Computational protocol: Identification of a Small Molecule that Increases Hemoglobin Oxygen Affinity and Reduces SS Erythrocyte Sickling

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Protocol publication

[…] To obtain the hemoglobin structure in complex with TD-1, a freshly prepared solution of TD-1 in DMSO was incubated with oxyHb for 1 h at 37 °C at a hemoglobin tetramer-compound molar ratio of 1:10. The mixture was saturated with carbon monoxide (CO) to generate carboxyhemoglobin (COHb). Crystallization was carried out with a solution of 20–30 mg mL–1 COHb, in a mixture of 3.0–3.4 M sodium monobasic phosphate and potassium phosphate dibasic, at pH values ranging from 6.4 to 7.6 using vacutainer tubes., One or two drops of toluene were added to the solution in each tube to facilitate crystallization. CO was bubbled into the tubes, and then sealed. X-ray-quality crystals grew in 2–3 days, with almost all the crystals as long rectangular needles, which are typical of quaternary R3-state crystals. In addition to the needle crystals, a few of the crystallization setups contained trigonal bipyrimidal (octahedral) crystals: the classical quaternary R-state crystals. Prior to study by X-ray diffraction, the crystals were washed in a cryoprotectant solution containing 50 μL mother liquor and 10–15 μL glycerol.Diffraction data of all crystals were collected at 100 K with a Rigaku IV ++ image plate detector using a Cu Kα X-rays (λ = 1.54 Å) from a MicroMax-007 source fitted with Varimax Confocal optics (Rigaku). The data sets were processed with the d*trek software (Rigaku) and the CCP4 suite of programs. [...] COHb in complex with TD-1 crystallized in the space group P4122 with approximate unit-cell constants of a = 62 Å, b = 62 Å, and c = 176 Å with one dimer (α1β1) per asymmetric unit, and expectedly isomorphous to the previously published high-salt R3 state COHb crystal.The isomorphous R3 state α1β1 dimeric COHb structure (PDB code 1YZI) was used as a starting coordinate to refine the structure of COHb in complex with TD-1 using software packages CNS and Phenix., Model building and correction were carried out using COOT. Initial difference map of the R3 structure identified four monomeric units (MUs) of the divalent TD-1 () and three toluene molecules (used in the crystallization experiment), which were modeled in the structure and refined (). The final crystallographic R-factor and R-free were 22.8 and 25.9, respectively. The X-ray data is summarized in . Structural figures in Figure and were prepared with PyMol and ChemBioDraw Ultra (PerkinElmer). […]

Pipeline specifications

Software tools CCP4, CNS, PHENIX, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Diseases Anemia, Sickle Cell, Hypoxia, Hemolysis
Chemicals Ethylmaleimide, Oxygen