Solution structure of human GammaC crystallin

Abstract

Purpose : GammaC crystallin (HGC) is one of the three major gamma crystallins in the human lens, yet its 3D-structure remains unsolved, primarily because of its apparent low solubility. Here we show that HGC is highly soluble and present its 3D structure.

Methods : Recombinant HGC was purified using size-exclusion and cation-exchange chromatographies at pH 4.5. Protein was not exposed to detergent during cell-lysis. For NMR experiments, the cells were grown either in the (i) M9 minimal medium containing 1 g/L [U-¹⁵N]NH₄Cl and 2g/L D-glucose (for [U-,¹⁵N] labeled protein), or in the (ii) M9 minimal medium containing 1 g/L [U-¹⁵N]NH₄Cl and 2g/L [U-¹³C₆] D-glucose [for U-,¹⁵N,¹³C labeled protein].Structure was determined using multidimensional triple resonance NMR spectroscopy at 25^oC, using distance restraints from unambiguously assigned ¹H-¹H NOE peaks and dihedral angle restraints.¹⁵N protein backbone dynamics was obtained by T₁ and T₂ relaxation rates and steady-state heteronuclear {¹H}-¹⁵N NOEs. H-D exchange to identify backbone amides was carried out in 100% D₂O and a series of 2D ¹H-¹⁵N HSQC spectra were measured over time. 3D structures were calculated using the torsion angle dynamics protocol in CYANA. Dipole moments were calculated using CHIMERA software.

Results : HGC was stable even above 200mg/ml at pH 7, enabling its 3D-structure determination. The protein has a 2-domain, predominantly beta-sheet structure typical of the gamma crystallins. Superposition of this structure on the x-ray structure of mouse gammaC crystallin shows an RMSD of 1.3Å. Overall rotational correlation time from NMR calculated from average T₁ and T₂ values is ~10.9 ns, indicating a monomeric protein. Significantly, slow H-D exchange for residues Q54,L56 and R58 on one side of the domain interface and Q142, L144, and R146 on the other, attests to a tight interdomain interface covering an area of ~1942 A². Thus, there is no loosening of this domain interface as proposed earlier for HGC. Finally, the magnitude and directions of static dipole moments of Gamma crystallins do not correlate with their solubilities.

Conclusions : NMR structure assignments now enable residue-specific analysis of structural perturbations arising from HGC mutations or modifications. The structure highlights a tight solvent-inaccessible interdomain interface. Cys residues or protein dipole moment do not have a special role in determining stability or solubility, and HGC is as souble as other crystallins.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.