Cytochrome-P450–Cytochrome-b5 Interaction in a Membrane Environment Changes 15N Chemical Shift Anisotropy Tensors

Abstract

It has been well realized that the dependence of chemical shift anisotropy (CSA) tensors on the amino acid sequence, secondary structure, dynamics, and electrostatic interactions can be utilized in the structural and dynamic studies of proteins by NMR spectroscopy. In addition, CSA tensors could also be utilized to measure the structural interactions between proteins in a protein-protein complex. To this end, we report the experimentally measured backbone amide-(15)N CSA tensors for a membrane-bound 16.7 kDa full-length rabbit cytochrome-b5 (cytb5), in complexation with a 55.8 kDa microsomal rabbit cytochrome P450 2B4 (cytP4502B4). The (15)N-CSAs, determined using the (15)N CSA/(15)N-(1)H dipolar coupling transverse cross-correlated rates, for free cytb5 are compared with those for the cytb5 bound to cytP4502B4. An overall increase in backbone amide-(15)N transverse cross-correlated rates for the cytb5 residues in the cytb5-cytP450 complex is observed as compared to the free cytb5 residues. Due to fast spin-spin relaxation (T2) and subsequent broadening of the signals in the complex, we could measure amide-(15)N CSAs only for 48 residues of cytb5 as compared to 84 residues of free cytb5. We observed a change in (15)N CSA for most residues of cytb5 in the complex, as compared to free cytb5, suggesting a dynamic interaction between the oppositely charged surfaces of anionic cytb5 and cationic cytP450. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions of cytb5 in the complex are -184.5, -146.8, and -146.2 ppm, respectively, with an overall average value of -165.5 ppm (excluding the values from residues in more flexible termini). The measured CSA value for residues in helical conformation is slightly larger as compared to previously reported values. This may be attributed to the paramagnetic effect from Fe(III) of the heme in cytb5, which is similar to our previously reported values for the free cytb5.