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.

Glycans modulate lipid binding in Lili-Mip lipocalin protein

“…S1. After this initial structural relaxation, further change in structure occur more gradually indicating equilibrium sampling in all the four systems including the G NL and NG NL where we removed the lipid substrate, indicating that the structural integrity is maintained even in the absence of ligand that conforms to previous findings 7 . Comparing all the 4 systems we observed that the glycosylated forms (G L and G NL) have relatively larger values of RMSD than the non- glycosylated forms.…”

“…At this point we must be cognizant of the fact that such transitions in lipid orientations can take place only if the barrel structure is sufficiently flexible. However former studies have shown that the glycans do not affect the overall pocket volume of Lili-Mip 7 . Hence a subtle re-arrangement of interior residue orientations might facilitate the lipid’s access to the buried residues.…”

“…The lipid binding pocket 5 is a characteristic β -barrel structure which bears similarities to extracellular lipid binding proteins (eLPBs). The fatty acids with carbon chain length between 14 and 18 have the propensity to occupy the barrel, with C18 fatty acid being one among the most preferred 6 though they have limited effect on the thermal stability of protein 7 . The protein also has four potential glycosylation sites occupied by varied degree and composition of glycans but it is found to be readily crystallizable 6 .…”