Ec POX activation by the membrane mimic SDS

Limited proteolysis was used in chapter 4.1.4 to investigate the activation process of EcPOX. Both applied assays required an established equilibrium of conformations SR II and SR III of the enzyme to generate activated EcPOX Δ23. Given that the proteolysis assay is an artificial system, the results previously observed were validated by stimulations of EcPOX with SDS. This activation system is probably more native-like since SDS mimics the cell membrane.

The activation model of SDS

It was reported that EcPOX shows an increased activity if detergents or lipids are present in in vitro assays [34]. The molecular mechanism of interaction of the enzyme with such amphiphiles is still uncertain due to the fact that no structures of EcPOX in complex with detergents or lipids are available. However, interactions of those amphiphilic molecules with the enzyme are supposed to be restricted to the membrane anchor, since activity of the proteolytically activated Δ23 variant failed to be further accelerated by addition of amphiphiles [18]. One could speculate that lipids or detergents stabilize the exposed membrane anchor of pyruvate-reduced EcPOX similar to the native membrane by induction of a helical structure.

A similar mode of activation was proposed for SDS-stimulation and proteolysis for EcPOX ( figure 28).

The enzyme is reduced with pyruvate leading to equilibrium of enzyme forms SR II and SR III. While the membrane anchor is still attached to the protein surface in SR II, possible interactions with SDS are supposed to maintain resting-state activity. Remarkably, only conformation SR III resembles a released membrane anchor which could be stabilized by SDS contacts leading to accelerated activity. The increase in activity can be directly correlated to the amount of SR III in equilibrium.

figure 28: Supposed model of SDS-mediated EcPOX activation. In the resting state (R) the Δ101 cleavage site is accessible whereas the Δ23 restriction site is protected. The substrate-bound state (SB) is induced by formation of covalent ThDP-intermediates, while FAD remains unaffected. Both protease cleavage sites are shielded. The substrate-reduced state (SR) is formed due to pyruvate-induced reduction of FAD. Equilibrium of two forms is established: SR II represents an enzyme species where the flavin cofactor is already reduced, but the Δ23 cleavage site is still protected. SR III resembles the enzyme after the second conformational change with an exposed Δ23 cleavage site and a released membrane anchor. Only state III is competent to be activated by SDS leading to helix formation of the membrane anchor. The functional tetramer of EcPOX is simplified illustrated as monomer for better visualization. Note that the alpha-peptide is displayed separately from the beta-peptide, although the alpha-peptide resembles the C-terminal part of the beta-peptide. Different conformations of the R- and SB-state as previously determined by proteolysis experiments (chapter 4.1.4) were omitted for reasons of clarity.

However, activation of EcPOX with SDS illustrates not only the transition S II→S III but also the property of the membrane anchor to form an amphipathic helix. One can assumes that only a properly folded helix can be stabilized by SDS resulting in the desired activity acceleration. Therefore, this method additionally allows analysis of the intrinsic helix propensity of the membrane anchor which might be altered due to amino acid substitutions in variants I554A/G, L565A/G, 2G and W570G.

SDS-mediated activation assay

To study the influence of different amino acid residues on the activation in a native-like system EcPOX is stimulated with SDS in an assay constructed similar to the proteolysis experiments. The enzyme is pre-incubated with pyruvate to establish SR II:SR III equilibrium. Addition of SDS leads preferentially to stabilization of the SR III-state which is therefore gradually removed from equilibrium.

After initiation of the activation process by addition of different amounts of SDS, the increase in activity is determined after distinct time points using the Q0 steady-state assay (figure 29 A and B). Afterwards, obtained activities are displayed in dependence of the SDS concentration (figure 29 C). Although this figure is not time-resolved, for one specific SDS concentration the lowest activity corresponds to an SDS incubation time of approximately one minute and the final activity to enzyme stimulated for 30 min with SDS. To compare the different variants the final activity is converted to relative activity by adjusting the

SDS

resting state (R) substrate-bound state (SB) substrate-reduced state (SR)

+ pyruvate

electron transfer

= pyruvate

= β-peptide (EcPOX 471-572)

= α-peptide (EcPOX 550-572)

= covalent ThDP intermediates (L-ThDP, HE-ThDP, Ac-ThDP)

= conformational change

α α

β

ββ

ββ β α

II

III SDS

β α

= α-peptide (helical form) ββ

Δ101 protected Δ23 accessible Δ101 and Δ23 protected

maximum activity to 100 %. Activation studied with different amounts of SDS allows determination of SDS0.5, the SDS concentration where 50 % of the protein is activated after half of the measuring time (figure 29 D). SDS0.5 describes the affinity of EcPOX for SDS molecules and was used as a measure to compare the different variants. Thus, an increase of SDS0.5 with respect to the wt enzyme can indicate a smaller amount of SR III in equilibrium or a disturbed helix formation.

SDS-mediated activation of EcPOX variants EcPOX wt:

Kinetics of SDS-mediated activation of EcPOX wt and variant L565A are exemplary shown in figure 29 A and B. Data of other variants are illustrated in the appendix (chapter 8.4) and summarized in figure 29 D.

The wt enzyme is completely activated in the presence of 100 µM SDS. At this concentration the activation process is already finished within the dead time of the assay (approximately one minute).

Intermediary activation is observed if 20-70 µM SDS are added. In those cases the activity is increased with longer SDS incubation time. SDS0.5 was determined to be 50 µM.

Putative signal transfer variants F465A, Y278F, Y549A and F260A:

Variant F465A is not activated by SDS which resembles results observed in the proteolysis reactions.

Since both assays rely on exposition of the membrane anchor in state SR III based on functional FAD reduction, the observed effects can be correlated to an impaired electron transfer to the flavin cofactor due to the substitution of Ala for Phe.

EcPOX Y278F shows nearly no activation if assayed with 30 µM SDS and complete activation only at 150 µM detergent. In comparison to the wt enzyme Y278F needs an approximately 1.5fold higher SDS amount to reach a similar activation level (EcPOX wt: SDS0.5 = 50 µM, Y278F: SDS0.5 = 70 µM). Since the amino acid sequence of the membrane-anchor is not altered in this variant, the observed results only correspond to a shift of the SR II:SR III equilibrium towards the SR II-state. The same effect was observed for proteolytic activation (chapter 4.1.4). Both experiments imply an important role of Tyr278 on the activation process starting from the reduced flavin cofactor.

A approximately 1.7fold decreased SDS0.5 is detected for Y549A. Although the exchanged residue of this variant is located at the N-terminal end of the membrane anchor an influence on helix formation cannot be excluded. However, it seems more likely that the lowered SDS0.5 is caused by a shift of the SR II:SR III equilibrium towards the membrane binding competent species SR III in the variant.

Wt-like activation is observed for EcPOX F260A with a SDS0.5 of 50 µM. As already indicated in proteolysis reaction Phe260 does not seem to play a central role in release of the membrane anchor and helix formation and is therefore not involved in the activation process.

figure 29: SDS-mediated activation of EcPOX variants. 160 nM EcPOX were pre-incubated with 200 mM pyruvate. The activation process was initiated by addition of different SDS amounts and activity was monitored by Q0 steady-state assay. A:

Kinetics of EcPOX wt activation. B: Kinetics of EcPOX L565A activation. C: Comparison of SDS-mediated activation of EcPOX wt and L565A in dependence of the desired SDS concentration. Each ensemble of data points at a given SDS concentration resembles one curve of A and B. The gray dashed line highlights SDS0.5. D: Comparison of SDS0.5 values of EcPOX variants.

SDS0.5 values are displayed as bar plot and by white numbers. Red color indicates a SDS0.5 smaller than wt whereas black color illustrates a SDS0.5 higher than wt. (* F465 was not activated with SDS. # EcPOX 2G was already completely activated without SDS)

Potential alpha-peptide binding variants 3x, I554A/G, L565A/G, 2G, W570G:

Variants supposed to be involved in hydrophobic stabilization of the membrane anchor (I554A, L565A/G, 2G) show a decreased SDS0.5 value compared with the wt enzyme. The most pronounced effect is observed for EcPOX 2G which is already completely activated in the resting state. Thus, one can suggest that the equilibrium between SR II and SR III is shifted towards the SDS-binding competent SR III-state in those variants. These findings are in line with proteolysis experiments and foster the idea that the membrane anchor is stabilized at the protein surface by hydrophobic interactions.

A similar observation can be made for variant EcPOX 3x which lacks the prominent electrostatic interactions and hydrogen bonds of the membrane anchor to the protein core. Compared to EcPOX wt the determined SDS0.5 value is 1.7fold decreased which reveals accumulation of state SR III in equilibrium.

However, this result is in contrast to proteolytic digestion approaches which show a wt-like equilibrium position. One could speculate that due to its amphiphilic nature SDS is able to provide electrostatic

interactions contrary to the protein surface. Thus, SDS could increase the amount of the exposed form of the membrane anchor which leads to accelerated activity compared to the wt enzyme.

An opposite effect is observed for EcPOX I554G and W570G. For both variants a SDS0.5 of 70 µM is determined. These findings are in contrast to proteolysis experiments where the equilibrium of SR II and SR III is shifted towards the SR III-state and a partially released membrane in the resting state is detected.

Therefore, the decreased SDS0.5 value is most likely caused by a disturbed helix formation due to the amino acid exchanges in both variants which leads to a decreased affinity for SDS molecules. One could propose that the SDS0.5 would be even lower but that the accumulation of SDS-binding competent SR III in equilibrium counteracts disturbed generation of the helix.