Domain V of m-calpain shows the potential to form an oblique-orientated alpha-helix, which may modulate the enzyme's activity via interactions with anionic lipid.
The activity of m-calpain, a heterodimeric, Ca2+-dependent cysteine protease appears to be modulated by membrane interactions involving oblique-orientated alpha-helix formation by a segment, GTAMRILGGVI, in the protein's smaller subunit. Here, graphical and hydrophobic moment-based analyses predicted that this segment may form an alpha-helix with strong structural resemblance to the influenza virus peptide, HA2, a known oblique-orientated alpha-helix former. Fourier transform infrared spectroscopy showed that a peptide homologue of the GTAMRILGGVI segment, VP1, adopted low levels of alpha-helical structure ( approximately 20%) in the presence of zwitterionic lipid and induced a minor decrease (3 degrees C) in the gel to liquid-crystalline phase transition temperature, TC, of the hydrocarbon chains of zwitterionic membranes, suggesting interaction with the lipid headgroup region. In contrast, VP1 adopted high levels of alpha-helical structure (65%) in the presence of anionic lipid, induced a large increase (10 degrees C) in the TC of anionic membranes, and showed high levels of anionic lipid monolayer penetration (DeltaSP = 5.5 mN.m-1), suggesting deep levels of membrane penetration. VP1 showed strong haemolytic ability (LD50 = 1.45 mm), but in the presence of ionic agents, this ability, and that of VP1 to penetrate anionic lipid monolayers, was greatly reduced. In combination, our results suggest that m-calpain domain V may penetrate membranes via the adoption of an oblique-orientated alpha-helix and electrostatic interactions. We speculate that these interactions may involve snorkelling by an arginine residue located in the polar face of this alpha-helix.
Brandenburg
Klaus
K
Division of Biophysics, Forschunginstitute Borstel, Germany.
Harris
Frederick
F
Dennison
Sarah
S
Seydel
Ulrich
U
Phoenix
David
D
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D006388:Hemagglutinins / Q000737:chemistry; D007477:Ions; D037102:Lectins; D008055:Lipids / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D006388:Hemagglutinins; D007477:Ions; D037102:Lectins; D008055:Lipids; D010446:Peptide Fragments; D010455:Peptides; D010940:Plant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; C442835:influenza virus-hemagglutinin peptide 2 (1-48); D002154:Calpain; C449000:m-calpain; D002118:Calcium