Crystal Structure of Chicken γS-Crystallin Reveals Lattice Contacts with Implications for Function in the Lens and the Evolution of the βγ-Crystallins

Abstract

Previous attempts to crystallize mammalian γS-crystallin were unsuccessful. Native L16 chicken γS crystallized avidly while the Q16 mutant did not. The X-ray structure for chicken γS at 2.3 Å resolution shows the canonical structure of the superfamily plus a well-ordered N arm aligned with a β sheet of a neighboring N domain. L16 is also in a lattice contact, partially shielded from solvent. Unexpectedly, the major lattice contact matches a conserved interface (QR) in the multimeric β-crystallins. QR shows little conservation of residue contacts, except for one between symmetry-related tyrosines, but molecular dipoles for the proteins with QR show striking similarities while other γ-crystallins differ. In γS, QR has few hydrophobic contacts and features a thin layer of tightly bound water. The free energy of QR is slightly repulsive and analytical ultracentrifugation confirms no dimerization in solution. The lattice contacts suggest how γ-crystallins allow close packing without aggregation in the crowded environment of the lens.

Keywords: crystallin; dimerization; lens; molecular dipoles; non-bonding interfaces.

Figure 1. Structure of Chicken γS-crystallin

A: Main chain trace and secondary structure elements. Repeated motif and domain structures are labelled. B: Alignment of residues around position 16 in the chicken crystal structure and the mouse NMR structure for γS. The Q>L substitution has little effect on local structure.

Figure 2. Lattice contact involving L16

A: Overview of monomer to monomer contact in the crystal lattice centered on L16. B: Detailed view of contact. L16 makes no direct hydrophobic contact but is substantially shielded from water by the neighboring monomer. Several intermolecular hydrogen bonds are shown between monomers in blue and green.

Figure 3. Lattice contact involving the N-arm

A: The N-arm aligns with the edge of a β-sheet of the N-domain of another monomer. The well-defined electron density for the N-arm is shown. B: A closer view of the lattice contact shows hydrogen bonding between monomers. There is one main chain backbone hydrogen bond interaction, between the peptide carbonyl of R2 and the peptide nitrogen of the adjacent Q21′ while a second interaction between G4 and R19′ is bridged by water. The side chain of R2 forms an ion-pair with D23′of the other monomer and is within hydrogen bonding distance of Y20′.

Figure 4. A large lattice contact between γS monomers contains tightly coordinated water

A, B: Two views of the monomer- monomer (red and green) contact are shown in secondary structure trace, with residues in the contact region shown as stick models. The contact involves two groups of residues from interdomain motifs 2 and 4 of the symmetry related neighbors. Contact residues are indicated with arrows in view B. C: Surface polar side chains in the lattice contact form networks including a thin layer of tightly bound water (red spheres). There are direct side chain interactions, such as His71-Arg145 and water bridges such as Arg63-Ser116. D: Another region showing tightly bound waters and water bridges. Hydrogen bonds are shown as dotted lines.

Figure 5. Conserved dimer-like interaction among β-crystallins and γS-crystallin

A–E show ribbon traces to illustrate different arrangements of monomeric γ-crystallins and multimeric β-crystallins in crystal structures. A: chicken γS monomer. B: human βB1 dimer with QR interface. C: human βB3 trimer. D: human βB2 domain-swapped dimer. E: The non-bonded crystal lattice tetramer of human βB2. F and G: Two views of a structural alignment of crystal dimeric or monomer-monomer interactions about the QR interface for γS (red), βB1 (green), βB2 (yellow) and βA4 (blue).

Figure 6. Residues of the QR interface

Top: Sequence alignment of β- and γ-crystallins exhibiting the QR interface. Human βB1 (GenBank: AAC50383.1); Human βB2 (GenBank: AB25691.1); Human βA4 (GenBank: AAC50970.1) Chicken γS (GenBank: ACX94083.1). Initiator methionines are not included in alignment or numbering. Protein sequences are aligned to emphasize the common four motif structure with the important G and S residues in blue. General positions of β-strands are indicated with a, b, c, d indicating the four strands of each motif. Residues involved in the QR interface, as defined by Contact are shown colored. Sequence positions are given for βB1 (above) and γS (below). Details of contacts are listed in Table S2. Below: Only one residue is completely conserved in the different QR interfaces. A: An example of a non-polar contact in the βB1 dimer (green) that is not present in the chicken γS QR interface (red). B: Arg145 of γS (red) and Arg201 of βB1 (green) are conserved, but adopt different conformations in the two proteins. C: Tyr144 (of γS) is completely conserved in all QR interfaces. A multiple alignment of γS and β-crystallin structures with the QR interface shows the close superposition of the conserved tyrosine equivalent to Y144 in γS (Y). Colors are as in fig 5.

Figure 7. Proteins which adopt QR have similar aligned and additive molecular dipoles

QR interactions for crystal and modelled structures for β- and γ-crystallins are shown in the same orientation. Calculated molecular dipole moments for monomers are shown as white vectors; dipoles for the dimer are shown in red. Magnitudes (in Debye units) are indicated by length and by the associated values.

Figure 8. AUC measurements show no evidence for multimer formation by chicken γS

A: Representative sedimentation equilibrium distributions of 7 μM γS at 12,000 (purple), 16,000 (blue) and 25,000 rpm (green). For clarity, only every third data point is shown (symbols). The solid lines are the global fit (including data at other concentrations, not shown) with a single-species model with best-fit molar mass of 20.2 kDa. B: Sedimentation velocity profiles of 2.9 mg/ml chicken γS at 50,000 rpm (symbols; for clarity, only every 10th data point is shown, and later scans are indicated by higher color temperature), fitted with a c(s) distribution (solid lines). Residuals are shown in the lower panel. C: Best-fit c(s) distributions obtained from the analysis of sedimentation velocity profiles of chicken γS at different concentrations. D: Isotherm of experimental weighted-average s-values as a function of concentration (symbols), fitted by using monomer dimer self-association model (solid line) including repulsive hydrodynamic non-ideality, leading to a best-fit KD value of 4.2 mM.