F1LBoundBimBH3

Vaccinia virus F1L bound to Bim BH3 (PDB ID: 4D2M) is a protein complex made up of viral anti-apoptotic protein F1L (PBD ID: 2VTY) from Vaccinia virus Ankara bound to Bim Bcl-2 homology domain 3 (BH3) from Homo sapiens. Apoptosis is the process of programmed cell death, which occurs when a cell is damaged or mutated. The Bcl-2 proteins are a family consisting of both pro-apoptotic and anti-apoptotic proteins, which are subdivided based on the Bcl-2 homology (BH) domains that they contain (1). One of these divisions contains pro-apoptotic proteins that only retain the BH3 domain, such as Bim. Bim carries out its function by either deactivating multidomain Bcl-2 anti-apoptotic proteins, such as Bak or Bax, or activating pro-apoptotic ones, such as Bcl-2 proper, by binding to a binding groove conserved on these mutidomain proteins (1,2). Viruses have developed orthologs of Bcl-2 proteins to counter the apoptosis of infected cells. These proteins retain the important structural features of Bcl-2 proteins, such as the folding patterns and domains, but not necessarily the primary structure (3). The vaccinia virus F1L protein is a Bcl-2-like protein that retains no clear structural homology with Bcl-2 anti-apoptotic proteins but is still able to engage and deactivate certain pro-apoptotic proteins such as Bim BH3 (4). The nature of the binding site between F1L and anti-apoptotic Bcl-2 proteins is important to researchers because it provides insight into the mechanism of pro-apoptosis deactivation.

The binding properties of F1L bound to Bim BH3 are determined by its structure. The molecular weight of F1L bound to Bim BH3 is 46346.62 Da and its isoelectric point is 4.77 (5). The complex is made up of 4 subunits: two Bim BH3 domains (Subunits B and D) and a F1L dimer (Subunits A and C). The Bim subunits each consist of 26 residues and have a single α helix (BH3 domain) while each F1L monomer consists of 168 residues, and its secondary structure consists of seven α helices (α1-α7) (6). The helices are numbered such that they correspond with the prototypical Bcl-2 protein structures. The tertiary structure of each monomer contains a helix core, made up of α2-α7, and a spatially separate α1. The distance between α1 and the helix core results in dimerization, as the α1 in each monomer is "swapped" into the neighboring subunit so that α1*, the swapped helix, is now part of the helix core of other monomer (7). This results in the dimer being tightly held together, as there are hydrophobic interactions between the helix core and α1*, as well as hydrophobic interactions between both α1 helices.Important residues involved in the swapped domain interactions include Val-62, Ala-65, Val-66, Tyr-69 and Met-70 from α1* and Tyr-89, Ile-146, Cys-174, Phe-178, Val-179, Ile-182 from the helix core. Also, residues binding the α1 helices to one another include Met-64, Tyr-68, Leu-75, Tyr-79 and Leu-82 (7).

While the interior of the F1L dimer is involved in hydrophobic interactions keeping the dimer together, the exterior, composed of the outer faces of α2-α5, forms a binding groove where Bim can fit. As with the interior interactions, the F1L:Bim binding consists of only hydrophobic interactions. Each Bim subunit contains four critical binding residues that protrude into four pockets in the binding groove: Ile-58, Leu-62, Ile-65 and Phe-69. In turn, residues Met-108, Met-111, Met-114, Trp-119, Ile-132, Asn-140 from F1L facilitate contact with the Bim residues (6). Although the binding site contains a fold similar to that of anti-apoptotic Bcl-2 proteins, one notable difference is the lack of an ionic interaction between F1L and its ligand, which is present in all Bcl-2:Bim interactions between Bim Asp-67 and an arginine residue in bcl-2 proteins (6). This results in an overall weaker F1L:Bim interaction.

Neither the C- nor N-terminus was included in the F1L sample used for this protein complex, as the F1L subunits used were composed of residues 18-186 of a 226-residue polypeptide. However, sequence analysis of the entire protein shows the presence of a hydrophobic domain surrounded by positively-charged residues and a short hydrophilic tail. This region binds to mitochondria membranes and proves that F1L localizes to mitochondria during infection and regulates apoptosis (8).

F1L bound to Bim BH3 contains three types of ligands: Bim BH3, chloride ions, and 2-methyl-2,4-pentanediol. Although being proteins themselves, Bim subunits are essentially ligands of the F1L dimer instead of being part of the protein's quaternary structure because Bim is derived from humans and was manually added to F1L. Also, the subunits of interest in this protein complex are the F1L subunits, and Bim is important only for containing a BH3 domain. The other two ligands have no functional purpose and are present from the crystallization steps. Specifically, 2-methyl-2,4-pentanediol and two chloride ions are found in the center of the dimer, helping to stabilize the F1L subunits, while the remaining two chlorides are located outside of the protein. The role of the latter chlorides becomes clear upon generating the full unit cell of the protein, which is actually a tetramer, where these ions stabilize the separate dimers. This conformation is not discussed in detail because the asymmetric unit, which is the dimer, carries out all of its functions independently of the other one, as the tetramer exists only in the crystal and does not add any function. The F1L:Bim BH3 crystals were grown using the sitting-drop method at room temperature in a mixture of 0.2 M KCl, 15% polyethylene glycol 400 (PEG400), 1.44% 2-methyl-2,4-pentanediol, and 0.1 ammonium citrate (pH 6.2) (6).

The F1L subunits in F1L bound to Bim BH3 have no close similarities with any other known protein, neither in sequence nor in tertiary structure, according to the results of the PSI-BLAST program and the Dali server. The PSI-BLAST, or Position-Specific-Iterated Basic Local Alignment Search Tool, is a program that compares the primary structure of a protein query with those of a database. The program assigns an E value based on the sequence homology between two proteins, taking into account mutations and gaps in the sequence. E values below 0.05 are considered significant for sequence similarity. The Dali server is a program that compares the three-dimensional structure of a protein query with those of a database. It works by comparing intramolecular distances, and yields a Z score, which must be above 2 to show significant structural similarity.

Based on the results for both programs, a suitable protein for comparison is Vaccinia virus F1L (PDB ID: 2VTY), which had an E value of 2 x 10^-133 and a Z score of 16.3 (9,10). Although this protein is almost identical to the F1L subunits in the protein of interest and does not show a different function or come from a different organism, the two servers found no other acceptable match, except for F1L bound to another Bcl-2 BH3 protein. Nevertheless, there do exist small but significant differences between the two F1L proteins that affect their crystallization and binding properties. The F1L protein found in the BH3-bound dimer contains a mutation at residue 125 in which isoleucine was replaced by a phenylalanine residue (6). This residue is located on the surface of the binding groove, and the mutation alters its shape, as phenylalanine is significantly larger than isoleucine. This was purposely done, after surveying various mutations, to increase the binding strength of F1L and Bim. Another major difference between the two proteins is their crystal structure. F1L bound to Bim has an ordered crystal structure from residues 51-186, while ligand-free F1L is ordered from residues 34-104 and 114-186 (6,7). The crystal structure of the ligand-free protein shows an additional α helix between residues 39 and 48, termed α0, which has no corresponding domain in Bcl-2 proteins, unlike the other helices. Also, the ligand-free protein has a completely disordered α3 helix, which becomes elucidated in the mutant variant (6).

Overall, this protein complex is useful in showcasing the specifics of viral anti-apoptotic protein binding. It is interesting to note how the F1L:Bim binding changes when just one residue in the binding pocket is changed. This binding is important because if the binding mechanism is understood, then it could be possible to disable the protein's function, thus preventing the survival of infected cells.