Protein expression, purification and characterization
The coding sequences of LaM2 and LaM4 were optimized based on favored codon usage in E. coli and were synthesized by Genewiz (Suzhou, China). For crystallization and binding assays, DNA encoding LaM2 and LaM4 was subcloned into the pET28a-SUMO vector with an N-terminal 6xHis tag followed by a SUMO tag or a pET21a-derived vector with an N-terminal 10xHis tag, respectively. The plasmids were transformed into E. coli strain BL21 (DE3) for expression. The bacteria were cultured in LB medium at 37 °C until the OD600 reached 0.8. Recombinant protein expression was induced by the addition of 0.2 mM isopropyl-D-1-thiogalactopyranoside (IPTG) and incubation for an additional 18 h at 18 °C. The cells were harvested and resuspended in NiA buffer containing 20 mM imidazole, 5% glycerol, 150 mM NaCl, and 100 mM Tris-HCl, pH 7.5. The His10-tagged recombinant LaM2/LaM4 and their respective mutants were initially purified by Ni-NTA affinity purification using a HisTrap HP column (Qiagen) and eluted with NiB buffer containing 300 mM imidazole, 5% glycerol, 150 mM NaCl, and 100 mM Tris pH 7.5. For crystallization, the His-SUMO tag was removed by incubation with recombinant ULP1 overnight at 4 °C. The cleaved tag fragment and ULP1 were removed by passing through a HisTrap HP column. LaM2/LaM4 were further purified by size exclusion chromatography on a Superdex75 Increase column (Cytiva), and the buffer was exchanged to gel filtration buffer: 10 mM HEPES, pH 7.4 and 100 mM NaCl. The purity and molecular weight of the target proteins were verified by SDS–PAGE. Detailed information on the expression and purification of recombinant proteins is provided in the supplementary data.
Site-directed mutagenesis
Site-directed mutagenesis was carried out by employing a PCR-based mutagenesis site-directed method (Vazyme 2x Phanta Master Mix) using His10-LaM2 and His10-LaM4 as the template. The sequences of the primers used to generate these mutants are displayed in supplementary Table S1. All site-directed mutagenesis constructs were confirmed by DNA sequencing (RuiDi, Shanghai, China).
Crystallization and data collection
Concentrated LaM2 or LaM4 was mixed with mCherry at a molar ratio of 2:1 and incubated for 1 h at 4 °C. The mCherry/LaM2 and mCherry/LaM4 complexes were separated from the excess LaM2/LaM4 by size exclusion chromatography using a Superdex75 Increase column (Cytiva). The mCherry/Nb complex was then concentrated to 15 mg/ml in gel filtration buffer containing 10 mM HEPES, pH 7.4 and 100 mM NaCl. Crystals of the mCherry/LaM2 complex were obtained by the sitting-drop vapor diffusion method at 293 K in drops containing a mixture of 1 μl of protein solution and 1 μl of reservoir solution, which consisted of 0.2 M magnesium chloride hexahydrate, 0.1 M Tris-HCl, pH 8.5, 25% w/v polyethylene glycol 350. Crystals of the mCherry/LaM4 complex were obtained in 0.2 M lithium sulfate monohydrate, 0.1 M Bis-Tris, pH 5.5, and 25% w/v polyethylene glycol 3350.
Cryoprotection was performed by adding glycerol to the reservoir buffer at a 20% concentration. X-ray diffraction data were collected at 100 K in beamlines BL17U1 [24] and BL19U1 [25], Shanghai Synchrotron Radiation Facility, Chinese Academy of Sciences.
Determination and refinement of protein structure
Diffraction images were indexed and processed by HKL2000 [26]. The structures of mCherry/LaM2 and mCherry/LaM4 were obtained by molecular replacement using the Phaser program from the CCP4 crystallography package [27] with mCherry (PDB ID: 2H5Q) and a GFP Nb (PDB ID: 3K1K) as the search model. Structure refinement was performed by Refmac [28] and Phenix [29]. The model was refined by COOT [30]. The crystallographic parameters of mCherry/LaM2 (PDB ID: 6IR2; 1.39 Å) and mCherry/LaM4 (PDB ID: 6IRI; 1.92 Å) are listed in Table 1. The related figures were drawn by PyMOL [31].
Isothermal titration calorimetry (ITC)
The thermodynamic parameters of the binding of LaM2/LaM4 and their respective mutants to mCherry were determined by ITC using a VP-ITC calorimeter (MicroCal VP-ITC, Malvern). In a typical experiment, each titration was performed by injecting a 12 μl aliquot of protein sample (0.15 mM to 0.52 mM) into the cell containing another reactant (0.015 mM to 0.029 mM) at a time interval of 120 s to ensure that the titration peak returned to the baseline. Altogether, 23 aliquots were titrated in each individual experiment. The stoichiometry of binding (n), the association constant Ka, and the binding enthalpy H were evaluated using MicroCal Origin 7.0 software with a one-site binding model.
Fluorescence-based Size Exclusion Chromatography (F-SEC)
The oligomeric state of the tested samples in buffers were recorded by F-SEC. We used 100 μl 0.1 mg/ml mCherry as control. For LaM2-mCherry and LaM4-mCherry complex, 50 μl 0.2 mg/ml of mCherry (about 7 μM) and 0.2 mg/ml (about 14 μM) of LaM2 or LaM4 were mixed in equal volume (the final concentration of mCherry was 3.5 μM, and the final concentration of LaM2/LaM4 was 7μM) and incubated on ice for 1 hour. For LaM2-LaM4-mCherry complex, 50 μl 0.2 mg/ml mCherry (about 7 μM) was mixed with 25 μl 0.4 mg/ml LaM2 and 25 μl 0.4 mg/ml LaM4 and incubated on ice for 1 hour. After high-speed centrifugation, the supernatants were loaded onto a Superdex 200 Increase size-exclusion column (Cytiva) equilibrated with SEC buffer (20 mM HEPES pH 7.0, 150 mM NaCl). The fluorescence of each sample was recorded by a fluorometer (excitation, 587 nm; emission, 610 nm for mCherry fluorescence). The data were processed and normalized by FSECplotter software.
Emission spectrum measurements
The emission spectra of mCherry (0.1 mg/mL) and mCherry-LaM2/LaM4 (mCherry 0.1 mg/mL with excess Nb) were recorded using a fluorescence spectrophotometer (Varian Cary Eclipse). The excitation wavelength was 587 nm. The emission spectrum was recorded between 550-700 nm. The spectra data were analyzed with Origin.
Dynamic light scattering assay
The particle size distribution of mCherry, LaM2-mCherry complex, LaM4-mCherry complex and LaM2-LaM4-mCherry complex were measured by Nano-size-Zeta potential analyzer (Malvern Instruments, ZS90-2026), the test temperature is 25 °C, the test angle is 90 degrees.