In our experience, TR LBD is a delicate protein that crystallizes only when carefully prepared fresh. Because of the protein instability, TR requires many iterative protein preparations for obtaining and optimizing crystals suitable for high-resolution structural analysis. In addition, TR is unstable without a ligand, which is absolutely required during protein purification at all stages. When stabilized by a bound ligand, TR LBD is a monomer in solution, although it is shown to form dimers and trimers on DNA in vivo
). To date, there is no crystal structure of any TR self-assembly, however extensive mutational analyses and homology modeling suggested that TR LBD might employ helix 11 to form dimers (21
). Independent studies showed that clustered charged surface residues in helices 7 and 8 might influence stability of the TR LBD and affect its dimerization on DNA and function on TR response elements (22
). In particular, a single surface mutation D355R in helix 8 was shown to enhance TR homodimer formation on DNA in the presence of hormone (22
). This observation was noteworthy because it revealed a curious improvement of protein stability without any obvious explanation. This mutation is remote from the predicted dimerization helix 11, therefore, the molecular mechanism of the assembly enhancement could not be explained by similarities with other NR homodimers (23
Any novel protein assemblies provide useful information of higher order complex architecture (26
). Moreover, homodimeric complexes are generally more stable and, therefore, amenable for protein crystallization (17
). In this work, we performed a detailed structural and functional analysis of D355R TR LBD and its dimeric assembly. Specifically, we determined the three-dimensional structure of this protein to visualize the mechanism of its dimerization. We complemented this structural study with analyses of the protein stability, behavior in solution and ultimately its utility for high throughput crystallographic techniques.
We found that in contrast to the wild type TR monomer, the mutant receptor LBD undergoes a ligand-dependent molecular assembly, forming a homodimer in addition to the monomer in certain preparative conditions. In our experiments, a typical size exclusion chromatography run for the purified D355R TR mutant protein resulted in separation of 60 kDa from 30kDa protein species, which correspond to the dimeric and monomeric states of TR LBD (). Repeated chromatography of the main protein fraction corresponding to the TR homodimer (~80% of total protein) demonstrated that the formed dimer is stable under the experimental conditions (). Altering the buffer composition such as removal of sulfate ions, resulted in loss of dimers, with only monomeric protein present in the chromatographic fractions (). In contrast, the TR homodimer formed very efficiently if sulfate ions were present in preparative buffers (). Other variations in protein buffers such as ionic strength, pH and the presence of detergents were also found to influence formation of the TR homodimer and the dimer/monomer partition in solution (data not presented). Based on these experiments, we found the protein preparation conditions that allow purification of the TR homodimer as a major protein fraction with high yield (specified in Experimental procedures). The ultimate step of protein purification employing FPLC was critical as it resulted in efficient separation of the dimeric protein from the TR LBD monomer, thus ensuring protein purity and homogeneity. After this final purification step, the TR homodimer was more than 95% pure as judged by gel electrophoresis. Furthermore, the D355R TR dimer was highly soluble and maintained its monodisperse dimeric state both at high (40mg/ml) and low (5mg/ml) concentrations as judged by native gels and size exclusion chromatography analyses. Notably, the TR LBD retained its dimeric state not only throughout protein preparation, but also after several freeze-thaw cycles, as judged by size exclusion chromatography. Based on these observations, we conclude that the dimeric form of the mutant increased stability of the TR LBD, which could tolerate 2-3 cycles of freezing – thawing and produce high quality crystals that diffracted to 2.0 - 2.5 Å resolution. The increased stability of TR LBD is a crucial improvement as wild type protein could not produce crystals upon storage or freezing. Furthermore, these improved protein and crystal properties are compliant with the demands of a high throughput crystallography (28
Size exclusion chromatography of D355R TR LBD
The structure of the TR homodimer was determined by the molecular replacement method and refined to 2.2 Å resolution with Rfree/R values of 24.5/21.7. The current model includes residues 210-460 and 211-460 for the first and the second TRIAC bound TR LBDs (). The unstructured loop residues 255-260 (chain A) and residues 256 and 257 (chain B) are omitted from the model due to the lack of electron density. There are 123 water molecules and one coordinated sulfate ion in the TR homodimer structure. Data collection and refinement statistics are summarized in .
Structure of the TRβ D355R mutant dimer
The crystal structure of TR homodimer showed that D355R mutation affected neither the tertiary structure of the nuclear receptor LBD nor its AF-2 surface as judged by comparison of the two LBDs from the complex with available structure of monomeric TR (PDB ID 1NAX). The structure of TR mutant revealed one homodimer in the crystal asymmetric unit (). The two monomers in the complex are related by two-fold non-crystallographic symmetry and are structurally very similar to each other, with r.m.s. deviation of only 0.23 Å for 169 Cα atoms. The dimer interface is extensive (~ 1330 å2) and is formed between mostly polar or charged residues (). Hydrogen bonds link the side chains of the interfacial residues through six water molecules that are shared at the interface. Although mostly composed of polar and charged residues, the dimer interface is additionally stabilized by hydrophobic interactions (). The residues forming contacts between monomers are mostly contributed from helix 8 and loops H6-H7 and H10-H11. There are two sites at the interface that are evidently crucial for the dimer formation. The first site includes a four-histidine cluster composed of H412 and H413 from the two TR LBDs. A single sulfate ion links the four His side chains within the cluster; His412 requires a water molecule to bridge to the sulfate ion (). The presence of the coordinated sulfate ion at the dimer interface is consistent with our biochemical data showing the necessity of sulfate for dimerization of the mutant TR LBD (). The second site is composed of residue R338 and mutated residue R355 of each monomer that form hydrogen bonds to the oppositely charged E324 and E326 of the partner LBD (). In wild type TR, side chain of D355 forms salt bridge with the side chain of R338, which is also hydrogen bonded with the side chain of D351. Thus R338, D351 and D355 are charged residues of a solvated 3 side chain intramolecular cluster. In the dimer, the polar interaction network is intermolecular.
Contacting residues at the TR homodimer interface
Based on the data described here, we conclude that with the strategically placed single surface mutation D355R we engineered a symmetrical dimer of TR LBD with an interface composed mostly of polar amino acid side chains. The polar side chains are partly solvated, the dimer interface contains 6 water molecules (). The interface has almost no significant hydrophobic interactions, with only 10% of the contacts formed between paired nonpolar atoms.
The mutant TR is characterized by the increased protein stability and crystallizability and thus is a good tool for crystal based screenings employing high throughput soakings of surface binding small molecules. We can envision other nuclear receptors being amenable for dimer engineering, making this approach a valuable technique for obtaining stable protein tools for high resolution crystallography. Predicting single site mutants that would form stable protein dimers is recognized to be challenging. The results presented here suggest that appropriate stabilizing residues for the protein of interest are better found by experiment with a biochemical assay for stability.