Figure 1.
Restricted volume effect of domain tethering.
(A) Crystal structure of Syt I in the absence of ligand (PDB 2R83). The C2A domain, nine-residue linker region, and C2B domain are colored purple, blue, and green, respectively. (B) Conceptual representation and illustrative calculation of the volume accessible to the C2B domain with respect to C2A. Because the C2B domain is tethered to C2A, the volume it can occupy is restricted. This significantly increases the local concentration of C2B. If, for instance, the volume is calculated using the length of the linker and width of C2B as an approximate hemisphere radius (Pymol measurement of roughly 57 Å), the accessible volume is 4×10−19 cm3. Assuming 1 molecule of C2B occupies this volume, its local concentration is equal to (1 molecule C2B/6.022×1023 molecules per mole)/(4×10−25 m3) or ∼4 M. The local concentration of the C2A domain can be approximated in an analogous way. The resulting concentrations would shift the association equilibrium in the direction of bound domains.
Table 1.
Concentration dependence controls for C2B and C2AB.
Figure 2.
Globally-fit denaturation of C2A (•) and C2B (○) using DSC (top) and FLT (bottom) methods.
Circles represent the raw data and lines are the fitted model. C2A denaturations displayed with permission from Biophysical Journal. Higher concentrations refer to DSC samples. (A, E) [C2A] = 13 µM and 0.75 µM, [C2B] = 13 µM and 0.80 µM in the presence of 500 µM EGTA. (B, F) [C2A] = 13 µM and 0.75 µM, [C2B] = 12 µM and 0.75 µM in the presence of Ca2+. [Ca2+] = 800 µM and 770 µM for C2A, [Ca2+] = 5.3 mM and 5.2 mM for C2B. (C, G) [C2A] = 13 µM and 0.75 µM, [C2B] = 15.0 µM and 3.2 µM in the presence of LUVs consisting of 60∶40 POPC:POPS. [Lipid] = 870 µM and 50 µM for C2A, [Lipid] = 1.1 mM and 240 µM for C2B. Higher [C2B] in FLT was needed under lipid conditions to better see transition. (D, H) [C2A] = 13 µM and 0.75 µM, [C2B] = 13 µM and 3.2 µM for C2B in the presence of both LUVs and Ca2+. For C2A, [Ca2+] = 800 µM and [lipid] = 870 µM, [Ca2+] = 770 µM and [lipid] = 50 µM. For C2B, [Ca2+] = 5.2 mM and [lipid] = 290 µM, [Ca2+] = 5.2 mM and [lipid] = 70 µM. Low [lipid] for C2B prevented precipitation in calorimeter, but also limited FLT data collection due to flocculation and light scattering [29].
Table 2.
Scan rate dependence controls for C2B and C2AB.
Table 3.
Thermodynamic parameters and their associated error from the global fit of DSC and FLT data.
Figure 3.
Raw DSC data for simultaneous thermal denaturation of separate C2A and C2B domains.
(A) 10 µM of each domain in the presence of 500 µM EGTA. (B) 15 µM of each domain in the presence of 5.2 mM Ca2+ conditions. (C) 15 µM of each domain in the presence of 2.2 mM PS. (D) 11 µM of each domain in the presence of 2.9 mM PIP2. (E) 12 µM of each domain in the presence of 1.6 mM PS and 5.2 mM Ca2+. (F) 10 µM of each domain in the presence of 2.8 mM PIP2 and 5.2 mM Ca2+ conditions. Note that in most instances, two separate peaks are seen representing the independent unfolding of each domain. If C2A and C2B within the C2AB construct did not communicate, the C2AB denaturation profile would more closely resemble the above heat capacity profiles.
Figure 4.
Thermal denaturation of C2AB using DSC (A–C, G–I) and FLT (D–F, J–L) methods.
Circles represent raw data and lines are the fitted model, excluding panels (H) and (I) wherein the line represents raw heat capacity data. Large and small concentrations refer to DSC and FLT concentrations, respectively. (A, D) 13 µM and 4.5 µM C2AB in the presence of 500 µM EGTA. (B, E) 12 µM and 0.75 µM C2AB in the presence of 5.2 mM and 5.1 mM Ca2+. (C, F) 12 µM and 0.75 µM C2AB in the presence of 1.7 mM and 110 µM PS. (G, J) 11 µM (3 replicates of DSC) and 0.75 µM C2AB in the presence of 2.9 mM and 210 µM PIP2. (H, K) 12 µM C2AB in the presence of 5.2 mM Ca2+ and 1.7 mM PS; 0.75 µM C2AB in the presence of 5.1 mM Ca2+ and 110 µM PS. (F, I) 11 µM (1 replicate of DSC) C2AB in the presence of 5.2 mM Ca2+ and 2.9 mM PIP2; 0.75 µM C2AB in the presence of 5.1 mM Ca2+ and 210 µM PIP2. Both calorimetric denaturations involving PIP2 had a limited number of replicates due to precipitation. In the absence of any interaction, the two domains would unfold independently. Instead, here the two domains unfold as one.
Figure 5.
Stability of C2AB as a function of temperature in the presence and absence of ligand.
Solid, dashed, dotted, and dash-dot-dash lines represent EGTA, Ca2+, phosphatidylserine, and phosphatidylinositol environments. Note that most proteins of comparable size have maxima in the range of 10–20 kcal/mole [30], [31].
Figure 6.
Conceptual representation of negative coupling.
In the absence of bound ligand (top), domains have basal level stability. When a ligand specific to the C2A domain binds (middle), C2A is stabilized and C2B is destabilized through negative coupling. When a ligand specific to C2B binds (bottom), the opposite effect is seen; C2B is stabilized and C2A is destabilized. Note that binding of domain-specific ligands lowers the probability of binding-competent conformers being occupied in the adjacent domain through domain destabilization, representing a form of allosteric regulation. The extent of negative coupling, like domain stability, changes depending on the types of ligand present.
Figure 7.
Location of tryptophan residues (orange) in C2A (purple) and C2B (green).
Two of three tryptophan residues (C2A’s and one of C2B’s) occupy superficial positions and may, as a result, be more solvent exposed in solution (left). The second tryptophan in the C2B domain is partially embedded in the core of the β-sandwich motif amongst several hydrophobic residues (yellow) (right). The differences in tryptophan environment likely give rise to unequal FLT signal contributions, with most of the signal coming from C2B’s β-sandwich residue.