Dynamics of Ligand Binding to a Rigid Glycosidase*

Abstract

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.

Keywords: NMR spectroscopy; dynamics; glycosidases; ligand binding; rigid fold.

Figure 1

BCX structural analysis of the crystalline and solution states for several steps along the enzymatic pathway. A) “Koshland” double displacement retaining mechanism. B) Crystal structure of resting state BCX (state E) shown as black Cα trace (PDB:2bvv). ^[10] The nucleophile (E78, red) and acid‐base (E172, green) catalytic dyad are shown in sticks. The location of the lanthanoid (blue sphere) in the CLaNP‐5 tag and the tensor Δχ (red arrows) defined by the anisotropic component of the magnetic susceptibility are shown. Cysteines used for CLaNP tagging are in sticks. C) Structures of BCX, from left to right, model of the ES complex based on the structure of XynII bound to X6 (PDB: 4hk8) ^[11] in which the substrate binding subsites are numbered (−3/+3); EI complex (PDB: 1bvv) ^[10] and EP complex (PDB:1bcx). ^[12] The RMSD values for the Cα atoms relative to the resting state are ≈0.1 Å for all structures. D) Correlation plots of the experimental PCS of the E, (ES)₁, EI and EP states fitted to the crystal structure of resting state BCX (PDB: 2bvv), indicating that this structure is a good model for these catalytic states in solution. Fitting to the crystal structures of the intermediate states does not improve the fit. E) BCX‐EI covalent complex formation by epoxyX2. The inactivation mechanism involves the attack of the ligand epoxide active center by the enzyme nucleophile, resulting in ring opening and the formation of a covalent bond between the enzyme nucleophile and the inhibitor, which emulates the EI state of the natural substrate hydrolysis reaction. The process is facilitated by protonation of the inactivator reactive center by the general acid/base residue.

Figure 2

BCX‐E78Q Michaelis complex formation. A) In the upper panel BCX‐E78Q ¹H‐¹⁵N HSQC ¹H slices are displayed for residue A115, showing that its amide resonance position and intensity changes with the appearance of its new peak in the (ES)₂ state upon titration with X6. Note that at the highest concentrations of X6 a general line broadening occurred due to an increase in solution viscosity (Figure S3C). In the lower panel, CSP of BCX‐E78Q backbone amides in the active site (AS) and SBS are shown next to simulated spectra using the binding model described in M & M and implemented in TITAN software ^[20] (see M & M and Figure S3). B) Amide groups with additional peaks representing the (ES)₂ state are shown in black spheres on BCX crystal structure. C) Correlations between the experimental PCS of the (ES)₁ and (ES)₂ states. D) Overlay between the free X6 ¹H‐¹³C HSQC spectrum (1500 μm in blue); in the presence of 480 μm (in green) and 1500 μm of BCX‐E78Q (in red). The 1D projections of the ¹H‐¹³C HSQC spectra of each condition are shown on the left side of the spectrum using the same color code. The emerging X6 peaks are numbered and connected to the 1D projections with dashed lines. The asterisk marks the residual water signal. On the basis of the binding parameters obtained from the global fit (Table S2), it is estimated that the concentrations (ES)₁ and (ES)₂ are 74 and 44 μm, and 192 and 115 μm for the samples with 480 μm and 1500 μm BCX‐E78Q, respectively (neglecting an allosteric effect).

Figure 3

Non‐covalent interactions enhance BCX millisecond time scale dynamics in the (ES)₁ and EP states. A,C) Overlay of the R _ex values of (ES)₁ (A) or EP (C) (red dots) and E states (blue dots) plotted versus the residue number. Several residues that show a prominent difference in R _ex are labeled. The secondary structure elements of BCX are represented by black arrows for β‐strands of sheet A and in white ones for sheet B and the α‐helix in rings. The “thumb” loop connects β‐strands 9 and 10. B,D) Amide nitrogen atoms used in the RD global fit for (ES)₁ (B) and EP (D) are shown in spheres on the BCX crystal structure (PDB ID: 2bvv) colored by their Δω between the ground and excited states, using a white/blue gradient. The Δω were derived from a global two‐state fit to the dispersion curves.

Figure 4

Proposed model for BCX enzymatic cycle. Blue sugar units indicate X6 distortion. Possible values for the microscopic rates k _{on, i} and k _{off, i} that yield the experimental dissociation rates, exchange rates, minor state populations and dissociation constants are given in Table S4. Dashed arrows indicate equilibria that could be present but for which no experimental evidence was obtained. In the model, the substrate X6 and X2 can bind in multiple ways, forming a major state and one or more minor state(s), indicated with an asterisk.