“Unveiling the Molecular Architecture: Demystifying the Structure of Type IV Secretion Systems (T4SS)”
Key Words: Type IV secretion system, Conjugative T4SS, Conjugation, Negative stain electron microscopy(NSEM)
Introduction:
In the intricate world of bacterial biology, where survival often hinges on the ability to interact with and manipulate their environments, the Type IV Secretion System (T4SS) stands as an astonishing example of microbial ingenuity. This sophisticated molecular machinery enables certain bacteria to not only communicate with their surroundings but also to transport a diverse array of molecules, ranging from proteins to genetic material, across their cell membranes. In gram-negative bacteria, conjugation is accomplished by a large transport apparatus called the conjugative type 4 secretion system (T4SS), produced by the donor cell and embedded in both its outer and inner membrane. The T4SS also elaborates a long extracellular filament called the conjunctive pilus which is essential for for DNA transfer. So why are we studying this structure?
It is because without T4SS DNA transfer among bacterial populations would not occur thus we can develop anti-conjugation strategies by analyzing these structures which could limit the spread of antibiotic-resistant genes among pathogens.
The overall structure of T4SS
T4SS is made up of 12 proteins, virB1-virB11 and virD-4 (where “vir” stands for virulence), out of which virB1 is non-essential. Three ATPases VirB4, VirB11, and VirD4 provide energy to the system. VirB7, VirB9, and VirB10 are formed by the outer membrane core complex(OMCC). The Inner membrane complex (IMC) is found beneath the OMCC. Conjugative T4SS must produce a conjugative pilus that makes contact with the recipient cell, VirB2-VirB11, and VirD4.
The Inner Membrane Complex.
The Inner membrane structure is hexameric which is a hexamer of dimers. The size of IMC: 1.32MDa in size and 295 angstroms in diameter. AAA+(AAA+ stands for a family of proteins that possess specific ATPase activity, where AAA means ATPases Associated with different cellular Activities)VirB4 ATPase is the main component of the IMC containing 12 subunits. The above structure has been obtained by Cryo-EM methods, where two kinds of VirB4, VirB4(central) and VirB4(outward as it protrudes outside) form the protomers which act as the building block of IMC(as the building block is a dimer and their number is 6, the structure is called hexamer of dimers). The dimer interaction between VirB4 central and VirB4 outward is mediated by the N-terminal domains of each subunit. By contrast, the interface between two adjacent VirB4 central is spread out over both N-terminal and C-terminal domains.
In the unbound state, it is observed that T4SS shows a trimer of dimers state giving rise to a double barrel structure, this is similar to the result of Negative Stain Electron Microscopy(NSEM).
There are also two other components of IMC, VirB3 and VirB8 tails. The VirB3 interacts only with VirB4 central. So, there are six VirB3 subunits. VirB8 tails interact with only VirB4 outward, and 3 copies of VirB8 interact with VirB4 outward, so they are 18 in number.
The Stalk, Arches, and OMCC
The stalk is a central conical structure with 0.29MDa size, with a diameter of 92 angstroms and a length of 216 angstroms. It protrudes from the inner membrane and is composed of a pentamer of VirB6 inserted into the inner membrane and a pentamer of VirB5 mounted upon the VirB6 base. However, in its pentameric form, VirB5 appears to have undergone a conformational change compared with the protein on its own, with its N-terminal part projected out in a manner reminiscent of pore-forming proteins. This function may be required to interact with the membrane of the recipient cell. Finally, one VirB5 binds between two VirB6 subunits. The Arches are composed of hexamers of VirB8(peri), forming a 177 angstroms diameter ring around the stalk.
The OMCC(Outer Membrane Complex Core) is composed of the O-layer embedded in the outer membrane and the I-layer located underneath within the periplasm mainly composed of VirB10 and VirB9.
