Binding affinities between XD and N TAIL constructs were established using 180-225 RU of immobilized XD and N TAIL concentrations ranging from 0.1 to 10 mM (see Materials and Methods). Dosage-dependent binding was observed in this range. Reactions conformed to a 1:1 ligand-substrate (Langmuir) binding model, exhibiting an excellent fit (i.e., a 2 value <1 and residuals within the range of 62) following global analysis of sensorgrams. Binding reactions between XD and N TAILHNFC exhibit an equilibrium dissociation constant of 80 nM (see Table 1 ). The XD binding affinity for N TAILD1 (50 nM) is similar to that of N TAILHNFC indicating that Box1 does not participate in binding. In contrast, removal of either Box3 alone or Box2 plus Box3 results in a strong decrease (three orders of magnitude) in the equilibrium dissociation constant, where N TAILD3 and N TAILD2,3 display similar binding affinities (see Table 1 ). The strong decrease in the affinity resulting from removal of Box3 clearly indicates that Box2 is not the sole region involved in binding to XD, and attests that Box3 also plays a role in the interaction with XD, as already suggested by SAXS studies.