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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.
Highlights
BAP29 and BAP31 are two homologous membrane proteins (,50% sequence identity in humans) that reside in the endoplasmic reticulum (ER) [1]
In the case of BAP29, it is clear from visual inspection of the spectra that the variant of death effector domain (vDED) domain contains little a-helix at pH 7.0 while a strong increase in a-helical structure is induced at pH 4.2 (Fig. 3a)
The C-terminal cytoplasmic region is rather poorly structured at pH 7.0, as indicated by the limited proteolysis results, whereas structure is acquired at pH 4.2, as observed for the vDED domain alone
Summary
BAP29 and BAP31 are two homologous membrane proteins (,50% sequence identity in humans) that reside in the endoplasmic reticulum (ER) [1]. BAP31 functions in quality control and sorting of a number of client membrane proteins including immunoglobulin D (IgD) [1], major histocompatibility (MHC) class I molecules [5,6], cystic fibrosis transmembrane regulator (CFTR) [7], beta-2 integrin [8], cellubrevin [9], several members of the MARCH family of membrane associated ubiquitin ligases [10] and the tetraspanins CD9 and CD81 [11] These proteins are likely recognized and bound as they enter the ER, as BAP31 interacts with components of the Sec translocon including Sec61b and TRAM, and has been shown to bind directly to translocon-locked CFTR [12]. To shed light on the structure and function of the vDED domains and full-length C-terminal cytoplasmic regions of BAP29 and BAP31, we have investigated their architecture at the secondary, tertiary and quaternary structure levels using a range of biophysical methods
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