Spatial organization via encapsulation of enzymes within recombinant nanocompartments may increase efficiency in multienzyme cascades. Previously, we reported the encapsulation of single cargo proteins within nanocompartments in the heterologous host Escherichia coli. This was achieved by coexpression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK, with a signal sequence EutC1-19 cargo protein fusion. Optimization of this system, leading to the targeting of more than one cargo protein, requires an understanding of the encapsulation mechanism. In this work, we report that the signal sequence EutC1-19 targets cargo to the interior of nanocompartments via a hydrophobic interaction with a helix on shell protein EutS. We confirm that EutC1-19 does not interact with other Eut BMC shell proteins, EutMNLK. Furthermore, we show that a second signal sequence EutE1-21 interacts specifically with the same helix on EutS. Both signal sequences appear to compete for the same EutS helix to simultaneously colocalize two cargo proteins to the interior of recombinant nanocompartments. This work offers the first insights into signal sequence-shell protein interactions required for cargo sequestration within Eut BMCs. It also provides a basis for the future engineering of Eut nanocompartments as a platform for the potential colocalization of multienzyme cascades for synthetic biology applications.
Bibliographical noteFunding Information:
The authors thank Gail Celio, Grant Barthel, and Mark Sanders of the University of Minnesota, Imaging Center for the technical support with microscopy. LC/MS data were collected by LeeAnn Higgins and Todd Markowski of the University of Minnesota, Center for Mass Spectrometry and Proteomics. Kurt Peterson of Fluorescence Innovations, Inc. collected FRET data. The authors thank Mike Autry of the University of Minnesota, Biophysical Technology Center, for the fruitful discussions. Also, thanks to Ammanuel Taye for the help with purification of nanocompartments and Jill Sampson and Mark Held for the help with cloning. This research was supported by National Science Foundation Grant MCB-12644429 and Defense Threat Reduction Agency Grant HDTRA-15-0004 (to CS-D).
© 2016, Springer-Verlag Berlin Heidelberg.
- Bacterial microcompartments
- Enzyme encapsulation
- Fluorescent proteins
- Spatial organization
- Synthetic biology