SITE-DIRECTED MUTAGENESIS AND STRUCTURAL INTERPRETATION OF THE NIDOGEN BINDING-SITE OF THE LAMININ GAMMA-1 CHAIN

A precise molecular map of the nidogen binding site of laminins was ob tained by site-directed mutagenesis and structural analysis of the 56 residue LE module gamma 1III4 of their gamma 1 chain. This demonstrate d the crucial importance of the sequence DPNAV (position 800-804) in t he disulfide-bonded loop a, with major contributions made by all resid ues except P801, Different substitutions of these residues emphasized the essential role of the negative charge (D800) and carboxamide group (N802) as well as their spacings and hydrophobic contacts (V804) for interaction, and predict direct contacts of these three residues with a complementary binding region of nidogen, An inactivating A803-V subs titution, however, may lead to a distorted loop structure, ii lower bu t still significant contribution originates from the non-contiguous li nk/loop c sequence LKCIY (positions 815-819) which is spatially close to the loop a sequence. The link residues (L815 and K816) provide main chain hydrogen bonds to N806 and a side chain hydrogen bond to the V8 04 carbonyl and thus stabilize the conformation of loop a. The side ch ains of I818 and Y819 together with P842 from loop d form hydrophobic contacts that provide further stability but could possibly also partic ipate in direct ligation. The nidogen binding epitope is therefore loc alized on a narrow ridge and has a length of similar to Angstrom. The data also indicate a strong conservation of the epitope in the laminin gamma 1 chains of several invertebrates.