Team:SCUT-China/Project/Docking Domain

Overview

The fidelity and efficiency of acyl transfer on the interfaces of the individual PKS proteins is thought to be governed by helical regions, termed Docking Domains (DD), which are located at the C-terminus of the upstream and N-terminus of downstream polypeptide chains. The length of docking domain is about thirty to ninety amino acids (Figure 1) ^[1]. It not only participates in the regulation of protein function, but also completes the transshipment of upstream intermediate, and forecasts its vast prospects in the development of new drugs ^[2].

There are two types of docking domains. Class 1 is from actinomycetes and mucous bacteria while class 2 is from cyanobacteria mostly ^[3]. Class 1 DD is also in the form of dimer just like the polyketide synthase. At present, the class 2 DD is seen to be a genetic engineering tool because of its shorter length and tight combination, and it will has great application prospect for deeper researches.

Docking domain consists of ACP-side docking domain (ACP DD) at the C-terminus of the upstream and KS-side docking domain (KS DD) at the N-terminus of downstream. The two parts locate in the downstream of ACP and upstream of KS respectively, and lie in the different modules. Each DD has formed specificity of the binding because it has specific connection site. Namely each docking domain is unique in the whole polyketone synthesis pathway. With this important feature, we can ensure the connection between adjacent modules does not have randomness, which makes the product unique.

ACP of class 2 DD is about 40 animo acids shorter than class 1, which cannot form a polymerization area. Having the similar length with class 1 DD, KS of class 2 is unable to form a spiral structure domain coiled coil due to its strong polarity. Therefore, the class 2 DD have no dimer, even though all of their structures are of even numbers.

There are two alpha helix existing in the upstream and downstream of the class 2 DD separately. KS has alpha A and alpha B at the head of domain, while ACP has alpha 1 and alpha 2 at its end (Figure. 2). In order to get closer and form tighter combinations, the alpha helices between alpha A and alpha B or between alpha 1 and alpha 2 form a sharp turn (sharp bend). This structure makes class 2 DD different from class 1 ^[3][4].

Figure 2: Structure of class 2 docking domain

Design:

As introduced, it is obvious that the Class 2 DD has more advantages, such as its shorter lengths, closer combinations, clearer effect of regulating protein function and higher transfer efficiency in completing upstream intermediate. Therefore, we have selected the Class 2 Docking Domain as our part. Because of its specificity as mentioned, the connections between different sets of ACP and KS are different. Typical PKS subunits are tightly homodimeric and contain one to six modules each. They are thought to associate with other multienzyme subunits through contacts at their C and N termini to form the overall PKS complexes. For example, the 6-deoxyerythronolide B synthase (DEBS), assembling the polyketide core of erythromycin A, contains three multienzyme subunits as DEBS 1, DEBS 2, and DEBS3, each of which includes two extension modules ^[1][5]. We get the interest sequence of six pairs of DDs (DD1, DD2, DD3, DD4, DD5, DD6) by sequencing synthesis, and we also add RFC 23 to insert restriction enzyme cutting sizes（Figure 3). At last, we built them to pSB1C3 vector.

Figure 3: The design of docking domains in our experiment. At each set of docking domain, the top was connected with ACP that its upstream joined in the PFC 23 and its downstream joined in the RFC 10. The following came close to KS that the upstream joined in the RFC 10 and the downstream joined in the RFC 23. The following has no linker generally.

Results

After long exploration, we have got the standard DDs that can be used in different connections of ACP and KS. All docking domain are class 2 in our project. At each set of docking domain, the top was connected with ACP that its upstream added the RFC 23 and its downstream added the RFC 10. The following came close to KS that the upstream added the RFC 10 and the downstream added the RFC 23. The following has no linker generally. With dedicated efforts, we have built out the different modules by using the same docking domains. With the upstream and downstream added for assembly, it has been constructed into pSB1C3 vector for further use.

Figure 4: Gel Electrophoresis of our amplified fragments needed for Docking Domains. Each fragment showed the different DD and the”M” is stand for 100bp maker. In the picture (a), number 1 to 3 refer to the” DD1上” . Number 1 to 4 refer to the “DD2上、DD2下、DD3下、DD3上” separately in the picture (b). For (c), number 1 to 5 refer to the “DD4上、DD4下、DD6上、DD6上、DD6下” separately.

Conclusion

In our initial design, we aimed to create the standard general DD that can connect different Module (get more information about Module, please click here). Now, we have got the standard biobricks of DD but without strict verification. Then, we will continue completing the remaining parts of the experiment.

Reference

[1]Broadhurst RW, Nietlispach D, Wheatcroft MP, Leadlay PF, Weissman KJ(2003) The structure of docking domains in modular polyketide synthases. Chem Biol 10: 723–731.
[2] Worthington, A. S., Hur, G. H., Meier, J. L., Cheng, Q., Moore, B. S.and Burkart, M. D. (2008) Probing the compatibility of type II ketosynthase-carrier protein partners,ChemBioChem 9, 2096 –2103
[3]onia JB, Todd WG, Frank EB Kevin AR, Janet LS, and David HS(2009)Structural Basis for Binding Specificity between Subclasses of Modular Polyketide Synthase Docking Domains. ACS Chem Biol Vol.4 No.1
[4]Jonathan RW, Sarah SS, Douglas AH, William CB, William HG,David HS and Janet LS(2013)Cyanobacterial PolyketideSynthase Docking Domains:A Tool for Engineering Natural Product Biosynthesis. Chem Biol 20:1340–1351.
[5][Tang, Y., Kim, C.-Y., Mathews, I. I., Cane, D. E. and Khosla, C. (2006)The 2.7-angstrom crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase,Proc. Natl. Acad. Sci. U.S.A. 103, 11124 -11129.