Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants

Abstract

Leucyl-tRNA synthetase 1 (LARS1) synthesizes Leu-tRNA^Leu for protein synthesis and plays an important role in mTORC1 activation by sensing intracellular leucine concentrations. Here, we describe a protocol for the purification, reductive methylation, binding affinity measurement by microscale thermophoresis, T _i value measurement by Tycho, and post-crystallization soaking and cooling in cryoprotectants to improve crystallization of LARS1. Collectively, this allowed us to build the RagD binding domain, which was shown to be a dynamic region of LARS1 refractory to crystallization. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).

Keywords: Protein Biochemistry; Structural Biology; X-ray Crystallography.

Graphical abstract

Figure 1

Overall Domain arrangement of LARS1 CD, catalytic domain; LSD, leucine-specific domain; CP, connective polypeptide; SC, stem-contact; CTD, C-terminal domain; RBD, RagD-binding domain; UNE-L, unnamed domain of LARS. Figure reprinted with permission from Kim et al. (2021).

Figure 2

Elution profile after each step purification of LARS1 (A) Elution profile at 280 nm after Histrap chromatography of LARS1. (B) Elution profile at 280 nm after HiTrap Q FF chromatography of LARS1

Figure 3

Elution profile at 280 nm after S200 gel filtration chromatography of LARS1 and methylated LARS1 The mass of methylated LARS1 (LARS1^methyl) is increased compared to the mass of LARS1 because reductive methylation elevates the mass of the protein by 28 Da for each lysine residue present or could impact the conformation of LARS1 leading to a more extended conformer that elutes earlier then unmethylated LARS1.

Figure 4

Binding affinity of leucine for LARS1 Binding affinity of leucine for LARS1 was determined using MST. The extract K_d values are listed in Figure 2 (n = 3; means ± SD). LARS1 WT binds to leucine, whereas LARS1 Y52A/Y54A/H91A does not bind to leucine. Figure reprinted with permission from Kim et al. (2021).

Figure 5

Inflection temperature (T_i) of LARS1 with leucine The first derivatives of the fluorescence ratio (350–330 nm) are plotted. LARS1 was incubated with leucine and the inflection temperature (T_i) was measured. The extracted T_i values are listed in Figure 3. Figure reprinted with permission from Kim et al. (2021).

Figure 6

Post-Crystallization Soaking & Cooling in cryoprotectant of LARS1-Leu^syn crystals (A) Pick up the LARS1-Leu^syn crystals with a cryoloop. (B) Transfer the LARS1-Leu^syn crystals to 9 Well Glass Plate filled with 20 μL of cryoprotectant solution and seal with Crystal Clear sealing tape. Stabilize at −20°C for 1 day. (C) Transfer the LARS1-Leu^syn crystals to a Uni-Puck under liquid nitrogen. (D) Transfer the Uni-Puck to a dry shipper filled with liquid nitrogen until X-ray diffraction data collection.

Figure 7

The ribbon model with electron density around RBD of LARS1 Comparison of electron density (2fo-fc map; 1.5 σ cutoff) of LARS1-Leu-AMS^syn (PDB entry: 6KIE) (A) and LARS1^methyl-Leu-AMS^syn (PDB entry: 6KR7) (B) The RBD region (946-1015) is represented as an orange cartoon model. The resolution has greatly improved to ∼3.3 Å from ∼7.0 Å and the moscaicity (0.5 to 1.0) is much smaller than that of the original crystals (>1.5) (Figure 8).

Figure 8

Photo image of X-ray diffraction Comparison of diffraction data for a crystal in cryoprotectant immediately (A) and that of a crystal stablized in cryoprotectant at −20°C for 1 day (B)

Figure 9

Elution data for LARS1 in ologomeric state with different buffer conditions (A) Elution data of LARS1 after reductive methylation using a buffer without Tween-20 (B) Elution data of LARS1 after reductive methylation using a buffer containing imidazole

Figure 10

Proper analysis of MST raw data profiles with capillaries scan (A) Protein is absorbed to the inner surface of capillaries. Double fluorescence peaks appear. (B) Protein is not absorbed onto the inner surface of capillaries after treatment with 0.05% Tween-20 and 0.05% BSA in buffer A. Symmetrical fluorescence peaks are clearly shown for suitable analysis.

Figure 11

Proper analysis of MST raw data profiles with fluorescence quenching upon ligand binding (A) The fluorescence intensity decreases with increasing concentrations of the ligand. (B) The fluorescence is identical in all capillaries.