Interaction between MMACHC and MMADHC, two human proteins participating in intracellular vitamin B₁₂ metabolism.
The identification of eight genes involved in inherited cobalamin (Cbl) disorders has provided insight into the complexity of the vitamin B₁₂ trafficking pathway. Detailed knowledge about the structure, interaction, and physiological functions for many of the gene products, including the MMACHC and MMADHC proteins, is lacking. Having cloned, expressed, and purified MMACHC in Escherichia coli, we demonstrated its monodispersity by dynamic light scattering and measured its hydrodynamic radius, either alone or in complex with each of four vitamin B₁₂ derivatives. Using solution-phase intrinsic fluorescence and label-free, real-time surface plasmon resonance (SPR), MMACHC bound cyanocobalamin and hydroxycobalamin with similar low micromolar affinities (K(D) 6.4 and 9.8 μM, respectively); adenosylcobalamin and methylcobalamin also shared similar binding affinities for MMACHC (K(D) 1.7 and 1.4 μM, respectively). To predict specific regions of interaction between MMACHC and the proposed partner protein MMADHC, MMACHC was subjected to phage display. Five putative MMACHC-binding sites were identified. Finally, MMADHC was confirmed as a binding partner for MMACHC both in vitro (SPR) and in vivo (bacterial two-hybrid system).
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Maria
M
Plesa
Jaeseung
J
Kim
Stéphane G
SG
Paquette
Hubert
H
Gagnon
Christopher
C
Ng-Thow-Hing
Bernard F
BF
Gibbs
Mark A
MA
Hancock
David S
DS
Rosenblatt
James W
JW
Coulton
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D042541:Intracellular Space / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010088:Oxidoreductases; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014805:Vitamin B 12 / Q000031:analogs & derivatives / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; C527938:MMADHC protein, human; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; C508359:MMACHC protein, human; D010088:Oxidoreductases; D014805:Vitamin B 12