Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

Abstract

While SecA is the ATPase component of the major bacterial secretory (Sec) system, mycobacteria and some Gram-positive pathogens have a second paralog, SecA2. In bacteria with two SecA paralogs, each SecA is functionally distinct, and they cannot compensate for one another. Compared to SecA1, SecA2 exports a distinct and smaller set of substrates, some of which have roles in virulence. In the mycobacterial system, some SecA2-dependent substrates lack a signal peptide, while others contain a signal peptide but possess features in the mature protein that necessitate a role for SecA2 in their export. It is unclear how SecA2 functions in protein export, and one open question is whether SecA2 works with the canonical SecYEG channel to export proteins. In this study, we report the structure of Mycobacterium tuberculosis SecA2 (MtbSecA2), which is the first structure of any SecA2 protein. A high level of structural similarity is observed between SecA2 and SecA1. The major structural difference is the absence of the helical wing domain, which is likely to play a role in how MtbSecA2 recognizes its unique substrates. Importantly, structural features critical to the interaction between SecA1 and SecYEG are preserved in SecA2. Furthermore, suppressor mutations of a dominant-negative secA2 mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG or the translocating polypeptide substrate. These results support a model in which the mycobacterial SecA2 works with SecYEG. IMPORTANCESecA2 is a paralog of SecA1, which is the ATPase of the canonical bacterial Sec secretion system. SecA2 has a nonredundant function with SecA1, and SecA2 exports a distinct and smaller set of substrates than SecA1. This work reports the crystal structure of SecA2 of Mycobacterium tuberculosis (the first SecA2 structure reported for any organism). Many of the structural features of SecA1 are conserved in the SecA2 structure, including putative contacts with the SecYEG channel. Several structural differences are also identified that could relate to the unique function and selectivity of SecA2. Suppressor mutations of a secA2 mutant map to the surface of SecA2 and help identify functional regions of SecA2 that may promote interactions with SecYEG.

The Structure of Clostridioides difficile SecA2 ATPase Exposes Regions Responsible for Differential Target Recognition of the SecA1 and SecA2-Dependent Systems

“…Our analysis was consistent with the findings of our modeling attempt, which among the available structures of SecY-bound SecA conformation lacked a suitable template for the interaction site of the target protein that connects the two β-strands of the PPXD to NBD1 in CDSecA2 ( Figure 3A,B and Figure S2). In addition, the SecY channel interaction region is located at the 2HF loop and its surrounding area [23,24,59], where high surface residue conservation suggests their conserved functional role ( Figures 3C and 5 and Figures S2 and S3). Namely, superimposition of our CDSecA2-SecY-bound conformation model with the T. maritima SecA-SecY structure (PDB ID 3DIN; [24]) showed structural similarities that would lead to the PPXD with its loops and the long helix of the HSD forming the contact surface with the SecY channel and the 9-residue-long and flexible 2HF loop inserting into the SecY channel (residues L742-P750; Figure 3C and Figures S2 and S3).…”

“…Furthermore, HWD interacts with C-terminal part of the SecY in crystal structures of SecA-SecY(EG) complexes [ 24 , 29 ] and with adjacent lipids [ 30 ]. Thus, the lack of HWD in SecA2 systems may enable binding of specific substrates by increasing solvent exposure of the target recognition site [ 23 ] and hamper binding of SecA2 to SecYEG. We showed that the conformation of CDSecA2 HWD closely resembles those of the general SecA systems ( Figure 1 C), thereby suggesting CDSecA2 may bind to SecYEG and participate in substrate recognition and translocation in a similar manner.…”

“…* marks the structure shown in Figure 1B or Figure 1C. In addition to closed conformation, the intermediate [23], open [7,19,[26][27][28] and SecY-bound conformations [24,29,30] were observed, with PPDX occupying varying positions in regard to the HWD and different ligands. To present variations in positions of the PPXD, representative structures from different species were superimposed and are shown in Figure 1B.…”

The Two Distinct Types of SecA2-Dependent Export Systems

“…In addition, M. tuberculosis SecAl and SecA2 share only 38% identity (54% sequence similarity). Thus, it was a surprise to discover broad similarity between the crystal structures of M. tuberculosis SecAl and SecA2 (80). However, compared to SecAl, the CTD of SecA2 is truncated, similar to what was found for SecA2 of the aSec system (Fig.…”

“…In addition, the helical wing domain (HWD) is missing in the mycobacterial SecA2. The lack of an HWD is a conserved feature of actinobacteria SecA2 proteins, but small HWD truncations may also exist in other SecA2 proteins (80). The significance of CTD and HWD truncations to SecA2 function remains to be investigated.…”