Composition and Activity of the Non-canonical Gram-positive SecY2 Complex

Mikaila Bandara,Robin A Corey,Remy Martin,J Mark Skehel,Ariel J Blocker,Howard F Jenkinson,Ian Collinson

doi:10.1074/jbc.m116.729806

Abstract

The accessory Sec system in Streptococcus gordonii DL1 is a specialized export system that transports a large serine-rich repeat protein, Hsa, to the bacterial surface. The system is composed of core proteins SecA2 and SecY2 and accessory Sec proteins Asp1–Asp5. Similar to canonical SecYEG, SecY2 forms a channel for translocation of the Hsa adhesin across the cytoplasmic membrane. Accessory Sec proteins Asp4 and Asp5 have been suggested to work alongside SecY2 to form the translocon, similar to the associated SecY, SecE, and SecG of the canonical system (SecYEG). To test this theory, S. gordonii secY2, asp4, and asp5 were co-expressed in Escherichia coli. The resultant complex was subsequently purified, and its composition was confirmed by mass spectrometry to be SecY2-Asp4-Asp5. Like SecYEG, the non-canonical complex activates the ATPase activity of the SecA motor (SecA2). This study also shows that Asp4 and Asp5 are necessary for optimal adhesion of S. gordonii to glycoproteins gp340 and fibronectin, known Hsa binding partners, as well as for early stage biofilm formation. This work opens new avenues for understanding the structure and function of the accessory Sec system.

Highlights

Human platelets and formation of vegetations at cardiac sites
We provide evidence that Asp4 and Asp5, and the intact accessory complex, are required for optimal adhesion of S. gordonii to glycoproteins gp3402 and fibronectin, as well as for early stage biofilm formation
The purified complex was subjected to SDS-PAGE analysis, alongside the E. coli canonical counterpart SecYEG (Fig. 3)

Summary

Results

Purification of the Non-canonical Translocon Complex— SecY2-Asp4-Asp was purified by nickel affinity and gel filtration chromatography (Fig. 2). Model—Sequence identity between the Gram-negative canonical SecYEG-SecA complex from Thermotoga maritima, of known structure [29], and the S. gordonii accessory Sec counterparts are ϳ40, 24, 20, and 20% for SecA, SecY, SecE, SecG, respectively. This enabled the construction of a homology model of the non-canonical complex (Fig. 7A). Compared with the WT, biofilm formation for the ⌬secA2 mutant strain was decreased by 56 and 51% for the 6- and 24-h time points, respectively (Fig. 9A). The S. gordonii ⌬asp4/(pAsp4ϩ) complemented strain produced biofilms comparable with the WT with no significant differences between the two (Fig. 9). There was no significant difference in binding levels between the WT and S. gordonii ⌬asp4/ (pAsp4ϩ) complemented strain (Fig. 11)

Discussion

Experimental Procedures

Relevant characteristics