*2.4. Interdomain Cleft*

β β β β α α α The cofactor binding site in each YqhD monomer spans the interdomain face. The residues of β6/β7 loop region act as the clamp at the mouth of the cofactor binding region, which remains in open conformation in the apo form [7] and closed in the holoenzyme crystal structures. The closed conformation of holoenzyme has hydrogen bond Asp159-Lys359/His363 involving residues D159 of β6/β7 loop region in the Rossmanntype domain and residues K359/H363 of α12 helix in the α-helical domain. For a better understanding of cleft opening/closing and its relationship with cofactor binding/release, we defined interdomain cleft using residue pair D159-H363 at the mouth of the cofactor

binding site, on the interface of the Rossmann-type and α-helical domains (see Figure 5a). In the crystal structure, the holoenzyme has a distance of 0.16 nm for cleft, while the apo-enzyme has distances of 1.25 nm for cleft [7], which indicates the involvement of cleft opening/closing in cofactor binding and release. Figure 5a shows the residue pair D159- H363 which is involved in interdomain cleft formation and clamps the NADP/H cofactor in the binding site. The interdomain cleft shows hydrogen bonding between residue pairs D159-K359/H363 and K160-H271/E272 in the crystal structure and simulations. YqhD has ~5% D159-H363 hydrogen bond existence in the NADPH-bound monomer trajectory; however, its occurrence is negligible in NADP-bound monomers.

Figure 5c shows the distribution of cleft distances using the residue pair D159-H363. Cleft distances observed in the crystal structures are shown in Figure 5c by vertical cyan and blue colored lines for the holo- and apo-enzymes, respectively. NADP-bound monomer shows a wider distribution range of cleft distances, from 0.14–2.0 nm, compared to those in NADPH: 0.14–1.7 nm. It can be seen in Figure 5c that the distribution of cleft distances shifts to the right with oxidized cofactor, reflecting wider openings of the interdomain cleft in NADP-bound monomers. Monomers sample conformations with interdomain cleft distances using residue pairs D159-H363: (i) less than 0.55 nm, representing closed cleft as observed in holoenzyme, (ii) ranges from 0.55–1.0 nm, for partially-closed cleft representing transition states and (iii) more than 1.0 nm, having open cleft as observed in protein. NADPH-bound monomers have two peaks in the distance distribution (Figure 5c) at 0.16 and 0.28 nm for cleft distances representing closed confirmations, and the third peak at 0.58 nm showing partially-closed conformations. However, NADP-bound monomers show a peak at 0.17 and a broader distribution of cleft distances, encompassing partiallyclosed and, mainly, open cleft confirmations. Changes in interdomain cleft distances occurred mainly due to a major shift in the positions of helix α12 and beta sheets β6 and β7 (see the circled region in Figure 5a), but it is a cooperative motion (involving the cleft forming regions of both domains). Cleft distance data indicate that residue D159 of cleft works as the clamp to monitor cleft opening and closing for cofactor binding and release, and its movement is affected by cofactor oxidation state.
