The crystal structure of Co(II)-isoDromeQC wild type soaked with the substrate QFRH In this experiment the aim was to obtain a structure of the QC in complex with a substrate or an

intermediate state of the glutaminyl cyclization reaction. As mentioned in the introduction, the glutaminyl cyclization correlates to the physiological function of QCs. No other substrate possessing an N-terminal glutamine leads to analyzable structure data. This substrate shows high affinity to the Zn(II)-isoDromeQC wild type and reveals a high catalytic efficiency (cf. Section 3.2).

The crystallization attempts with QFRH and Zn(II)-isoDromeQC were without success.

Interestingly, the Co(II)-substituted isoDromeQC provides analyzable crystal structures of the enzyme substrate complex.

The analysis of Co(II)-isoDromeQC wild type crystals soaked with substrate QFRH showed that varying ligand orientations in both the molecules of the unit cell are possible. In one of the molecules the γ-amido oxygen of the first-position glutamine acts as the fourth coordination partner of the Co(II) ion (cf. Figure 3.11 A, B and C). Different structure refinements show coordinative bond lengths around 2.0 ± 0.1 Å.

87 with the α- and γ-amino nitrogen of the N-terminal glutamine. The γ-carboxylate oxygen atoms of D228 may form a hydrogen bond to the γ-amino nitrogen of the N-terminal substrate glutamine. The backbone of D292 (not shown) acts as a further binding motif. The main chain nitrogen of D292 interacts with the main chain oxygen of the second amino acid in the substrate sequence and the main chain oxygen from D292 interacts with the main chain nitrogen from the phenylalanine via hydrogen bond. This binding motif occurs also when an N-terminal glutamate (cf. Figure 3.12 B) or pyroglutamic acid (cf. Figure 3.13 B) is bound to the active center. Even if the inhibitor AFA is bound (cf. 3.14 B), both hydrogen bond interactions occur.

An alternative orientation of the substrate is shown in Figure 3.11 (D: view from above, and E:

side view). In this position, the hydrogen bond mediated contact between the substrate and the enzyme is bridged by six water molecules. The distance between the γ-amido moiety of the N-terminal glutamine and the metal ion is too large for chemical bonding.

Nevertheless, the N-terminal glutamine residue of the substrate is directed into the acidic binding pocket of the QC molecule. The measured electron density for residues in the second shell around the metal ion is barely perceptible.

88

Figure 3.11 Crystal structure of Co(II)-isoDromeQC wild type in complex with the substrate QFRH The soaked synthetic substrate QFRH (surrounded by a 2F_o-F_c electron density map (sigma contour level = 1.0 (yellow) was found in two different localizations. The first exhibits the γ-amido oxygen of the N-terminal glutamine in a cobalt-bound state along with potential hydrogen bond interactions (dashed line) (A, B and C). The monodentate ligand coordinates a tetrahedral Co(II) ion. The second position of the substrate shows an outlying ligand (D and E). The contact between substrate and QC is mediated by water molecules. The resolution of this structure is 1.31 (work = 0.143, R-free = 0.182).

A

B C

D E

89 The following crystallization attempt was performed to analyze the structure of the complex between the Co(II)-isoDromeQC wild type and a substrate containing an N-terminal glutamate.

Substrates with N-terminal glutamate are involved in the pathophysiological side reaction leading to neurodegenerative process in the brain of humans [23].

In this experiment again, the use of Co(II)-substituted isoDromeQC wild type leads to analyzable structure data.

The analysis of the XDS-processed and Phenix-refined structure data showed an observed electron density, which indicates that the active center might be occupied by EFRH as well as by the reaction product pEFRH (cf. Figure 3.12 A). The appearance of the product in this structure is obviously due to the substrate conversion during the co-crystallization-procedure. Despite the minimal rate constant for this substrate (kcat = 1/10000 s^-1, measured with hQC), apparently the high concentration of EFRH (approx. 7.5 mM) and the typical high QC concentration in the crystal lattice has led to sufficient product conversion. The most reliable statistic values, such as the R-work and R-free factors, were obtained when the PHENIX refinement definitions were assessed to an occupancy nEFRH = npEFRH = 0.5. This indicates that the ratio of the protein-bound substrate and product molecules, respectively, is one to one.

The Co(II)-isoDromeQC in complex with the product pEFRH is described in a separate Section below (cf. Section 3.5.5). In this section, only the binding mode of EFRH will be described.

The flexibility of the side chain of phenylalanine (the second residue of EFRH) is apparently too high. This leads to a lack of electron density (B). The corresponding averaged B-factor of the phenylalanine side chain is approx. 45 Ų and is hence increased by a factor of two compared to the N-terminal glutamate residue (Bfaveraged = 25 Ų).If a substrate with an N-terminal glutamine is bound to the active center (cf. Section 3.5.3), the Co(II) ion is four-coordinated. In stark contrast thereto, in this structure the Co(II) ion is five-coordinated (cf. Figure 3.12. B). This is the result of the bidentate coordination of the γ-carboxylate moiety belonging to the N-terminal glutamate side chain. The coordinative bond lengths between the Co(II) ion and both oxygen atoms is 2.2 Å.

Further substrate protein interactions are displayed in Figure 3.12 B. Next to possible polar contacts between the γ-carboxylate moiety and H318 and W317 respectively, and the α-amino moiety and the carboxylate moiety of E190, the backbone of E292 and one water molecule provide hydrogen bond donators as well acceptors.

As mentioned above, clear differences in the binding mode of the glutamate substrate is the five-coordinated Co(II) ion and the missing polar contact between the N-terminal glutamate side chain (due to the absence of γ-amido nitrogen) and D228.

90

Figure 3.12 Crystal structure of Co(II)-isoDromeQC wild type co-crystallized with the N-terminal glutamate substrate EFRH The resolved structure of a crystal co-crystallized with EFRH (orange rods) resolution: 1.42, R-work = 0.154, R-free = 0.1940) provides an observed electron density of the substrate and accumulated reaction product pEFRH (green rods) (A). The γ-carboxylate oxygens of the N-terminal substrate glutamate indicate bidentate coordination of Co(II)(dark salmon sphere) (B) Both ligands are surrounded by a 2Fo-Fc electron density map with a sigma contour level of 1.0 σ.