The rhomboid protease GlpG has weak interaction energies in its active site hydrogen bond network

Abstract

Intramembrane rhomboid proteases are of particular interest because of their function to hydrolyze a peptide bond of a substrate buried in the membrane. Crystal structures of the bacterial rhomboid protease GlpG have revealed a catalytic dyad (Ser201-His254) and oxyanion hole (His150/Asn154/the backbone amide of Ser201) surrounded by the protein matrix and contacting a narrow water channel. Although multiple crystal structures have been solved, the catalytic mechanism of GlpG is not completely understood. Because it is a serine protease, hydrogen bonding interactions between the active site residues are thought to play a critical role in the catalytic cycle. Here, we dissect the interaction energies among the active site residues His254, Ser201, and Asn154 of Escherichia coli GlpG, which form a hydrogen bonding network. We combine double mutant cycle analysis with stability measurements using steric trapping. In mild detergent, the active site residues are weakly coupled with interaction energies (ΔΔG _Inter) of ‒1.4 kcal/mol between His254 and Ser201 and ‒0.2 kcal/mol between Ser201 and Asn154. Further, by analyzing the propagation of single mutations of the active site residues, we find that these residues are important not only for function but also for the folding cooperativity of GlpG. The weak interaction between Ser and His in the catalytic dyad may partly explain the unusually slow proteolysis by GlpG compared with other canonical serine proteases. Our result suggests that the weak hydrogen bonds in the active site are sufficient to carry out the proteolytic function of rhomboid proteases.

Figure 1.

Hydrogen bond network in the active site of the intramembrane protease GlpG of E. coli. Structure of GlpG (PDB accession no. 3B45) showing the location of the active site and the crystallographic water molecules. Ser201 and His254 form a catalytic dyad. The conserved residue Asn154 forms the oxyanion hole together with the backbone amide group of Ser201 and another conserved residue His150 (data not shown).

Figure 2.

Measuring thermodynamic stability of GlpG using steric trapping. (a) Principle of steric trapping. Left: Thiol-reactive biotin derivative with a fluorescent reporter group employed in this study (Guo et al., 2016). Right: When biotin tags are conjugated to two specific residues that are spatially close in the folded state but distant in the amino acid sequence, the first mSA binds either biotin label with the intrinsic binding affinity (ΔG^o_Bind). Because of steric hindrance, the second mSA binds only when native tertiary contacts are unraveled by transient unfolding. Hence, binding of the second mSA is attenuated depending on the stability of the target protein (ΔG^o_Bind + ΔG^o_U). By adjusting the biotin affinity of mSA by mutation, unfolding and binding reactions can be reversibly controlled, and ΔG^o_U of the target protein can be obtained by monitoring binding of the second mSA or protein unfolding. Binding of mSA to biotin labels on GlpG was measured by FRET-based assay using BtnPyr label (donor) and mSA-labeled with nonfluorescent dabcyl quencher (acceptor; Guo et al., 2016). Thiol-reactive dabcyl (DAB-maleimide) was conjugated to a unique cysteine residue (Cys82) engineered in the active subunit of mSA (denoted as mSA_DAB). (b) Binding isotherms between double-biotin variants of GlpG (95/172_N-BtnPyr₂) and mSA_DAB variants with a reduced biotin binding affinity monitored by quenching of pyrene fluorescence. The backbone in cyan: N subdomain (residues 87–198); the backbone in orange: C subdomain (residues 199–276; Guo et al., 2016). Errors in ΔG^o_U denote ±SD from fitting. The mSA variant mSA_DAB-S27A (left, K_d,biotin = 1.4 ± 0.9 nM) was used when ΔG^o_U of more stable GlpG mutants were measured while mSA_DAB-S45A (right, K_d,biotin = 9.0 ± 4.3 nM) was used for less stable GlpG mutants.

Figure 3.

Double mutant cycle analysis to measure the side-chain interaction energies in the active site of GlpG. All energy values have units of kcal/mol. The values adjacent to the arrows indicate ΔΔG°_U induced by the designated mutations. Errors denote ±SD from fitting.

Figure 4.

Cooperativity profiling of the active site residues of GlpG. (a) Binding isotherms between double-biotin variants of GlpG (172/267_C-BtnPyr₂) and mSA_DAB variants to measure ΔG^o_U of the C subdomain. (b) The cooperativity profiles of the active site residues.