A second determinant of binding to the p75 neurotrophin receptor revealed by alanine-scanning mutagenesis of a conserved loop in nerve growth factor.

In the neurotrophin family, variable regions contain solvent-accessible residues important for receptor binding specificity, whereas many of the conserved residues are buried in hydrophobic cores or in the dimer interface. A stretch of six amino acids (from Asp-72 to Asn-77) in nerve growth factor (NGF) represents an exception to this general rule. These residues are highly conserved and yet form an exposed hydrophilic loop region away from other known determinants of receptor binding. We have investigated the functional importance of this region in NGF using alanine-scanning mutagenesis. Individual mutation of Asp-72, Lys-74, or His-75 to alanine (mutants D72A, K74A, and H75A, respectively) reduced the binding affinity for the p75 neurotrophin receptor by 4-10-fold. Only the D72A mutant showed an additional impairment in binding to the TrkA receptor, which was accompanied by reduced biological activity in PC12 cells, indicating a structural and/or conformational effect of this mutation. Replacement of Ser-73 or Asn-77 with alanine (mutants S73A and N77A, respectively) had no measurable effects on receptor binding. The triple mutant K74A/H75A/N77A exhibited properties that were consistent with the combined effects of the individual mutations, namely impaired binding to p75 without deficits in its interaction with TrkA. In contrast, in the triple mutant D72A/S73A/K74A, the simultaneous replacement of Asp-72 and Lys-74 with alanine had a compensatory effect such that binding to both p75 and TrkA was comparable to that of wild-type NGF, despite the deficits seen in the individual replacements. This molecule, however, was produced at low levels, and its biological activity in sympathetic ganglion explants was reduced, which, together with results from TrkA phosphorylation assays, indicated a reduced stability during prolonged culture conditions. Taken together, these data reveal a second region of interaction with the p75 receptor in NGF with the positively charged residues Lys-74 and His-75 as candidate points of contact. In addition, Asp-72 appears to be a structurally important side chain for stabilizing the conformation of the loop through interactions with neighboring residues.