Structural and biochemical insights into human zinc finger protein AEBP2 reveals interactions with RBBP4

Figure S1. Sequence alignment of orthologs of AEBP2 (Residues Asn258 Asp396) protein from diverse vertebrate species. The cysteines and histidines coordinating zinc ions are marked with stars. The residues corresponding to DNA binding zinc fingers involved in DNA interaction are marked with triangles. The RRK rich motif are highlighted with yellow background, with crucial residues contributing to RBBP4 recognition marked with red circles.

. NOE columns showing inter-zinc finger interactions between ZF1 and ZF2. A, Representative 1 H-1 H spectral columns from 3D 15 N-NOESY spectrum for loop regions.
All NMR spectra were collected at 298K on a Bruker DMX600 spectrometer

NMR structure calculation
Structure calculation of all three AEBP2 zinc fingers was performed in CYANA using fully automated NOESY assignment (Guntert, 2003). Distance restraints were generated and calibrated automatically by CYANA using 2D 1H-1H NOESY, 15 N-and 13 C-edited NOESY-HSQC peak lists. The restraints for backbone dihedral angles were determined using TALOS+ (Shen et al., 2009). Additional restraints were introduced for distances between the zinc ion and zinc-coordinating atoms to enforce tetrahedral geometry of the zinc center. The structure was recalculated by CNS software (Brunger et al., 1998) and 20 lowest-energy conformers were selected. The quality of the ensemble of 20 lowest energy structures was assessed using PROCHECK-NMR (Laskowski et al., 1996) and Protein Structure Validation Server (PSVS) (Bhattacharya et al., 2007). NMR calculation and refinement data are displayed in Table 1. The structure figures were prepared with MOLMOL (Koradi et al., 1996) and PyMOL (DeLano, 2002) (DeLano Scientific San Carlos, California USA).

RBBP4 protein preparation.
The

Isothermal Titration Calorimetry (ITC) Experiments.
ITC measurements were carried out at 25°C using a MicroCal iTC200 titration calorimeter (GE Healthcare). RBBP4 and AEBP2 peptides were dissolved separately in ITC buffer (25 mM Tris, pH 7.4, 150 mM NaCl). The AEBP2 peptides were injected into RBBP4 at time intervals of two minutes between each 2 µl injection (20 injections in total). The data were analyzed using one-site binding model via the MicroCal Origin 7.0 software package (provided by the manufacturer).

Crystallization and Structure Determination.
Concentrated RBBP4 protein (10 mg/ml) was incubated overnight at 4°C with AEBP2 379-390 peptide at a molar ratio of 1:2 . Additionally, 1 μl RBBP4-AEBP2 complex drops were mixed with 1 µl of crystallization solution using the sitting drop vapor diffusion method. Single crystals were obtained at 8-12°C for a week in 0.2 M Ammonium sulfate; 0.1 M MES monohydrate pH 6.5; 30% w/v Polyethylene glycol monomethyl ether 5,000 (HAMPTOM RESEARCH). Crystals were harvested, soaked in mother liquor supplemented with 30% glycerol and then flash frozen in liquid nitrogen. X-ray diffraction data were collected on beamline 17U1 at the Shanghai Synchrotron Radiation Facility (SSRF). The initial data were processed using HKL2000 (Battye et al., 2011). The structure was then solved by molecular replacement using RBBP4 (PDB ID: 3GFC) (Xu and Min, 2011) as a search model via MOLREP (Vagin and Teplyakov, 2010). The model was further built and refined using Coot (Emsley et al., 2010), Refmac (Murshudov et al., 2011) and Phenix (Afonine et al., 2012). Crystal diffraction data and refinement statistics are displayed in Table 2.

Coordinates
Coordinates, chemical shifts and peaks of AEBP2 three zinc fingers have been