*2.3. Cluster Analysis of YqhD Enzyme*

Conformational differences observed due to the cofactor oxidation/reduction state were further quantified using cluster analysis on monomers (see Section 4.4 for details). In the combined trajectory, a total of 54 (NADP) and 43 (NADPH) clusters were obtained for the monomer using an RMSD cutoff of 0.13 nm. Figure 4 represents the first three clusters of NADP-bound and NADPH-bound monomers in red, blue and green colored ribbons, respectively. These first three clusters comprise 62.7% (30.8%, 19.3%, and 12.6%) and 72.9% (46.0%, 13.8%, and 13.1%) of the total monomer conformations occupied by YqhD for NADP- and NADPH-bound enzyme, respectively. **2021**, , x FOR PEER REVIEW 7 of 17

**Figure 4.** The representative conformations of the first five clusters (colored by red, blue, green, tan, and gray color, respectively) of homodimer (**a**) NADP-bound and (**b**) NADPH-bound are superimposed and represented as ribbons. The dotted arrows show the monomer- and domain- interface. The region involved in the opening/closing of the interdomain cleft is highlighted. (**c**) Interdomain cleft residues D159 and H363 are represented in the NADPH-bound monomer of crystallographic structure. Cluster 1 (in red color) shows partially closed conformations in both NADP/H bound- Yqhd monomer, while Cluster 2 (in blue color) is open confirmation in NADP-bound and closed in NADPH-bound monomer.

β β α α α By looking at the superimposed mean cluster structures and crystal structure illustrated in Figure 4, it can be seen that the primary differences in monomer conformations arise from the position of the β6/β7 loop region (circled) of the Rossmann-type domain and

β β

the α8/α12 helix of α-helical domain. Figure 5a shows the superimposed crystallographic structures of apo- and holo- proteins. The β6/β7 loop region is in open conformation in the apo form of the YqhD crystal structure [7] (and closed in the holoenzyme), indicating that they comprise an interdomain cleft which opens and closes for cofactor binding and release. In this side-by-side comparison of the monomer, it can be seen that, with both cofactors, these loops are significantly dynamic. The highly populated conformations of NADPH-bound involve both monomers within the dimer remaining with a partially closed cleft (cluster 1), closed cleft (cluster 2), and open cleft (cluster 3). On the other hand, when oxidized NADP cofactor is bound, each monomer within the dimer samples more conformations with open cleft. All three of the highly populated conformations of NADPbound enzyme are open cleft, indicating that cofactor oxidation state has an effect on the structures and populations of the open and closed conformation. Figure 5b represents the distribution of distances using the center of mass of two domains in the simulations as a global measure of the opening and closing of the domains. Domain distances observed in the crystal structures are shown in Figure 5b by vertical cyan and blue colored lines for the holo- and apo- enzymes, respectively. The NADPH-bound monomer remains in more closed and partially closed conformation, indicated by two main peaks for the distances between domains at 2.68 nm and 2.73 nm, respectively. A third peak is also observed at 2.83 nm, which is close to the one observed in the apoprotein, at 2.86 nm. In contrast, the NADP-bound monomer has the main peak at 2.90 nm for distances between domains, indicating a population of more open conformations, and a second small peak at 2.72 nm for sampling partially-closed structures; the molecular details are discussed later. **2021**, , x FOR PEER REVIEW 8 of 17

α **Figure 5.** (**a**) Crystallographic structure of the YqhD monomer as apoprotein and holoprotein is in tan and sky-blue colored cartoon representation, respectively, with bound NADP (green licorice representation) and Zn2+ (violent sphere) in the active site [7]. Cleft residues and Zn2+ binding residues are labeled. (**b**) The distribution of distances between Rossmann-type and α-helical domains is shown using the center of mass. (**c**) The distribution for cleft minimum distances using residue pair D159-H363 is shown for NADP- and NADPH-bound monomers during the simulations. The cleft distances observed in the apo- and holo- YqhD protein crystal structures are marked with blue (apo) and cyan (holo) bars.
