At the second position, Pro seems to be strictly required based on several observations: (1) there is absolutely no variation in the alignments at this position, (2) the chronic granulomatous disease-associated mutation within CYBA (P156Q) hits this second position and is known to abrogate binding, (3) the Pro at this position (equivalent to P299 within the AIR of NCF1) makes contacts with both domains of the tandem SH3 [19,20]. This evidence concerns the gene NCF1 and chronic granulomatous disease.