Amer Alam1, Jae-Sung Woo1, Jennifer Schmitz1, Bernadette Prinz1, Katharina Root2, Fan Chen2, Joël S. Bloch1, Renato Zenobi1 and Kaspar P. Locher1
- Department of Biology, Institute of Molecular Biology and Biophysics, ETH Zurich, 8093 Zurich, Switzerland.
- Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland
Cellular uptake of vitamin B12 (cobalamin) requires capture of transcobalamin (TC) from the plasma by CD320, a ubiquitous cell surface receptor of the LDLR family. Here we present the crystal structure of human holo-TC in complex with the extracellular domain of CD320, visualizing the structural basis of the TC-CD320 interaction. The observed interaction chemistry can rationalize the high affinity of CD320 for TC and lack of haptocorrin binding. The in vitro affinity and complex stability of TC-CD320 were quantitated using a solid-phase binding assay and thermostability analysis. Stable complexes with TC were also observed for the disease-causing CD320ΔE88 mutant and for the isolated LDLR-A2 domain. We also determined the structure of the TC-CD320ΔE88 complex, which revealed only minor changes compared with the wild-type complex. Finally, we demonstrate significantly reduced in vitro affinity of TC for CD320 at low pH, recapitulating the proposed ligand release during the endocytic pathway.
CovalX Technology Used (Click each option to learn more)
The human gene for TC that contains the R209Q mutation was obtained and the protein was expressed and purified. The protein complexes (10 mg ml-1) were mixed with a 10% glutaraldehyde solution in a 10:1 ratio and then the cross-linking reaction was allowed to occur at room temperature for one hour in buffer solutions. The mixtures were diluted using the original protein buffer solution or water.
0.5 μL of matrix (sinapic acid (20 mg ml-1) in water/acetonitrile/TFA (49.95/49.95/0.1)) was spotted on a MALDI target and allowed to dry under ambient conditions. 0.5 μL of sample was spotted on top of the matrix spots and dried again under ambient conditions before 0.5 μL of matrix was spotted again. The samples underwent analysis on a mass spectrometer that had been modified with a CovalX HM2 detection system.