title: A histidine/tryptophan pi-stacking interaction stabilizes the heme-independent folding core of microsomal apocytochrome b5 relative to that of mitochondrial apocytochrome b5.
abstract: The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is considerably more stable than its microsomal counterpart (Mc b5), whereas the corresponding apoproteins (OM and Mc apo-b5) exhibit similar stability. OM and Mc apo-b5 are also similar in that their empty heme-binding pockets (core 1) are highly disordered but that the remainder of each apoprotein (core 2) displays substantial hololike structure. Core 1 residue 71 is leucine in all known mammalian OM b5's and serine in the corresponding Mc proteins. Replacing Leu-71 in rat OM (rOM) b5 with Ser has been shown to (1) decrease apoprotein thermodynamic stability by >2 kcal/mol and (2) extend conformational disorder beyond core 1 and into core 2, as evidenced in part by loss of a near-UV circular dichroism signal associated with the side chain of invariant residue Trp-22. Herein we report identification of a conserved Mc b5 core 2 packing motif that plays a key role in stabilizing apoprotein conformation in the vicinity of Trp-22, thereby compensating for the presence of Ser at position 71: a pi-stacking interaction between the side chains of Trp-22 and His-15 that is extended by hydrogen bonding between the side chains of His-15, Ser-20, and Glu-11. The corresponding conserved packing motif in OM b5's differs in having arginine at position 15 and glutamate at position 20. We also present evidence indicating that the conserved Mc b5 packing motif noted above contributes to the unusually extensive secondary structure exhibited by bovine Mc apo-b5 in the urea-denatured state.
authors.affiliation: Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
authors.forename: Lijun
authors.initials: L
authors.lastname: Wang
authors.forename: Na
authors.initials: N
authors.lastname: Sun
authors.forename: Simon
authors.initials: S
authors.lastname: Terzyan
authors.forename: Xuejun
authors.initials: X
authors.lastname: Zhang
authors.forename: David R
authors.initials: DR
authors.lastname: Benson
mesh_terms: D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D015786:Cytochromes b5 / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006418:Heme / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology*; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D017931:DNA Primers; D006418:Heme; D015786:Cytochromes b5