P450's Proximal FCG Alignment Method

Localcolabfold (model1_ptm,  recycle=6, centerd and rotated around proximal Cys)runner_P450align.py -f CYPs/CYPXXXA1.fasta -nm 5 -mr 6 -alirunner_P450align.py -f CYPs/CYPXXXA1.fasta -nm 5 -mr 6 -ali -upAlign crystal struvture at FXXXXXXCXGaligin_P450.py -i XXXX.P450 -al1 "residue No. F" -al2 "residue No. G" -al3 "residue No. C"

Objective

   Alphafold2 predict the folding of various proteins with high quality. Most of protein structures of major bio-species such as E. coli, Glycine max, human, were recorded on the Alphafold protein structure database. 
   Alphafold is only supported for single poly-peptide without cofactors such as DNA, Flavin, Metal ion and heme. However, cofactor bound crystal structures were also used for the training of the alphafold parameters (model1-5, model1-5_ptm). The predicted structure was cofactor-bound structure in high possibility, because cofactor makes protein stable under the crystallization process. P450 posses heme molecule on the conserve cysteine residue. The heme is important for the oxidation reactions. P450 researchers require the heme-bound structure to predict the reactivity of substrates in the computational science.
    Docking simulation of metal complex is difficult owing to the distance restriction of coordination bond between metal ion and amino-acid ligand. Therefore I try the alignment of P450 around the heme. Finally the Cα atoms of proximal ligand cysteine (C), phenylalanine (F) 7th behind C, and Glycine (G) 2nd ahead  C were aliened on the xy  plane shown below. 

Method


1) The midpoint of C was shift to origin (0, 0, 0). 2) y values of F was align to 0 by the rotation of z axis3) z values of F was align to 0 by the rotation of y axis4) z values of G was align to 0 by the rotation of x axis5) The shift and three rotations were apply to all atoms. 
The result position of heme was also align around (-2, 2, 3.5). The z values of CHA, CHB, CHC, CHD, NA, NB, NC, and ND were also about 3.5Å. It means the aligned-heme is lying at z=3.5 plane.
Heme molecules of 23 family P450s were aligned by FCG residues. (PDB: 1CPT, 1IO7, 1N40, 1Z8O, 2IJ2, 2JJN, 3VE3, 2WHF, 2X5W, 2XKR, 2ZBX, 2ZWU, 3MGX, 3NC3, 4RM4, 4YT3, 5HDI, 5LI8, 5OFQ, 6DCD, 6LDL, 6T0H, 7OW9)

Exception

Mutation of F No ProblemCYP156A1, CYP158A1, CYP167A1, CYP176A1, & CYP190A1
Mutation of G No ProblemCYP141A1, CYP156A1, CYP160A1, CYP170A1, & CYP181A1
1 aa Interion between F and C No ProblemCYP124A1 & CYP125A1 
Long Interion between F and C (& FQ Mutation) No PorblemCYP152A1
Long Insertion UnfoldingCYP149A1
C-terminal frame shift Cys was not existCYP131A1
1aa Deletion post C region Orientation was differentCYP157A1 & CYP182A1
Alignment Alphafold structure of "Insertion between F and C"Insertion: CYP124A1, CYP125A1, CYP152A1Conserved: CYP101A1, CYP102A1, CYP105A1, CYP106A1
Alignment Alphafold structure of "Deletion of post C region" Deletion: CYP157A1, CYP182A1Conserved: CYP101A1, CYP102A1, CYP105A1, CYP106A1

Accuracy of Heme Positioning


1) CYP101A1 (38 WT crystal structures binding with heme cofactor)
  PDBID Fe-Heme S-Cys Fe-S ResolutionA1 2ZWU -1.99, 2.43, 3.13 -1.88, 2.60, 0.77 2.37 1.3ÅAverage -1.96, 2.30, 3.34Std. dev. 0.11, 0.10, 0.19
2) CYP101 family enzymes 
  PDBID Fe-Heme S-Cys Fe-S ResolutionA1 2ZWU -1.99, 2.43, 3.13 -1.88, 2.60, 0.77 2.37 1.3ÅC1 3OFT -2.23, 1.91, 3.64 -2.28, 2.32, 1.41 2.27 1.9ÅD1 4C9M -2.30, 2.30, 3.43 -2.18, 2.56, 1.12 2.33 1.8ÅD2 3NV6 -2.22, 2.30, 3.33 -2.21, 2.52, 1.11 2.23 2.2ÅJ1 5KYO -2.17, 2.22, 3.57 -2.17, 2.48, 1.19 2.39 1.8ÅAtcu 6WPL -2.10, 2.22, 3.33 -2.08, 2.58, 1.08 2.28 2.1Å