Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis

Abstract

Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes.

Keywords: Cytochrome P450; P450 systems; normal mode analysis; protein dynamics; protein superfamily.

Figure 1

Conserved structural elements of CYP proteins shown in the structure of the bacterial protein CYP eryF (1Z8O). (a) Alpha helices A through L and the meander motif are labeled; (b) Beta sheets β1 through β4 and the meander region are labeled.

Figure 2

The multiple sequence alignment (MSA) of five representative CYP proteins, one from each class of CYP proteins. The MSA was obtained from the Clustal Omega program. The highly conserved heme-ligating cysteine residue and EXXR motif are highlighted in green and the P450 signature motif FXXGX(H/R)XCXG is boxed.

Figure 3

Five CYP proteins, each from a different class of CYP electron transport system. Proteins are designated as their PDB codes.

Figure 4

DCCM plots obtained from the combined 200 modes showing correlations between the motions of C_α atoms in each representative protein from different P450 systems. Both axes of a matrix are the amino acid residue index. Each cell in a matrix shows the correlation between the motions of two amino acid residues (C_α atoms) in the protein on a range from −1 (anticorrelated, blue) to 1 (correlated, red), with 0 conferring no correlation. The residue number 1 in the DCCM of 1Z8O, 3MVR, 1BVY, 3MZS, and 2RCH corresponds to residue number 3, 28, 20, 6, and 52, respectively.

Figure 5

DCCM plots showing correlated motions between the C_α atoms of residues within 5 Å of the heme cofactor of the five CYP proteins. Each protein belongs to a different class of CYP proteins. The location of the invariable cysteine residue in each matrix is shown as a black square along the diagonal.

Figure 6

Atomic displacement fluctuations of five CYP proteins, each belongs to a different class of CYP proteins. The x-axis is the residue index according to the aligned protein sequences. The y-axis represents the normalized fluctuation score. Locations of helices A through L and the meander region of each protein are indicated by horizontal red lines in each plot. The critical cysteine residue and EXXR sequence motif are also located along the residue index for each protein.