Structural and Biophysical Characterization of the Cytoplasmic Domains of Human BAP29 and BAP31

Abstract

Two members of the B-cell associated 31 (BAP31) family are found in humans; BAP29 and BAP31. These are ubiquitously expressed receptors residing in the endoplasmic reticulum. BAP31 functions in sorting of membrane proteins and in caspase-8 mediated apoptosis, while BAP29 appears to mainly corroborate with BAP31 in sorting. The N-terminal half of these proteins is membrane-bound while the C-terminal half is cytoplasmic. The latter include the so called variant of death effector domain (vDED), which shares weak sequence homology with DED domains. Here we present two structures of BAP31 vDED determined from a single and a twinned crystal, grown at pH 8.0 and pH 4.2, respectively. These structures show that BAP31 vDED forms a dimeric parallel coiled coil with no structural similarity to DED domains. Solution studies support this conclusion and strongly suggest that an additional α-helical domain is present in the C-terminal cytoplasmic region, probably forming a second coiled coil. The thermal stability of BAP31 vDED is quite modest at neutral pH, suggesting that it may assemble in a dynamic fashion in vivo. Surprisingly, BAP29 vDED is partially unfolded at pH 7, while a coiled coil is formed at pH 4.2 in vitro. It is however likely that folding of the domain is triggered by other factors than low pH in vivo. We found no evidence for direct interaction of the cytoplasmic domains of BAP29 and BAP31.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

“…In our studies we attempted to confirm that the two factors bound independently to E5 using human cell lines. All cells we examined were found to express Bap31 and this is consistent with previous reports (Quistgaard, Low et al 2013). While many cells express A4, 293 embryonic kidney cells were found to be A4 deficient.…”

Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31

“…Because sequence identity between the target and template proteins amounts to higher than 80%, good structural models for both antibodies could be obtained in the homology modeling. The structure of epitope was extracted from the X-ray crystal structure of BAP31 (PDB ID: 4JZL) [21], which comprises ten amino acids in the sequence of QVLAMRKQSE. Gasteiger-Marsilli atomic charges were assigned for all receptor and ligand atoms to calculate the electrostatic interaction in the antibody-antigen complexes [22].…”

Interaction between human BAP31 and respiratory syncytial virus small hydrophobic (SH) protein

“…The C-terminal cytoplasmic domain (endodomain) of human BAP31 (UniProt: P51572, residues 124-242) corresponding to two consecutive coiled-coil forming domains, was cloned, expressed and purified as described previously (Quistgaard et al, 2013).…”