Table 1.

Dissociation of Ca²⁺ and Mn²⁺ from the EF-hand calcium-binding domains in wild-type and mutant S100 proteins.

S100 protein	EF1	EF2
Ca2+ binding
S100B (wt)	>350 μMa,c	56 ± 9 μMb,c
S100B (E31A)	>500 μMa	>500 μMa
S100B (E72A)	480 ± 130 μMa	>500 μMa
S100B (E31A + E72A)	>2 mMa	>2 mMa
S100B (+p53)	—	20 ± 3 μMa
S100B (E31A, +p53)	—	21 ± 7 μMa
S100B (E72A, +p53)	—	18 ± 4 μMa
S100B (E31A + E72A, +p53)	—	>300 μMa
S100B (wt, +TRTK12)	—	12 ± 7 μMc
S100A1 (wt)	—	27 ± 2 μMd
S100A1 (wt, +TRTK12)	—	8 ± 3 μMe
S100A2 (wt)	—	470 ± 50 μMf
S100A3 (wt)	—	∼4 mMg
S100A4 (wt)	—	∼2.6 μMh
S100A4 (wt, +p37)	—	∼0.2 μMh
S100A5 (wt)	160 μMi	∼0.2 μMi
S100A6 (wt)	—	∼3.0 μMj
S100A7 (wt)	—	∼1.0 μMk,l
S100A11 (wt)	—	∼0.5 mMm
S100A12 (wt)	—	∼50 μMn
S100A13 (wt)	∼400 μM	∼8 μMo,p
S100A16 (wt)	no binding	0.43 mMq
S100P (wt)	∼800 μM	∼2.0 μMr
S100Z (wt)	> 1 mM	∼0.2 μMs
Mn2+ binding
S100B (wt)	—	71 ± 12 μMa,c
S100B (wt, +p53)	—	27 ± 4 μMa,c
S100B (wt, +TRTK12)	—	6.0 ± 2.0 μMa,c

^aThe value listed is from previously published papers [109, 113], so direct comparisons of binding constants using similar methods/conditions could be made (+/− target, Figure 3). Several others report binding constants using different methods and varying conditions for EF1 (200 μM ≤K_D≤ 500 μM) and for EF2 (10 μM ≤K_D≤ 60 μM) [58, 78, 86, 87, 114–120].

^bThe dissociation rate constant for wild-type S100B was determined via stopped-flow methods and is k_off = 60 ± 22 s⁻¹. The off-rate together with the K_D enables the calculation of a macroscopic on-rate value of k_on = 1.1 × 10⁶ M⁻¹ s⁻¹ that includes calcium-association plus a large conformational change. The K_D value for the mutants was also determined using competition studies of Ca²⁺ with the respective Tb³⁺-bound S100B mutant in the absence and presence of p53 peptide. The dissociation constants together with the calcium off-rate values measured for the E31A and E72A mutants of 7.1 ± 3.7 s⁻¹ and 6.8 ± 2.0 s⁻¹, respectively, were sufficient to calculate on-rate values of 3.4 ± 2.0 × 10⁶ M⁻¹ s⁻¹ and 3.7 ± 1.3 × 10⁶ M⁻¹ s⁻¹ for the mutants [109, 113].

^cFrom Charpentier et al. (2010) [34].

^dFrom Wright et al. (2005) [61].

^eFrom Wright et al. (2009) [59]. S100A1 has also been shown to bind the full-length ryanodine receptor at 100 nM free Ca²⁺ [60, 67].

^fFrom Franz et al. (1998) [89].

^gFrom Fritz et al. (1998). A tenfold weaker affinity was reported when purified under aerobic conditions [90, 121].

^hFrom Dukhanina et al. (1998). A weaker affinity was reported under different conditions in Pedrocchi et al. (1994) when S100A4 was originally discovered [122, 123].

ⁱFrom Schäfer et al. (2000). For a direct comparison of Ca²⁺ and Zn²⁺ binding to S100A5 to those of other S100 proteins (i.e., S100B, S100A2, S100A3, S100A4, S1006, and S10011), under identical conditions and Methods, also see Schäfer et al., (2000) [124].

^jFrom Kuznicki and Filipek (1987) and Mani and Kay (1990). Kordowska et al. also measured Ca²⁺-binding for S100A6 under different conditions (^CaK_D ∼18 μM) and found that binding to the target caldesmon (CaD) increased the affinity of S100A6 for Ca²⁺ by approximately 6-fold [96, 125, 126]. Other measurements under higher salt and other varying conditions are also reported with weaker affinities for S100A6 [68, 124].

^kFrom Schäfer et al. (2000) [124]. Weaker binding to Ca²⁺ has also been reported for this protein in other conditions [127].

^lNo data is available for S100A8/A9, and S100A10 does not bind Ca²⁺.

^mFrom Allen et al. (1996) and Schäfer et al., (2000) [124, 128]. Note the affinity for Ca²⁺ increases by 10-fold upon the addition of a target molecule as found with other S100 proteins [128].

ⁿFrom Dell'Angelica et al. (1994). Note that Zn²⁺-binding to S100A12 significantly increases Ca²⁺-binding affinity for this protein in the presence of Zn²⁺ (EF2: ^CaK_D = 40 nM, EF1: ^CaK_D = 15 μM) [129].

^oFrom Ridinger et al. (2000). This protein is unique among S100 family members in that it does not bind to the hydrophobic binding dye, TNS, upon the addition of Ca²⁺ [130].

^pNo data is yet available for S100A14, and there is no S10015 [131].

^qFrom Sturchler et al. (2006). The value in the table is for human S100A16, mouse S100A16 bound one calcium too, only weaker (^CaK_D = 0.75 mM) [132].

^rFrom Becker et al. (1992) and Gribenko et al., (1998) [92, 133]. In Gribenko et al., (1998), the effects of Mg²⁺ binding on Ca²⁺ affinity are also rigorously addressed.