Figure 4. The interaction between P113 and the N-terminal region of RH5 is specific and 10-fold stronger in the context of full-length RH5.

(a) RH5Nt bound P113 specifically and with low affinity. Purified monomeric RH5Nt was serially diluted before being injected over 780 RU of monobiotinylated P113 immobilized on a streptavidin-coated sensor chip. The binding traces suggested the presence of a small amount of multimeric material (see Methods); consequently, binding equilibrium was not quite achieved even though long (>60 s) injection times were used; the dashed line (inset) marks the time point used for equilibrium binding. From these data, an equilibrium binding constant (K_D) of 3.0±0.5 μM (mean±s.d., n=2) was calculated. (b,c) RH5FL binds P113 with a ten-fold higher affinity than RH5Nt. A kinetic analysis of P113 binding RH5Nt (b) or RH5FL (c) using SPR; inset gels demonstrate the homogeneity and integrity of each RH5 analyte preparation. Serially diluted RH5 analytes were injected over 780 RU of immobilized P113. Binding data (black lines) fitted a simple 1:1 binding model well (red lines), and were used to determine binding constants. (d) Gel filtration elution profiles of the entire ectodomain (EE) and an N-terminal subfragment (Y1-N653) of recombinant tagged P113 demonstrated that P113 EE can form multimers. The lettered dashed lines correspond to fractions collected over the resolved peaks; the elution volumes of gel filtration mass markers are indicated. (e) Native and denaturing (SDS–PAGE) gels corresponding to aliquots of the fractions indicated by the letters in d. P113 EE but not the Y1-N653 fragment resolves at an approximately fourfold higher mass under native when compared with denaturing conditions. The Y1-N653 fragment resolved as two species by native PAGE. Multimeric forms of P113 EE exhibited complex multivalent binding behaviour by SPR (f) compared with P113 Y1-N653 (g). Sensorgrams in (f,g) show a 120 s pulse of purified 8 μM P113 analyte injected over 510 RU of immobilized RH5Nt.