ΔN₁₂₃-GBD-CD2 calculated from EDA (A), NMA (B), and derived from X-ray (C). The color scale from orange to blue corresponds to discrete correlation coefficient values (DCC) from -1.0 to +1.0. The structural domains of ΔN₁₂₃-GBD-CD2 are shown using the same color code as in Fig 3. Parts marked by the rectangles a-j are discussed in the main text.

Fig 7. Large scale collective motions from normal or essential dynamics modes.

View of the three first normal modes of ΔN₁₂₃-GBD-CD2, corresponding respectively to the twist (I a), bend (II a) and wobble (III a) modes and three first essential dynamics modes corresponding respectively to the bend (I b), wobble (II b) and twist (III b). The black arrows point to the motion direction for each mode and the length of the arrows illustrates the amplitude of the movements. The orange spheres indicate the location of the catalytic gorge.

Fig 8. Free energy landscape (FEL) of ΔN₁₂₃-GBD-CD2.

FEL in free form (A) and in complex with sucrose (B) were determined using as reaction coordinates the projection of the first and second principal components. The bottom legend shows the color scale of the logarithm of FEL in J. mol^-1. Structural snapshots taken from the low or high energy regions pointed by the arrows are shown. The top center structural motif represents the subdomain H1-H2 structures from energetic wells of free-ligand (light blue) or complex (cyan) simulations. An empirical angle formed between the Cα atoms of N7 (domain V), the E912 (domain A) and N738 (domain C) was defined for each conformation to illustrate the bending mode.

Fig 9. Main interactions between sucrose and amino acid residues in the active site.

Map of hydrogen bonding interactions occurring between ΔN123-GBD-CD2 and sucrose in clusters taken from the energy landscape basins (ΔG < 100 J. mol^-1) (A). The x and y axis indicate the atoms from sucrose and the enzyme involved in hydrogen bonding interactions. The color code indicates the hydrogen bond occurrence percentage over the cluster. In (B) panel are illustrated the important interactions observed between sucrose and amino acids in the catalytic pocket.