Variability of Phenylalanine side chain conformations facilitates promiscuity of Fatty acid binding in Cockroach milk proteins.

Abstract

The pacific beetle cockroach, Diploptera punctata, is a viviparous cockroach that produces a milk-like substance to support the growing embryo with a brood sac. The structure of the in vivo grown crystals present in the gut of the embryo showed that the milk-derived crystals are heterogenous and are made of three proteins (called Lili-Mips). Multiple fatty acids could be modeled into the active site, and we hypothesized that each of the three isoforms of the protein bound to a different fatty acid. We previously reported that the recombinantly expressed Lili-Mip2 has a structure similar to the structure of the protein determined from in vivo crystals, and this single isoform also binds to several fatty acids. In this study, we aimed to probe the specificity and affinity of fatty acid binding and test the stability of different isoforms. We show that all the isoforms can bind to different fatty acids with very similar affinities, and the local abundance of a fatty acid determined bound fatty acid ratios. Lili-Mips thermostability is pH dependent, where stability is highest at acidic pH and declines as the pH increases to physiological levels near 7.0. The measurement of the pH in the gut lumen and the gut cells suggests that the pH in the gut is acidic and the pH inside the gut cells is closer to neutral pH. We propose that the protein has evolved to be highly stable in the acidic gut lumen and, when absorbed inside the gut cells, becomes less stable to enable the breakdown of the glycosylated lipo-protein complex to provide essential metabolites for survival and development of the embryo. The different orientations of Phe-98 and Phe-100 control the binding pocket volume and allow the binding of different chain-length fatty acids to bind with similar affinities.