Integrative structure determination reveals functional global flexibility for an ultra-multimodular arabinanase

Abstract

AbnA is an extracellular GH43 α-L-arabinanase from Geobacillus stearothermophilus, a key bacterial enzyme in the degradation and utilization of arabinan. We present herein its full-length crystal structure, revealing the only ultra-multimodular architecture and the largest structure to be reported so far within the GH43 family. Additionally, the structure of AbnA appears to contain two domains belonging to new uncharacterized carbohydrate-binding module (CBM) families. Three crystallographic conformational states are determined for AbnA, and this conformational flexibility is thoroughly investigated further using the "integrative structure determination" approach, integrating molecular dynamics, metadynamics, normal mode analysis, small angle X-ray scattering, dynamic light scattering, cross-linking, and kinetic experiments to reveal large functional conformational changes for AbnA, involving up to ~100 Å movement in the relative positions of its domains. The integrative structure determination approach demonstrated here may apply also to the conformational study of other ultra-multimodular proteins of diverse functions and structures.

Fig. 1. The crystal structure of AbnA and its complexes.

a The pincer-shaped structure of AbnA-Conf1, composed of the catalytic Domain1 (in red), Domain2 (green), Domain3 (blue), and Domain4 (orange). b The structure of AbnA-Conf1-A5, where an arabinopentaose (A5) molecule was found bound to Domain4. c The structure of the AbnA-D123 truncation mutant in complex with an arabinooctaose (A8) molecule in the active site. d Superposition of the AbnA-Conf1-A5 structure (purple) with the AbnA-D123-A8 structure (yellow), revealing that the arabinosaccharides present in the two binding sites of AbnA are bound in a perpendicular orientation to one another.

Fig. 2. The three crystal conformations determined for AbnA.

a Comparison between the AbnA-Conf1 and the AbnA-Conf2 structures, revealing an opening-closing movement of Domain4. b Superposition of the AbnA-Conf1 (in purple) and AbnA-Conf2 (in red) structures, revealing a ~13 Å downward movement in the relative position of Domain4. This comparison shows that only in the AbnA-Conf2 structure, unlike the AbnA-Conf1 structure, there are hydrogen bond interactions between Domain1 and Domain4. c Superposition of the AbnA-Conf3 (in color) and the AbnA-conf2 (in gray) structures one on the other, viewed from the side (alignment based on Domain 1) and from above (alignment based on Domain4). Such superposition reveals an internal rotation of Domain4 by about 45° relative to the AbnA-Conf2 structure. d Superposition of the AbnA-Conf2 (in blue) and AbnA-Conf3 (in red) structures reveals that the two structures possess different interactions between Domain1 and Domain4.

Fig. 3. Prediction of conformational change by normal mode analysis (NMA).

The NomadRef server predicted a a “sideways” motion of Domain4 (orange to gray) for the lowest frequency mode, and b an “upward” motion of Domain4 for the second lowest frequency mode. c, d The iModS webserver predicted for the lowest frequency mode a combination of “sideways” and “opening-up” motions for Domain4.

Fig. 4. Large conformational changes detected by MD and metadynamics simulations.

a An all-atom classical MD simulation of the AbnA-Conf2 crystal structure over 500 ns. The distance between Domain1 and Domain4 is plotted as a function of time. b The free energy landscape obtained by metadynamics for the conformational dynamics of AbnA as a function of (i) the distance between Domain1 and Domain4 and (ii) the dihedral angle between Domain1, Domain2, Domain3, and Domain4. Coloring corresponds to differences in energy values ranging from 0 (blue) to 15 kcal mol⁻¹ (red). Contour lines are contoured at 1.4 kcal mol⁻¹. Three energy minima are observed, as well as a small energy minimum shoulder located on an energetic plateau. The structural clusters, representative of each energy minimum, are presented, and the relative difference in energy between them is shown. Stars 1,2,3 indicate the locations of the AbnA-Conf1, AbnA-Conf2, and AbnA-Conf3 crystal structures on the energy landscape.

Fig. 5. SAXS data agree with metadynamics results.

a Qualitative fit of the AbnA-D12 (taken from the AbnA-Conf1 crystal structure), AbnA-D123 (taken from the EM4 metadynamics cluster), and AbnA-WT (taken from the EM3 metadynamics cluster) structures inside their averaged SAXS envelopes. b Successful fitting of the AbnA-WT SAXS scattering curve (in black) is achieved when considering 2- or 3-state models composed of the clusters representative to the energy-minima obtained in the metadynamics free energy conformational landscape of AbnA, with population weights as shown in (c). In contrast, such a fit is much worse (high χ values) when considering only the crystallographic structures.

Fig. 6. Two point mutations change the DLS diameter and kinetic activity of AbnA.

a DLS measurements reveal a hydrodynamic diameter of 16.9 ± 0.2 nm for AbnA-WT and a diameter of 9.1 ± 0.3 nm for AbnA-E420C-W758C, when oxidized by 2 mM GSSG. After the addition of 10 mM DTT to the oxidized AbnA-E420C-W758C, the diameter changed to 14.4 ± 0.2 nm. b Kinetic measurements of AbnA-WT (k_cat = 13 ± 2 s⁻¹) and oxidized AbnA-E420C-W758C (k_cat = 0.050 ± 0.003 s⁻¹) on branched arabinan demonstrate a x260 reduction in the catalytic activity.

Fig. 7. The integrative structure determination of AbnA.

The integrative structure determination of AbnA was conducted through four stages: (1) gathering of X-ray crystallography and SAXS data, where three crystallographic conformational states were determined; (2) sampling additional conformational states using normal mode analysis, molecular dynamics, and metadynamics; (3) scoring the energetic and biological relevance of these conformational states with metadynamics and SAXS data; (4) validating the large conformational changes sampled and scored in stages (2) and (3) with SAXS, dynamic light scattering (DLS), mutagenesis, and kinetic experiments.