Towards a Molecular Understanding of Phase Separation in the Lens: a Comparison of the X-ray Structures of Two HighTcγ-Crystallins, γE and γF, with Two LowTcγ-Crystallins, γB and γD

Abstract

γ-Crystallins, although closely related in sequence, show intriguing differences in their temperature-dependent interactions: those that have a high or intermediateTcfor phase separation are cryoproteins whereas lowTcγ-crystallins are not. To address the molecular basis of phase separation, X-ray crystallography has been used to define the structural differences between high and lowTcγ-crystallins. A pre-requisite for this study was to clarify the assignment of bovine gene sequences to bovine γ-crystallin proteins used for biophysical measurements. Based on nucleotide sequence analyses of γE and γF bovine crystallin genes, γF corresponds to the previously crystallised highTcprotein bovine γIVa and γE corresponds to the highTcbovine protein fraction previously known as γIIIa. The γF sequence has enabled the completion of the refinement of the bovine γF crystal structure which shows that the molecule has an additional surface tryptophan explaining why γF has different spectroscopic properties from γB. A highTcprotein from rat lens, γE crystallin, has been crystallised and the X-ray structure solved at 2.3 Å resolution. Comparison of the X-ray structures of two highTcproteins, rat γE and bovine γF, with the structures of two lowTcproteins, bovine γB and bovine γD, shows that the main conformational change between high and lowTcproteins is in thecdsurface loop of motif 3. All four structures have numerous ion pairs on their surfaces leading to a high surface charge density, yet with low overall charge. Comparison of the lattice contacts of the two highTcproteins with the two lowTcγ-crystallins indicates that these highTcproteins utilise more amino-aromatic interactions such as between histidine and arginine. Comparison of the sequences of all the γ-crystallins which have been characterised for phase separation temperature indicates that only residue Arg/Lys 163 uniquely distinguishes cryo from non-cryo γ-crystallins and it is close to the altered surface loop. Although this region probably contributes to phase separation,Tcis likely to be a function of an overall global property that is responsive to overall charge distribution. Calculated dipole moments of native γ-crystallins, lowTcγ-crystallin sequences threaded into highTcγ-crystallin structures, andvice versa, show how both sequence and 3D structure contribute to this overall property. HighTcγ-crystallins have on average higher Arg/Lys ratios and higher histidine content. It is hypothesised that this increases the proportion of surface static paired charged networks which thus reduces the repulsive hydration force and so increases the attractive interactions of the protein-rich phase in binary liquid phase separation.