Understanding Biological Processes – Membrane protein delivery

Membrane proteins are essential for many biological processes in organisms ranging from bacteria to humans. In the bacterium Escherichia coli (E. coli), a protein called YaeT selects and folds other proteins before inserting them into the outer membrane of the cell. Tim Knowles and his colleagues at the University of Birmingham have been using Diamond Light Source to study protein solution structures in order to understand the processes by which they are targeted in the cell, and how they are folded at their destinations. This is the first time that the full structure of YaeT’s soluble domain has been visualized due to difficulties in creating crystals.

In Detail

Membranes of Gram-negative bacteria, mitochondria and chloroplasts receive and fold b -barrel trans-membrane proteins through the action of polypeptide transport-associated (POTRA) domains of YaeT and its relatives. In E. coli, folding substrates are inserted into the outer membrane by the protein YaeT, which is essential for viability as its depletion leads to severe Outer Membrane Protein (OMP) biogenic defects.

This study combined Small Angle X-ray Scattering (SAXS) with Nuclear Magnetic Resonance (NMR) spectroscopy to reveal the structure of all five POTRA domains of YaeT. Previous studies have indicated that all five POTRA domains conserved in YaeT proteins play crucial and interrelated roles in the assembly and functioning of the b -barrel folding apparatus. The elucidation of this solution structure shows that the POTRA domains form an elongated structure that differs from the crystallized state reported earlier in Science by Daniel Kahne and colleagues.

Small-angle X-ray scattering of YaeT’s soluble domain. A. Solvated molecular envelope of the five POTRA domains calculated from the SAXS data

For the first time the orientation of the essential fifth POTRA domain, the linker orientations and inter-domain interfaces and the substrate binding mechanism have been visualized. This paper reports the NMR structure of tandem POTRA domains of YaeT and the SAXS conformation of all five POTRA domains, and reveals how folding substrate sequences pair with alternating sides of the mixed b sheets of adjacent domains. This provides the first mechanistic understanding of how OMP precursors could be transferred into the core folding machinery by the intact POTRA assembly, thereby revealing fundamental insights into processes required not only for bacterial cell survival but also for the survival of human and plant cells.

Timothy J. Knowles, Mark Jeeves, Saeeda Bobat, Felician Dance, Darren McClelland, Tracy Palmer, Michael Overduin, Ian R. Henderson, Fold and function of polypeptide transport-associated domains responsible for delivering unfolded proteins to membranes, Molecular Microbiology 68 (5) , 1216–1227
doi:10.1111/j.1365-2958.2008.06225.x