Extended Data Figure 2. Two point mutations in mouse CD40L coding sequence impair binding to CD40.

a, Sequence alignment of human and mouse CD40L proteins. Due to the lack of crystallographic data describing mouse CD40-CD40L complex, we identified residues potentially engaged in CD40 binding based on information available for the human CD40-CD40L complex. Residues in human CD40L sequences engaged in interaction with CD40 based on crystallographic data are highlighted in blue. Among these, residues for which a charge reversal mutation was shown to affect CD40 binding are boxed. Filled boxes identify the residues in mouse CD40L for which a charge reversal mutation was performed (K142E and R202E). Mutations at equivalent locations in the human CD40L coding sequence (K143, R203) have also been detected in hyper-IgM patients. CD40L carrying both mutations (K142E and R202E) is identified as CD40L*. b, Binding of CD40 to CD40L-SrtA and CD40L*-SrtA. 293T cells were transfected with CD40L-SrtA or CD40L*-SrtA, incubated with CD40-Fc protein and analyzed by flow cytometry. Histogram plots show severe impairment of CD40 binding to CD40L*-SrtA. c, B cell activation by CD40L-SrtA and CD40L*-SrtA. Primary murine B cells were cultured on a monolayer of 293T cells expressing CD40L-SrtA or CD40L*-SrtA. CD86 surface expression was analyzed by flow cytometry eighteen hours later. Histogram plots show reduced upregulation of CD86 in B cells stimulated with CD40L*-SrtA. Data representative of two independent experiments.