Abstract
In bacteria the biogenesis of inner membrane proteins requires targeting and insertion factors such as the signal recognition particle and the Sec translocon. YidC is an essential membrane protein involved in the insertion of inner membrane proteins together with the Sec translocon, but also as a separate entity. YidC of Escherichia coli is a member of the conserved YidC (in bacteria)/Oxa1 (in mitochondria)/Alb3 (in chloroplasts) protein family and contains six transmembrane segments and a large periplasmic domain (P1). We determined the crystal structure of the periplasmic domain of YidC from E. coli (P1D) at 1.8 A resolution. The structure of P1D shows the conserved beta-supersandwich fold of carbohydrate-binding proteins and an alpha-helical linker region at the C terminus that packs against the beta-supersandwich by a highly conserved interface. P1D exhibits an elongated cleft of similar architecture as found in the structural homologs. However, the electrostatic properties and molecular details of the cleft make it unlikely to interact with carbohydrate substrates. The cleft in P1D is occupied by a polyethylene glycol molecule suggesting an elongated peptide or acyl chain as a natural ligand. The region of P1D previously reported to interact with SecF maps to a surface area in the vicinity of the cleft. The conserved C-terminal region of the P1 domain was reported to be essential for the membrane insertase function of YidC. The analysis of this region suggests a role in membrane interaction and/or in the regulation of YidC interaction with binding partners.
Highlights
Of inner membrane proteins (IMPs)2 is predominantly accomplished in a co-translational manner and involves three distinct steps (2): (i) membrane targeting, mediated by the signal recognition particle (SRP) and the SRP-receptor FtsY (3); (ii) insertion into the lipid bilayer by the Sec translocon, consisting of the protein-conducting channel SecYEG, the accessory complex SecDFYajC, and the ATPase SecA (4 – 6); and (iii) folding and final assembly into a lipid-embedded functional structure
Primary structure analysis revealed that the N-terminal region of the P1 domain contains a low complexity region predicted to be unstructured according to multiple sequence alignments and secondary structure predictions
The C-terminal part of the linker region is important for the YidC insertase function and is conserved in YidC homologs in chloroplasts and mitochondria, suggesting a functional role conserved within the YidC/Oxa1/Alb3 family
Summary
Structure Determination and Refinement—The cloning, overexpression, purification, and crystallization of P1D (residues 56 –329 of E. coli YidC) and its selenomethionine (SeMet) derivative were performed as described (73). The structure of P1D was determined using single wavelength anomalous diffraction data collected on a SeMet crystal at the peak wavelength ( ϭ 0.9790 Å) and the PHENIX (Python-based Hierarchical Environment for Integrated Xtallography) program suite (33, 34). Phase extension, and automatic model building were carried out using a remote data set collected at 1.8 Å resolution and previously scaled to the peak wavelength data using SCALEIT (35). Residues interacting with the polyethylene glycol (PEG) molecule are according to LIGPLOT analysis (43). The PEG/protein and protein/protein interactions within the crystal packing were analyzed with the PISA server (44). Sedimentation velocity studies were carried out at 35,000 rpm at 20 °C in three different buffers (buffer A, buffer A ϩ 1 mM CaCl2, and buffer A ϩ 50 mM CaCl2)
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