Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding

Abstract

Caspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a β-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/deuterium exchange data in the L2' and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK _a values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.

Keywords: apoptosis; cysteine protease; hydrogen exchange mass spectrometry; molecular dynamics; neurodegeneration.

FIGURE 1.

Caspase-6 undergoes helix-strand transition upon substrate binding. The overall fold of canonical caspases before and after substrate binding is represented by the superimposition of the unliganded (orange; PDB code 1K86) and the peptide-based substrate mimic DEVD-bound (green; PDB code 1F1J) structures of caspase-7 (middle). Highlighted regions are the active site cysteine (blue), the dimer interface, and the substrate binding loops 1–4 (L1–L4). Caspase-7, like all other caspases, adopts a canonical strand conformation in its 130's region in both the unliganded state (orange; PDB code 1K86) and the peptide-based substrate mimic DEVD-bound (green; PDB code 1F1J) states (right). In contrast, caspase-6 can adopt a noncanonical extended helical conformation in its 130's region in the unliganded state (orange, PDB code 2WDP) but recovers the canonical strand conformation upon binding to a peptide-based substrate mimic VEID-aldehyde (green; PDB code 3OD5) (left).

FIGURE 2.

H/D exchange heat map of the relative deuterium incorporation. For each peptic peptide of the unliganded and the peptide-based substrate mimic-bound states of caspase-7 (A) and caspase-6 (B), the percentage relative deuterium level for each H/D exchange incubation time (0.17, 1, 10, 60, and 120 min) is mapped onto its corresponding linear sequence. The percentage relative deuterium incorporation is calculated by dividing the observed deuterium uptake by the theoretical maximum deuterium uptake for each peptide. The H/DX-MS experiments followed 64 peptides common to both unliganded and DEVD-bound caspase-7 that covers 93% of the linear sequence. Likewise, H/DX-MS experiments followed 70 peptides common to both unliganded and VEID-bound caspase-6 that covers 91% of the linear sequence. Peptic peptides with no H/D exchange data at any given incubation time are colored white. All caspase-6 and caspase-7 variants used in the H/DX-MS experiments were cleaved, active forms lacking both the prodomain (residues 1–23 in both caspase-6 and caspase-7) and linker (residues 180–193 in caspase-6; residues 199–206 in caspase-7). The secondary structural elements are also shown above the caspase-6 and caspase-7 sequences. The percentage relative deuterium level of each peptic peptide represents the average values of duplicate experiments performed on two separate days.

FIGURE 3.

Caspase-6 shows distinctive conformational dynamics in its 130's region. Shown is the difference in deuterium uptake (Da) of the corresponding peptic peptides in the unliganded and the peptide-based substrate mimic-bound states of caspase-7 (A) and caspase-6 (B) at the indicated time points of exposure to deuterium in solution. The residue numbers for each peptic peptide are listed with corresponding secondary structural elements. For these data, a deuterium uptake difference of >0.6 Da is considered significant at a 98% confidence interval. The intensity of the blue color represents the peptides that undergo a significant decrease in H/D exchange (less solvent-exposed, less flexible) upon peptide-based substrate mimic binding. The intensity of the red color represents the peptides that undergo significant increase in H/D exchange (more solvent-exposed, more flexible) upon peptide-based substrate mimic binding. C, representative deuterium incorporation plots for peptic peptides covering the 60's, 90's, and 130's regions of caspase-7 (top) and caspase-6 (bottom) in both unliganded (black lines) and peptide-based substrate mimic-bound (blue lines) states. The representative MS spectra of the highlighted peptic peptides are shown in supplemental Fig. S7. Error bars, S.D. of duplicate H/DX-MS measurements done on two separate days. The residue numbering is listed for the homologous regions in caspase-7 and caspase-6, which have different numbering for the structurally homologous regions due to differences in the lengths of their respective subunits. D, difference in deuterium uptake between the unliganded and the DEVD-bound states of caspase-7 after 2-h incubation mapped onto the structure of caspase-7 (PDB code 1K86) shown in both ribbon and surface representations. E, difference in deuterium uptake between the unliganded and the VEID-bound states of caspase-6 after 2-h incubation mapped onto the structure of caspase-6 (PDB code 2WDP) shown in both ribbon and surface representations.

FIGURE 4.

Protonation state of Glu-135 is critical to the dynamics of the 130's region. A, the unliganded (orange) and VEID-bound (green) states of caspase-6 present different local charge states of four identified residues in close proximity and within the 130's region, which are deprotonated upon substrate binding (shown in black). B, calculated local pK_a values of the four amino acid residues that undergo differential protonation states between the unliganded (helical) and the VEID-bound (strand) states of caspase-6. C, plot of RMSF (Å) of the backbone atoms of each amino acid in caspase-6. A 1.2-ns-long simulated annealing-based molecular dynamics simulation was performed in all capase-6 protonation variants. The unliganded helical protonation variant represents the control ensemble of the triply protonated state (HisH-52, GluH-135, GluH-221) of caspase-6. The VEID-bound (strand) protonation variant represents the control ensemble of the triply deprotonated state (His-52, Glu-135, Glu-221) of caspase-6. Unliganded with deprotonated E135, protonation variant of caspase-6 that has deprotonated Glu-135 but remains protonated at the other two residues (HisH-52 and GluH-221). Residue-by-residue fluctuations are shown for caspase-6 in the unliganded (helical) state (black), VEID-bound (strand) state (red), and the unliganded caspase-6 with deprotonated Glu-135 (blue). The RMSF profile of unliganded caspase-6 with deprotonated Glu-135 behaves similarly to the VEID-bound (strand) state in the 130's region (inset). D, variation of the RMSF (Å) along key regions of caspase-6 mapped onto the structure of unliganded caspase-6 (PDB code 2WDP) highlighting only the 60's, 90's, and 130's regions.

FIGURE 5.

E135Q has kinetics and pH profile similar to those of WT caspase-6. A, the Michaelis-Menten kinetic parameters of caspase-6 WT and the “constantly protonated” variant, E135Q. The values are reported as mean ± S.E. of three independent trials performed on three separate days. B, the activity of caspase-6 WT (black line) and E135Q (blue line) variants as a function of pH. For normalization, the highest and the lowest relative fluorescence response for each data set was set to 100 and 0%, respectively, and reported as fractions. Error bars, S.D. of duplicate measurements on two separate days. All caspase-6 activity assays used fluorescence-based measurements following cleavage of a peptide-based substrate mimic, VEID-AMC, by caspase-6.

FIGURE 6.

The stabilizing effect of constantly protonated E135Q variant is more pronounced at higher pH. A, the CD spectra of caspase-6 WT (black line) and the E135Q variant (blue line) measured at different pH levels. B, CD thermal denaturation profiles of caspase-6 WT (black dotted line) and E135Q (blue dotted line) at the indicated pH levels. Normalization of the CD signal was achieved by setting the highest and the lowest values for each data set as 100 and 0%, respectively. The thermal denaturation data were then fitted to the Boltzmann sigmoidal equation (black solid line in WT and blue solid line in E135Q), where the midpoint of the curve was determined to be the apparent T_m. C, the expected percentage protonation of Glu-135 at different pH was determined based on the calculated pK_a values in Fig. 4B following the Henderson-Hasselbalch equation. D, interactions between Glu-135 and adjacent residues that impact the helix-strand interconversion. E, the individual apparent T_m values and the differences in the apparent T_m values of caspase-6 WT and E135Q at different pH are tabulated. The apparent T_m values are reported as mean ± S.D. of duplicate measurements on two independently prepared samples performed on two different days.

FIGURE 7.

A model showing caspase-6 undergoes a helix-strand transition upon substrate binding. Before substrate binding, caspase-6 continuously interconverts between the noncanonical (helical) conformation and the canonical strand conformation and fully transitions to the strand conformation upon substrate binding. This transition may be facilitated by the presence of a dynamic hydrophobic patch (light orange, inset view) that could also function as an exosite for substrate binding. After substrate cleavage and release, caspase-6 exists in a dynamic equilibrium between the helical and strand states.