Biliverdin Reductase B Dynamics Are Coupled to Coenzyme Binding

Abstract

Biliverdin reductase B (BLVRB) is a newly identified cellular redox regulator that catalyzes the NADPH-dependent reduction of multiple substrates. Through mass spectrometry analysis, we identified high levels of BLVRB in mature red blood cells, highlighting the importance of BLVRB in redox regulation. The BLVRB conformational changes that occur during conezyme/substrate binding and the role of dynamics in BLVRB function, however, remain unknown. Through a combination of NMR, kinetics, and isothermal titration calorimetry studies, we determined that BLVRB binds its coenzyme 500-fold more tightly than its substrate. While the active site of apo BLVRB is highly dynamic on multiple timescales, active site dynamics are largely quenched within holo BLVRB, in which dynamics are redistributed to other regions of the enzyme. We show that a single point mutation of Arg78➔Ala leads to both an increase in active site micro-millisecond motions and an increase in the microscopic rate constants of coenzyme binding. This demonstrates that altering BLVRB active site dynamics can directly cause a change in functional characteristics. Our studies thus address the solution behavior of apo and holo BLVRB and identify a role of enzyme dynamics in coenzyme binding.

Keywords: coenzyme; dynamics; enzyme; network; nuclear magnetic resonance.

Fig. 1.

Structural and biological comparison of BLVRB and BLVRA. (a) The holo enzyme structures of BLVRB (PDB accession 1HE4, protein in white and NADP⁺ in red) with both the relatively conserved SDR domain and variable C-terminal domain shown. (b) BLVRB protein levels dominate in RBCs relative to BLVRA, as assessed by two independent methods [3].

Fig. 2.

BLVRB binding to both coenzyme and FAD substrate. (a) HSQC spectrum of apo BLVRB (black) superimposed with holo BLVRB (red). (b) HSQC spectrum of holo BLVRB (red) superimposed with addition of 3.2 mM FAD (green). (c) Binding isotherm of apo BLVRB to the reduced NADP⁺ coenzyme monitored through ITC at 20°C. (d) Binding isotherms of holo BLVRB to FAD monitored through NMR titration for amides of Thr15 (red), Lys178 (green), Leu74 (purple), Ser88 (black), Arg174 (blue), and an example of the CSPs of Thr15. Binding isotherms were globally fit to extract the K_D. (e) CSPs between apo BLVRB and holo BLVRB with 10 mM NADP⁺. (f) CSPs between holo BLVRB and holo BLVRB titrated with 3.2 mM FAD. (g) CSPs between apo and holo BLVRB versus the distance of each residue's amide to the coenzyme and mapped onto the X-ray crystal structure (purple) for CSPs >0.2ppm (PDB accession 1HE2). (e) CSPs between holo BLVRB and holo BLVRB titrated with 3.2 mM FAD substrate versus the distance of each residue's amide to the substrate and mapped onto the X-ray crystal structure (purple) for CSPs >0.2ppm (PDB accession 1HE4). Five proline residues comprise the FAD binding substrate pocket that includes 118, 122, 123, 151, and 152, and thus, there are no closer distances to amide than 6.5 Å. All NMR data were collected at 900 MHz at 20 °C.

Fig. 3.

BLVRB secondary structure propensities and changes upon coenzyme binding. (a) CA chemical shift differences between apo BLVRB and CA resonances within a random coil (δΔ CA). (b) CA chemical shift differences between holo BLVRB and a random coil. (c) The absolute difference in CA chemical shift differences between apo BLVRB and a random coil and between holo BLVRB and a random coil. CA chemical shift propensities were calculated by subtracting random coil CA resonance reported at the BMRB (http://www.bmrb.wisc.edu/). (d) An example of the 3D-HNCA spectra for both apo and holo BLVR showing no change for Asn93 CA resonances and large changes for Arg39 CA resonances. (e) These CA chemical shift differences >0.4ppm in (d) are mapped onto the X-ray crystal structure of BLVRB (orange) with unassigned resonances also shown (pink). For secondary structure, positive values indicate α-helical secondary structure, and negative values indicate β-strand secondary structure. Cartoon representation of secondary structure from the X-ray crystal structure is shown at the top (PDB accession 1HE4).

Fig. 4.

BLVRB dynamics are altered upon coenzyme binding. (a) ¹⁵N R1 relaxation rates for both apo BLVRB (black) and holo BLVRB (red). Inset: blowup of ¹⁵N R1 relaxation rates for the region of 72–86. (b) Residues with ¹⁵N R1 relaxation rates greater than one SDEV above the mean for apo BLVRB (left, yellow) and holo BLVRB (right, cyan) are mapped onto the X-ray crystal structure (PDB accession 1HE2). (c) R2 relaxation rates for both apo BLVRB (black) and holo BLVRB (red) extracted for each residue from ¹⁵N amide R2-CPMG dispersions at the lowest CPMG field of 50 Hz. (d) Residues exhibiting R2-CPMG dispersions greater than 1 Hz at a static field of 900 MHz are mapped onto the holo BLVRB structure for apo BLVRB (yellow) and for holo BLVRB (cyan). (e–i) R2-CPMG dispersions for residues within apo BLVRB (black) and holo BLVRB (red) individually fit (fitted line). All atoms from residues exhibiting elevated R1 relaxation rates and R2-CPMG dispersions are shown based on their respective ¹⁵N relaxation data.

Fig. 5.

Functional and dynamic impact of BLVRB R78A mutation. (a) Arg78 “clamps” over the coenzyme (red) to form a hydrogen bond with the backbone carbonyl of Thr12. Atoms are shown as balls (orange) in holo BLVRB (PDB accession 1HE2). (b) Blow-up of the active site and the hydrogen bond between Arg78 and Thr12. (c) ITC titration for BLVRB binding to NADP⁺ with wild type (black) and R78A (orange) with an extracted K_D of 0.5 ± 0.2 μM and 2.8 ± 0.2 μM, respectively. (d) ZZ-exchange buildup curves for exchange peaks of Val52 with 500 μM ¹⁵N-labeled wild type BLVRB or the R78A mutant in the presence of 250 μM NADP+. Left: Spectrum of each is shown for a 1.08-s delay, with arrows indicating the exchange peaks. Right: Build-up curves shown for exchange peaks with extracted rates derived from fits to all four intensities of k_on=1.3 ± 0.4 (s μM)⁻¹/k_off = 1.1 ± 0.5 s⁻¹ and kon = 4.6 ± 0.6 (s μM)⁻¹/k_off = 30 ± 4 s⁻¹. (e) R1 relaxation rates of the R78A mutant for both apo (black) and holo (red) forms. We note that in the holo R78A mutant, that residues 36–38 are absent as 36–37 are poorly fit and Ser38 is overlapped.

Fig. 6.

Dynamics within BLVRB are distally coupled to the active site. (a) R2-CPMG dispersions for both wild type BLVRB (black) and R78A (orange) are shown for Asp80, Lys105, Val107, Ala108, Cys109, Ser111, Leu125, and Tyr156. (b) Changes between wild type BLVRB and the R78A mutant R2-CPMG dispersions mapped onto the structure of holo BLVRB (orange). Two orientations are shown along with the largest distance between coupled residues (red arrows), and all atoms are depicted for those residues in which the full ¹⁵N R2-CPMG dispersions are shown.

Fig. 7.

The BLVRB active site is inherently dynamic. (a) Monitored changes to disorder within the ns-ps timescale. Entire residues are colored if their ¹⁵N R1 relaxation rates are diminished (yellow) or increased (cyan) greater than 0.5 SDEV (0.029 s⁻¹) above the average change (0.032 s⁻¹) for all residues, and the active site is denoted by an arrow (blue) to Arg78. (b) All three loops surrounding the BLVRB active site of residues 10–15, 76–82, and 109–114 exhibit μs–ms motions quenched upon coenzyme binding (yellow) as monitored through R2-CPMG dispersion. Only residues Asp36, Ser37, and Arg39 exhibit an increase in exchange upon coenzyme binding (cyan). Only the backbone atoms are colored. (c) The BLVRB loop region of 75–82 (orange) that comprises Arg78 is dynamic on multiple timescales and becomes ordered upon coenzyme binding. For wild type BLVRB, an exchange rate of this loop region estimated from a global fit of several adjacent residues (Fig. S6b) is shown and the microscopic exchange rates between apo and holo BLVRB are shown (from ZZ-exchange spectroscopy, see Fig. 5d). Two structures of apo BLVRB were modeled using harmonic constraints using Chemistry at Harvard Macromolecular Mechanics (CHARMM, version 36b1) for all backbone residues except for residues within the Arg78 loop. These include residues 75–84 that comprise elevated R1 relaxation rates for 76–82. Although R1 relaxation rates for Leu74, Pro83, and Thr84 were not determined due to overlap and the absence of a proline amide, these residues were not constrained as they reside within this loop region.

Fig. 8.

Evolutionary swapping of human Arg78 within the BLVRB active site. The phylogenetic tree graphic is derived from the tree shown in Figure S7, but focused on the Primates. Multiple BLVRBs from the Macaques, New World Monkeys, and Lemurs are shown as filled triangles on the tree. As in SFX, the two focal amino acids, at positions 14 and 78 (numbered relative to human positions), are shown along with the surrounding 3 amino acids on either side in the Ensembl amino acid alignment. The sequence examples shown illustrate the double substitution at sites 14 and 78 from the common ancestor of haplorrhynes to the common ancestor of new and old world monkeys (Simiiformes), along with the general conservation of the surrounding sites. The human BLVRB structure is shown with positions 14 and 78 in spacefill to illustrate their relationship to the rest of the structure. The lemur sequence shown is from the mouse lemur, Microcebus murinus.