A 2ND 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-accessibl e residues important for receptor binding specificity, whereas many of the conserved residues are buried in hydrophobic cores or in the dime r interface, A stretch of six amino acids (from Asp-72 to Asn-77) in n erve growth factor (NGF) represents an exception to this general rule, These residues are highly conserved and yet form an exposed hydrophil ic 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 4-1 0-fold, Only the D72A mutant showed an additional impairment in bindin g to the TrkA receptor, which was accompanied by reduced biological ac tivity in PC12 cells, indicating a structural and/or conformational ef fect of this mutation, Replacement of Ser-73 or Asn-77 with alanine (m utants S78A and N77A, respectively) had no measurable effects on recep tor binding, The triple mutant K74A/H75A/N77A exhibited properties tha t were consistent with the combined effects of the individual mutation s, namely impaired binding to p75 without deficits in its interaction with TrkA, In contrast, in the triple mutant D72A/S73A/K74A, the simul taneous replacement of Asp-72 and Lys-74 with alanine had a compensato ry effect such that binding to both p75 and TrkA was comparable to tha t of wild-type NGF, despite the deficits seen in the individual replac ements, This molecule, however, was produced at low levels, and its bi ological activity in sympathetic ganglion explants was reduced, which, together with results from TrkA phosphorylation assays, indicated a r educed 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 cand idate points of contact, In addition, Asp-72 appears to be a structura lly important side chain for stabilizing the conformation of the loop through interactions with neighboring residues.