Abstract
Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic α-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis.
Highlights
Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes
This observation is supported by molecular dynamics (MD) simulations, which spot the formation of a variety of YidC-Pf3 complexes, whose conformations differ in how the cytoplasmic α-helical hairpin or/and the hydrophilic groove of YidC interact with the Pf3 polypeptide
Our simulations observe the cytoplasmic α-helices of YidC to contribute to the initial binding of the Pf3 polypeptide and that the positively-charged residues K401, R384 and R394 guide the polypeptide along multiple pathways to the hydrophilic groove, which transfers the polypeptide to the transmembrane state
Summary
Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic α-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane α-helical proteins. While hydrophobic transmembrane segments of nascent polypeptides can spontaneously insert into cell membranes, the passage of hydrophilic polypeptide residues through the hydrophobic core of the membrane is thermodynamically unfavorable[1] To overcome this free-energy barrier of translocation, the majority of membrane proteins require the assistance of insertases and/or translocases to catalyze their insertion and supervise their folding process[2,3,4,5].
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