Table 6.

Dissociation Constants for MIIA Binding to S100A4

MIIA construct	Kd (μM)
S100A4 + MIIA1338-1960 (rod, low salt)	2.7 ± 0.6a
S100A4 + MIIA1851-1960 (coiled coil + tailpiece, low salt)	0.41 ± 0.01b
S100A4 + MIIA1851-1960 (coiled coil + tailpiece, high salt)	0.50 ± 0.01b
S100A4 + MIIA1893-1923 (coiled coil, low salt)	0.34 ± 0.02c
S100A4 + MIIA1893-1923 (coiled coil, high salt)	1.1 ± 0.1d
S100A4 + MIIA1908-1923 (minimal S100A4 site, low salt)	0.50 ± 0.20e
S100A4 + MIIA1908-1923 (minimal S100A4 site, low salt)	0.89 ± 0.12f
S100A4 + MIIA1908-1923 (minimal S100A4 site, high salt)	1.7 ± 0.2f

^aThis value is from published studies (29). The dissociation constant for the Ca²⁺-dependent interaction between S100A4 and MIIA rod filaments was determined in a cosedimentation assay under low-salt conditions [20 mM Tris (pH 7.5), 20 mM NaCl, 2 mM MgCl₂, 0.3 mM CaCl₂, 1 mM DTT, 0.02% NaN₃]. Binding could not be examined under high-salt conditions (150 mM KCl) since S100A4 disassembles the MIIA rods under these conditions.

^bThe dissociation constant for the Ca²⁺-dependent interaction between S100A4 and MIIA^1851-1960 was determined in competition studies with fluorescently labeled MIIA^1908-1923 using a fluorescence polarization assay under low- and high-salt conditions [20 mM Tris (pH 7.5), 20 or 150 mM KCl, 1 mM DTT, 0.02% NaN₃, and 0.5 mM CaCl₂]. MIIA^1851-1960 comprises part of the coiled-coil domain and the C-terminal tailpiece of MIIA.

^cThe dissociation constant for the Ca²⁺-dependent interaction between S100A4 and MIIA^1893-1923 was determined using ITC under low-salt conditions [20 mM Tris (pH 7.5), 20 mM KCl, 250 μM TCEP, and 0.8 mM CaCl₂]. MIIA^1893-1923 comprises part of the coiled-coil domain of MIIA.

^dThe dissociation constant for the Ca²⁺-dependent interaction between S100A4 and MIIA^1893-1923 was determined in competition studies with fluorescently labeled MIIA^1908-1923 using a fluorescence polarization assay under high-salt conditions [20 mM Tris (pH 7.5), 150 mM KCl, 1 mM DTT, 0.02% NaN₃, and 0.5 mM CaCl₂].

^eThe dissociation constant for the Ca²⁺-dependent interaction between S100A4 and MIIA^1908-1923 was determined using ITC under low-salt conditions [20 mM Tris (pH 7.5), 20 mM KCl, 250 μM TCEP, and 0.8 mM CaCl₂]. MIIA^1908-1923 comprises a portion of the coiled-coil domain of MIIA but is not predicted to form a coiled coil.

^fThe dissociation constant for the Ca²⁺-dependent interaction between S100A4 and FITC-MIIA^1908-1923 was determined as described previously using a fluorescence polarization assay (71) under low- or high-salt conditions [20 mM Tris (pH 7.5), 20 or 15 mM KCl, 1 mM DTT, 0.02% NaN₃, and 0.5 mM CaCl₂].