Presentation of gp12
During the first step of the lytic cycle, the short tail fibers gp12 attach the virion to the host cell.
Thanks to a three-dimensional cryoelectron microscopy, a reconstruction of the baseplate was determined to a resolution of 12 Angstrom. It has shown that the total length of the gp12 density is inferior at 340 Å. [1]
This technique was also a way to determine the interactions between the different proteins and their localization. Thus, interactions of the short tail fibers with each other and with the gp11 maintain the hexagon shape stability. The gp11 is also associated with gp10, which is clamped between the three fingers of gp11. This association between gp10
Short tail fibres consist of the single protein gp12 . This protein forms a parallel, in-register, homo-trimer of 527 residues per subunit [2]. 18 monomers are on the baseplate. 1ocy is a monomer of the short tail fibres. A monomer of gp12 has a mass of 55.3 kDa. The gp12 N-terminal domain is bound to the baseplate. While the C-terminal globular domain is supposed to bind the bacterial host cell [2].
Gp12 can be divided into two fragments. One fragment with a mass of 33kDA and a second with a mass of 45kDa.
The 33kDa Fragment [2] [3]
The 33kDa fragment (PDB:1H6W) was generated in the presence of EDTA [2]. This fragment contains the residues 85-395 and 518-527 . The residues 397-517 are lacking because of internal deletion.
The 33kDa fragment can be further sub-divided into two subunits [2]. The neck (residue 333-341) and the collar (residues 342-396 plus 518-527). The neck connects the body of the fibre to its C-terminal collar and receptor-binding site. It consists of a triple alpha-helix which is built by the residues 333-341.
The collar domain is a small globular domain. It contains six beta-strands and an alpha-helix [2].
The 45kDa Fragment [2]
For generating the 45kDa fragment the full length gp12 was co-expressed with its chaperone gp57 and purified [2]. Like the 33kDa fragment it also starts with the amino acid Leu85. The 45kDa fragment contains the residues 397-517 , which are in the 33kDa fragment internal deleted [2] .
Like the 33kDa fragment the 45kDa fragment can also be divided into two subunits. These two subunits are called head (residues 397-446 and residues 487-517 ) and bonnet (residues 447-487). On the border between the head and the bonnet subunit there is a metal-binding site [2].
Receptor-Binding Domain [2]
Gp12 is fixed with its N-terminal domain to the baseplate. So the C-terminal domain has to be involved in LPS-binding. To detect where the receptor-binding domain is, full-length gp12, 33kDa fragments and 45kDa fragments were immobilised in micro-plate wells and were allowed to bind to bacteria [2]. The result was that the 33kDa fragment did never bind to a bacteria [2]. The 45kDa fragment did bind. So the receptor-binding domain is absent in the 33kDa fragment but present in the 45kDa fragment. The residues which are present in the 45kDa fragment and lacking in the 33kDa fragment are the residues 397-517. They are referred to be part of the receptor-binding domain.
The receptor-binding domain can be sub-divided into head and bonnet. On the border between these two subdomains there is a metal-binding site [2]. This site binds presumably to zinc. Two His amino acids (His445 and His447) from each monomere are octahedrally coordinated around the zinc.
The receptor-binding domain can be compared to a flower bud. This flower bud has got 12 petals which are organised in a 3-fold symmetry [2]. At the bottom there are the residues 406-432 they form the first petal , just above there are the residues 489-504 which form the second petal . The third petal is formed by the residues 450-470 and at the top there are the residues 470-480 and form the fourth petal . The complete and active receptor-binding domain is built by the trimeric protein. This trimeric proteine structure is stabilised by many inter-domain hydrogen bounds [2]. These hydrogen bounds can have the profiles: main-chain-main-chain, main-chain-side-chain and side-chain-side-chain.
The LPS-Bindind Site [2]
The exact position of the lipo-polysaccharide(LPS)-binding site is not known. Proteolysis experiments showed, that it contains to the domain with the residues 397-517. Compared with the homologous bacteriophage T4-like strain AR1 sequence, which also binds to the same LPS core molecule like gp12, there can be some possible binding residues be assumed.
Basic putative binding residues:
Lys446, Lys422, Arg504, Arg424, Arg513
Aromatic putative binding residues:
Tyr454 ,Phe451, Trp477 ,Phe468 ,Phe460 ,Phe420 ,Tyr488,Tyr444 , His408, Tyr433
Aromatic and basic residues
It can not be ruled out that also other amino acids are important for binding ! [2] Further it can not be ruled out that not all of these named amino acids are important for binding.
The mentioned amino acids are starting point for site-directed mutations and /or other biochemical experiments to clarify the location of this binding site.
Ligands and their Binding-Sites
So4
In the gp12 there are two molecules of SO4 ligands. One of them SO4 (1529) interacts with the citric acid. The blue molecule is the SO4. The other SO4 molecule (1530) interacts with Ser387 . It builds two hydrogen bounds to Ser287 with the distances 2.66 Å and 3.20 Å.
Citric Acid
The citric acid is drawn in green. It interacts with following amino acids: Arg465 (distance 3.01 Å), Asp455(distance 2.42 Å) and Tyr454 (distance 2.94 Å). Here you can see the interactions between citric acid, SO4, Asp465, Asp455 and Tyr454. These interactions are caused by hydrogen bounds.
Zinc
The zinc is located in the centre of the receptor-binding domain. The zinc-binding site lies on the border between the head and the bonnet of the 45kDa domain. It interacts with the amino acids His445 and His447 of each monomer. The distances of the zinc ion to the NE2 of His445 and His447 are 2.22 Å and 2.25 Å. The normally found distances between His and zinc are shorter. The explaination that these distances are longer than normally found is the octahedral coordination of the zinc in this structure [2].
The role of the zinc ion is probably absolute of structural nature. It increases the stability of the C-terminus of gp12 against proteases, but it also raises the stability of the C-terminus in general [2].
- ↑ Leiman PG, Arisaka F, van Raaij MJ, Kostyuchenko VA, Aksyuk AA, Kanamaru S, Rossmann MG. Morphogenesis of the T4 tail and tail fibers. Virol J. 2010 Dec 3;7:355. doi: 10.1186/1743-422X-7-355. PMID:21129200 doi:10.1186/1743-422X-7-355
- ↑ 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 2.09 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 Thomassen E, Gielen G, Schutz M, Schoehn G, Abrahams JP, Miller S, van Raaij MJ. The structure of the receptor-binding domain of the bacteriophage T4 short tail fibre reveals a knitted trimeric metal-binding fold. J Mol Biol. 2003 Aug 8;331(2):361-73. PMID:12888344
- ↑ van Raaij MJ, Schoehn G, Jaquinod M, Ashman K, Burda MR, Miller S. Identification and crystallisation of a heat- and protease-stable fragment of the bacteriophage T4 short tail fibre. Biol Chem. 2001 Jul;382(7):1049-55. PMID:11530935 doi:10.1515/BC.2001.131
