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Noda Institute for Scientific Research GRANT
2012 Research Grant

Molecular mechanisms of the enhancement of protein exportby the membrane protein complex SecDF
Hiroyuki Mori
Institute for Virus Research, Kyoto University

Research aims
The SecYEG translocon and the SecA ATPase cooperate
to facilitate protein export across the bacterial cytoplasmic
membrane. In addition to these essential core components,
SecDF, a complex containing two membrane-integrated Sec
factors, plays important roles in efficient protein export invivo. We determined the crystal structure of SecDF fromThermus thermophilus at 3.3 Å resolution and proposed aworking hypothesis based on biochemical and biophysicalstudies that were designed using the protein structure (SeeFigure 1).1) According to the model, SecDF forms a complex with the SecYEG translocon, captures a substratepolypeptide emerging from the translocon by its P1 (thefirst periplasmic) domain and undergoes conformationalchanges by using the proton motive force (PMF) to facilitate forward movement of the polypeptide. However,modes of interaction between SecDF and Sec-related factors (including SecYEG) and substrate polypeptides remain
largely unknown. To elucidate these mechanisms, we performed systematic site-directed in vivo photo-cross-linking
analysis targeted to Escherichia coli SecD.
Methods
The site-specific in vivo photo-cross-linking proceduredeveloped by Schultz’s group is an innovative method thatallows for analysis of transient protein-protein interactions
that occur in living E. coli cells.2) In this system, a photoreactive amino acid, p-benzoyl phenylalanine (pBPA), can beincorporated at any position in a target protein using an engineered tRNA synthetase and mutated tRNA pair, and in-teractions can be detected as UV irradiation-dependent generation of cross-linked products. Previously, we have successfully applied this method to identify SecA neighboringsites in SecY.3) Based on the Thermus thermophilus SecDF(TtSecDF) structure, we designed E. coli SecD mutations tointroduce pBPA on the molecular surface of the protein. We
constructed 55 SecD derivatives for cross-linking experiments. Cross-linked proteins were identified by immunoblotting analysis using specific antibodies against candidates
including Sec related proteins.
Results and discussion
Our photo-crosslinking experiments gave the following
results:Cross-linking with SecF (colored in magenta in Fig. 2)
When pBPA was introduced at several positions in theSecD transmembrane regions located at the interface withSecF on the TtSecDF crystal structure, cross-linked products between SecD and SecF were generated. This supports
the idea that E. coli SecDF assumes a conformation similar
to TtSecDF in the membrane.

1)2) Cross-linking with periplasmic chaperones DegP andSkp (in red, Fig. 2)
SecD derivatives containing pBPA in an alpha helix inthe P1 domain facing the periplasmic space generated several cross-linked bands. We identified partner proteins as
Skp and DegP, periplasmic chaperones that prevent aggregation of newly translocated outer membrane proteins andfacilitate their delivery to the outer membrane. An interest-Fig. 1. Working model for SecDF-mediated facilitation of protein translocation

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Intramolecular cross-linking (in orange, Fig. 2)
The P1 domain is composed of head and base sub-domains. Our previous biochemical studies showed that SecD
could assume two different conformations, designated as Iand F, by rigid body movement of the P1 head subdomain.
Upon UV-irradiation, the SecD (Arg268pBPA) mutant generated a band that migrated slightly slower than SecD. Thismutant protein was presumably formed by intra-molecularcross-linking that occurred when the P1 domain was in theform I conformational state. Interestingly, when cells expressing this SecD derivative were treated with CCCP (aproton ionophore) prior to UV-irradiation to collapse thePMF across the membrane, the amount of cross-linked
product was dramatically reduced. Furthermore, the introduction of an additional SecD or SecF mutation that abolished both the proton conductance and the protein exportenhancing activities of SecDF resulted in almost complete
disappearance of the cross-linked product. These results arein agreement with our model, which suggests that proton
conductance of SecDF couples with the movement of theP1 domain.
4)

Fig. 2. Summary of site-directed in vivo photo-cross-linking experiments targeted to E. coli SecD. TtSecD amino acid residues corresponding
to those in E. coli SecD that generated cross-linked products when substituted with pBPA are shown as colored spheres on the TtSecDF crystal
structure (F form). Spheres colored in magenta, red, green, and orange indicate amino acid residues that are in close proximity to SecF, periplasmic
chaperones (DegP and Skp), maltose binding protein and P1 head subdomain in I form, respectively. Spheres in white show amino acid resides
that generated no cross-linked product when substituted with pBPA.

ing hypothesis is that the P1 domain could act not only as abinding site for a translocating substrate (see below), butalso as a docking platform for the periplasmic chaperones
to promote translocation and folding of the substrate polypeptide.
3) Cross-linking with maltose binding protein (MBP)(in green, Fig. 2)
When SecD derivatives having pBPA in a narrow crevasse in the P1 domain were used, SecD-MBP cross-linked
products were formed. Although it remains to be shown
whether this cross-linking represents interaction of translocating MBP with SecD, our results raise the possibility thatthe crevasse in the P1 domain functions as a binding site
for translocating substrate polypeptide chains.

Conclusion
We succeeded in identifying several SecD-neighbor factors by systematic, site-directed in vivo photo-cross-linking
analysis targeted to E.coli SecD. These results strongly
support our model of SecDF function. Further study will
provide us with important insights into the molecularmechanisms of PMF-driven SecDF function.
References
1) Tsukazaki, T. et al. (2011) Structure and function of a membranecomponent SecDF that enhances protein export. Nature 474: 235–238.
2) Chin, J. W. et al. (2002) Addition of a photocrosslinking amino acidto the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. USA.
99: 11020–11024
3) Mori, H. and Ito, K. (2006) Different modes of SecY-SecA interactions revealed by site-directed in vivo photo-cross-linking. Proc.
Natl. Acad. Sci. USA. 103: 16159–16164