Part:BBa_K3646008
FKBP CDS (FK506 Binding Protein)
The 12 kDa human FK506 binding protein (FKBP12) and the 100-amino acid domain of the kinase Target of Rapamycin (TOR) known as the FKBP-rapamycin binding domain (FRB) do not directly interact but dimerize in the presence of the chemical rapamycin. This interaction has been exploited for chemically induced activation of e.g. signalling cascades, changes in subcellular localisation and regulation of protein stability in mammalian and yeast systems.
In our project, we aim to use the FKBP-Rapamycin-FRB system as regulatory modules to control the strength of the interaction between our proteins of interest. This part works in concert with BBa_K3646004 to achieve this modulation.
A closer look at Protein-protein Interactions (PPIs)
Protein−protein interactions (PPIs) play decisive roles in almost all cellular processes. After more than a decade into the genomic era, efforts to experimentally determine the protein interactomes in humans and model organisms are a pressing research goal. Quantifying affinity is a critical step for live‐cell PPI measurements. Affinity defines the occupancy of a given binding site as a function of local concentrations. Quantitative information on affinities is also of great importance for modelling. There are various methods for quantification which have different advantages and disadvantages. They differ in terms of the type of interactions they can detect, the type of proteins they can be used with, the number of false positives and false negatives, instrumentation needed, and so on.
Most biochemical PPI reporter systems consist of two parts: one part is linked to one of the proteins of interest (sometimes called the “bait” protein) and the other one to the other protein of interest (sometimes called the “prey” protein). When both proteins interact with each other, the two parts of the reporter system are brought into close proximity and have a clearly measurable effect. The terms “bait” and “prey” are normally used if there is a protein of interest and the objective is to “hunt” for interacting partners. However, in many cases both partners are already known and the objective is to study the interaction in more detail.
There are also reporters based on gene expression (i.e., the PPI drives reporter expression) but unfortunately, these are not capable of following dynamic changes in the interaction, and can only be quantified by absorption and by using a colour producing enzyme.
Enough said about PPIs and their measurement, let us move on to understand where the CID system comes into the picture.
FKBP-FRB and Chemically Induced Dimerization : A Tool with great potential in Biology
Proximity plays an important role in biochemical processes. The probable answer to the fact of discrete biological response w.r.t localization is that the probability of an effective collision between two molecules is a third-order function of distance. Protein fusions (as we are using in our project FRaPPe) can lead to steric hindrances blocking functional responses. CID seems to offer more robust responses in these situations. It also allows precise mathematical modelling. The fundamental concept of effective molarity—that a localized concentration within solution may differ from the bulk concentration—underlies the rationale and practicality of using chemically induced proximity to study complex biological mechanisms.
Rapamycin binds with high affinity (Kd ) 0.2 nM) to the 12-kDa FK506 binding protein (FKBP12) as well as to a 100-amino acid domain (E2015 to Q2114) of the mammalian target of rapamycin (mTOR) known as the FKBP-rapamycin binding domain (FRB). The interaction between FKBP and rapamycin has been well characterized (Kd ) 0.2 nM), and early experiments suggest that formation of a ternary complex including FRB is quite favorable (Kd ≈ 2.5+/- 12 nM). Fluorescence polarization, surface plasmon resonance, and NMR spectroscopy based data (Banaszynski et al., 2004) shows that rapamycin binds to FRB with moderate affinity (Kd ) 26 +/- 0.8 μM. The FKBP12 + rapamycin complex, however, binds to FRB 2000-fold more tightly (Kd ) 12 +/- 0.8 nM than rapamycin alone.
A major challenge for any method of regulated gene expression is the steep dose-response curve induced by rapamycin. The use of nontoxic dimerisers such as abscisic acid provides a more graded dose-response and could be useful for precise dosage control. Because of high affinity and slow dissociation of rapamycin to its protein binding partners, rapamycin-induced dimerization is essentially irreversible. Moreover, because rapamycin also binds to endogenous mTOR and FKBP proteins, it can produce off-target effects, leading to undesirable biological activities including immunosuppression and induction of autophagy, by supressing the kinase activity of mTOR. Since signal transduction often operates in a reversible manner, it is important to be able to reversibly control cellular processes, in order to decipher signalling networks. Reversing the dimerization through photocleavage or competitor binding are some ways that have been explored. In addition, in our system, we will be using controls that will enable us to normalise off-target effects, if any. Moreover, since fluorescence readout is to be used as the measurable signal, which directly comes only from the expressed proteins, we believe that the measure will not have discrepancies, enabling us to utilise the benefits conferred by the CID system to the fullest.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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