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Table 1

Dissociation Constants for the Binding of Staphylococcal Enterotoxin Mutants to the TCR 14.3.d β Chain and Stimulatory Activity of these Mutants

SAG mutantKd analytical centrifugeKd BIAcoreΔΔGStimulatory capacity
  × 10−6 M  × 10−6 M  kcal/mol  ng/ml 
SEC3 wild-type         4.5    3.0          200 
SEC3 T20A        42   49       1.4         4,000 
SEC3 N23A  ⩾150*  NB  >2.5  >30,000§ 
SEC3 Y26A        86   60       1.7         3,000 
SEC3 N60A        25   48       1.3         2,000 
SEC3 Y90A  ⩾150*  240  >2.5         9,000 
SEC3 V91A       117  130       2.1         6,000 
SEC3 G102A         4.6  ND       0.1        400 
SEC3 K103A         5.6    9.4       0.4         1,000 
SEC3 G106A         3.4  ND       0.1        400 
SEC3 F176A        92  110       1.9        30,000 
SEC3 Q210A  ⩾150*  NB  >2.5  >30,000§ 
SEB wild-type       120  140           20 
SEB L20T       100  120           30 
SEB V26Y        26   12            5 
SEB Y91V       140  160          100 
SEB L20T, V26Y, Y91V  ⩾150  NB           7,000 
SAG mutantKd analytical centrifugeKd BIAcoreΔΔGStimulatory capacity
  × 10−6 M  × 10−6 M  kcal/mol  ng/ml 
SEC3 wild-type         4.5    3.0          200 
SEC3 T20A        42   49       1.4         4,000 
SEC3 N23A  ⩾150*  NB  >2.5  >30,000§ 
SEC3 Y26A        86   60       1.7         3,000 
SEC3 N60A        25   48       1.3         2,000 
SEC3 Y90A  ⩾150*  240  >2.5         9,000 
SEC3 V91A       117  130       2.1         6,000 
SEC3 G102A         4.6  ND       0.1        400 
SEC3 K103A         5.6    9.4       0.4         1,000 
SEC3 G106A         3.4  ND       0.1        400 
SEC3 F176A        92  110       1.9        30,000 
SEC3 Q210A  ⩾150*  NB  >2.5  >30,000§ 
SEB wild-type       120  140           20 
SEB L20T       100  120           30 
SEB V26Y        26   12            5 
SEB Y91V       140  160          100 
SEB L20T, V26Y, Y91V  ⩾150  NB           7,000 

Affinity measurements by sedimentation equilibrium and BIAcore, as well as the biological activity assays, were performed as described in Materials and Methods. Differences in free energy changes are calculated as the differences between ΔGs of mutant and wild-type SAGs (ΔΔG = ΔGmutant − ΔGwild-type). The values of the individual ΔGs are calculated from the average Kds obtained from sedimentation equilibrium and BIAcore measurements according to the equation ΔG = −RT ln (1/Kd), where R is the universal gas constant and T is the absolute temperature in Kelvin. For the three weakest binding mutants, N23A, Y90A, and Q210, a ΔΔG value of >2.5 kcal/mol (which corresponds to a 70-fold decrease in affinity) is estimated, as these mutants bind at least 50–100-fold weaker than wild-type SEC3. The stimulatory capacity of different SAGs was estimated from the dose (ng/ml) required to induce proliferation 100-fold above background. Background cpm were typically less than 100. NB, no detectable binding observed in the BIAcore measurements at TCR-β chain concentrations up to 128 μM; ND, not determined.  

*

 150 mM should be considered as a lower limit for these mutants.  

 Value for SEB wild-type was taken from the earlier work by Malchiodi et al. (15).  

§

 For these SEC3 mutants, SAG concentrations of 30,000 ng/ml were not sufficient to induce proliferation 100-fold above background.  

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