BIOCHEMICAL AND CRYSTALLOGRAPHIC ANALYSES OF A PORTAL MUTANT OF THE ADIPOCYTE LIPID-BINDING PROTEIN

A number of crystallographic studies of the adipocyte lipid-binding pr otein have established that the fatty acid binding site is within an i nternalized water-filled cavity. The same studies have also suggested the existence of a region physically distinct from the fatty acid-bind ing site which connects the cavity of the protein with the external so lvent, hereafter referred to as the portal. In an effort to examine th e portal region, we have used site-directed mutagenesis to introduce t he mutations V32D/F57H into the murine ALBP cDNA. Mutant protein has b een isolated, crystallized, and its stability and binding properties s tudied by biochemical methods. As assessed by guanidine-HCl denaturati on, the mutant form exhibited a slight overall destabilization relativ e to the wild-type protein under both acid and alkaline conditions. Ac cessibility to the cavity in both the mutant and wild-type proteins wa s observed by stopped-flow analysis of the modification of a cavity re sidue, Cys(117), by the sulfhydryl reactive agent 5,5'-dithiobis(2-nit robenzoic acid) at pH 8.5, Cys(117) Of V32D/F57H ALBP was modified 7-f old faster than the wild type protein. The ligand binding properties o f both the V32D/F57H mutant and wild-type proteins were analyzed using a fluorescent probe at pH 6.0 and 8.0. The apparent dissociation cons tants for 1-anilinonaphthalene-8-sulfonic acid were approximately 9-10 -fold greater than the wild-type protein, independent of pH. In additi on, there is a 6-fold increase in the K-d for oleic acid for the porta l mutant relative to the wild type at pH 8.0. To study the effect of p H on the double mutant, it was crystallized and analyzed in two distin ct space groups at pH 4.5 and 6.4. While in general the differences in the overall main chain conformations are negligible, changes were ob served in the crystallographic structures near the site of the mutatio ns. At both pH values, the mutant side chains are positioned somewhat differently than in wild-type protein. To ensure that the mutations ha d not altered ionic conditions near the binding site, the crystallogra phic coordinates were used to monitor the electrostatic potentials fro m the head group site to the positions near the portal region. The dif ferences in the electrostatic potentials were small in all regions, an d did not explain the differences in ligand affinity. We present these results within the context of fatty acid binding and suggest lipid as sociation is more complex than that described within a single equilibr ium event.