9.A.66 The Mistic (Mistic) Family
Mistic is an unusual 110 residue
hydrophilic integral membrane protein that folds autonomously into the
membrane, bypassing the cellular translocon machinery. Mistic can be
used for high-level production of other membrane proteins in their
native conformations, including many eukaryotic proteins that have
previously been intractable to bacterial expression. The 3-D structure
of Mistic has been solved revealing a 4-helix bundle with an unexpected
polar lipid-facing surface (Roosild et al. 2005).
The functional core of Mistic consists of 84 moderately conserved
residues that are sufficient for membrane targeting and integration (Roosild et al. 2006). Cytoplasmic homologues that polymerize and form fibrils are present in Bacillus leicheniformis (Dvir et al. 2009).
Isolated Mistic associate tightly with the bacterial membrane when expressed recombinantly in E. coli, and the monomeric detergent-solubilized protein binds tightly to micelles. Mistic targets proteins to the membrane, and when fused at its C-terminus to other proteins, it targets them to the membrane, facilitating insertion. The gene encoding it, mstX, overlaps by 4 nucleotides the yugO gene.
Mistic may facilitate insertion of YugO (TC# 1.A.1.13.4). MstX and YugO are regulators of biofilm formation in B. subtilis (Lundberg et al. 2013). Expression of mstX and the downstream putative K+ efflux channel gene, yugO, is necessary for biofilm development in B. subtilis, and overexpression of mstX induces biofilm assembly. Transcription of the mstX-yugO operon is under negative regulation by SinR, a transcription factor that governs the switch between planktonic and sessile states. Furthermore, MstX regulates the activity of Spo0A through a positive autoregulatory loop involving KinC, a histidine kinase that is activated by potassium leakage. The addition of potassium abrogated MstX-mediated biofilm formation (Lundberg et al. 2013).
The interaction of Mistic with the bacterial membrane promotes the overexpression of other membrane proteins. The third helical fragment of Mistic interacts only with micelles but does not partition into lipid bilayers, but the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full-length Mistic as N-terminal fusions to achieve overexpression of a human G-protein-coupled receptor in E. coli (Marino et al. 2015). A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. A highly conserved Shine-Dalgarno sequence in the operon mstX-yugO is important for downstream translation of the potassium ion channel YugO (Marino et al. 2015).