# Advances in the Assembly Model of Bacterial Type IVB Secretion Systems
## 3 Mosaic Structure Model of T4 BSS
### Substrate Recognition Protein  
In addition to the robust central channel protein DotG, DotF also has a substrate recognition function [24], which recognizes the effector RalF secreted by the Dot/Icm system. [PERSON] et al. [25] explored the ability of DotF to recognize other effectors and the results indicate that DotF can only bind to certain types of substrates, that is, DotF is not the main substrate recognition protein in the Dot/Icm system. IM AAA+ ATPase DotL is a member of the type IV coupling protein that is functionally similar to other T4 SS coupling proteins [26]. Structurally, the C-terminal cytoplasmic domain of DotL has a conserved Walker A motif and the N-terminus forms a hexameric assembly in the IM, which is related to the FtsK/SpoIIIE family of DNA translocation motors [27; 28]. As key components of T4 SS, coupling proteins are mainly responsible for the localisation of substrates at transmembrane receptors of the secretion system [29] and the hydrolysis of ATP to provide energy for substrate transfer [30]. DotL also forms a subcomplex with DotM, DotN, IcmS, and IcmW [31]. The C-terminal tail of DotL bound to DotN and binary complex IcmSW separately [32] and the recruitment of effectors independent of IcmSW is mediated by a secretion signal sequence rich in Glu residues and located at the C terminus, which is proved that DotM acts as a potential binding platform for the recruitment of acidic Glu-rich,  
Figure 3: Crystal structure and amino acid sequences alignment of T4 SS proteins and their structural homologs. **(a)** ATPases of _Legionella pneumophila_ DotB\({}_{\rm L}\) (PDB id: 6 GEB), _Agrobacterium tumefaciens_ VirB1A\({}_{\rm A}\)_, _Vibrio fluvialis_ GspE\({}_{\rm V}\); **(b)** Outer membrane proteins of \(L\). _pneumophila_ DotD\({}_{\rm L}\) (PDB id: 3 ADY), _A. tumefaciens_ VirB7\({}_{\rm A}\)_, _Vibrio fluvialis_ GspD\({}_{\rm V}\), and _Escherichia coli_ EscC (PDB id: 3 GR5); **(c)** Periplasmic proteins of \(L\). _pneumophila_ DotH\({}_{\rm L}\), _A. tumefaciens_ VirB9\({}_{\rm A}\); **(d)** Membrane-spanning proteins of \(L\). _pneumophila_ DotG\({}_{\rm L}\) and _A. tumefaciens_ VirB10\({}_{\rm A}\); **(e)** Inner membrane (IM)-associated proteins of \(L\). _pneumophila_ DotO\({}_{\rm L}\) and _A. tumefaciens_ VirB4\({}_{\rm A}\); **(f)** IM proteins of \(L\). _pneumophila_ DotI\({}_{\rm L}\) and _A. tumefaciens_ VirB8\({}_{\rm A}\); **(g)** Inner membrane proteins of \(L\). _pneumophila_ DotI\({}_{\rm L}\), and _E. coli_ TssL\({}_{\rm E}\) (PDB id: 3U66); **(h)** IM proteins of \(L\). _pneumophila_ IcmI\({}_{\rm F}\) and _E. coli_ TssM\({}_{\rm E}\) (PDB id: 4Y7M); **(i)** N-terminal domain of \(L\). _pneumophila_ DotD\({}_{\rm L}\) and _A. tumefaciens_ VirB7\({}_{\rm A}\); **(j)** Residues 168–227 of \(L\). _pneumophila_ DotH\({}_{\rm L}\) and N-terminal domain of _A. tumefaciens_ VirB9\({}_{\rm A}\); **(k)** C-terminal domain of \(L\). _pneumophila_ DotG\({}_{\rm L}\) and _A. tumefaciens_ VirB10\({}_{\rm A}\). IcmSW-independent effectors [33; 34]. In view of the fact that DotL binds directly to DotM, it is suggested that DotM may regulate the expression of DotL. DotN is a IM/cytoplasmic protein rich in cysteine with a small molecular weight that can stabilize the structure of DotL and DotM complexes, although its specific mechanism of action remains unclear [35]. IcmS and IcmW are small molecular weight soluble cytosolic proteins, often found in IcmSW binary complexes. There is a binding site for IcmSW in the C-terminus of DotL. When this binding site is mutated, the transport capacity of IcmSW-dependent substrates such as SdeA, SidD, and VipA decreases. Conversely, there is no effect on the transport of some non-IcmSW-dependent substrates such as RalF, LnaB, and LidA [31; 35]. When the expression levels of IcmS or IcmW are reduced or even eliminated, the infection ability of DotL decreases, indicating that IcmSW may be used as a targeting factor to recruit substrates to a location near DotL. OM protein LvgA is approximately 27 kDa in size.