The Scaffold of the Carrier Protein, 10th of April 2003
-Docking onto site of delivery-
This topic is about research on the structure and function of carrier protein GGA, which we have already introduced several times. The carrier protein GGA is composed of 3 domains, such as the VHS domain, which confirms the label of a cargo and the GAE domain, which is ear-shaped. On this Website, we have already introduced VHS domains on the page "Carrier Protein in Cells, -Capturing the moment of taking cargo-" and GAE domains on the page "Ear of Carrier Protein, -Determination of 3-D structure-". Today, we will describe central GAT domains, which are still undetermined.

Carrier Protein is working with fixed scaffold
In cells, there are small rooms (organelles) surrounded by membranes and each organelle is a site where basic activities of cells are performed. The working site of GGA is an important organelle called the trans-Golgi network where the materials for intracellular transport are assorted. The membranes that surround organelles in cells have a double membrane structure, in which bilayer hydrophobic lipids face each other and fold together. ARF, a go-between protein, helps it, when the carrier protein GGA docks on the membrane. ARF has anchor-shaped lipids and is fixed to the membrane by digging the anchor into the bilayer lipid membrane. The carrier protein can make a scaffold at the working site by binding to ARF fixed to the membrane.

Role of the GAT domain
How does the carrier protein GGA bind to ARF? In fact, a GAT domain located at the center of GGA plays this important role. A GAT domain binds to a go-between protein ARF and fixes the scaffold firmly. The 3-dimentional structure of the GAT domain has not been elucidated but the Structural Biology Group of Professor Soichi Wakatsuki as a leader in KEK has just solved the 3-D structure of ARF-GAT domain complex using synchrotron radiation of the synchrotron radiation research facility (Photon Factory). For this, the mechanism of the docking of the carrier protein GGA to the membrane has been clarified. These results will be published in the May issue of Nature Structural Biology and has already appeared online on 8th of April. Two other research groups, the MRC group in Cambridge University, the UK and the NIH group in the US also solved the GAT domain structure at almost same time (Figure 3), but only the KEK group has successfully determined the structure of its complex with ARF.

GAT domain structure alone and its complex with ARF
As shown in Figure 2a, the GAT domain itself is composed of 3 helical parts (alpha helix). The first helix is about twice the length of the other helices. The dot indicates a part with no fixed structure. Figure 2b describes the structures of part of the GAT domain (a part interacts to ARF) and GAT-ARF1 complex. Reds indicates the common parts and the dot circle in Figure 1 corresponds to where one part slides over another. After forming a complex with ARF, the part of no fixed structure in GGA forms 2 helices, alpha helices. The other protein that interacts with ARF lacks such manner of recognizing ARF by using these 2 alpha helices that are almost parallel and this is the first time to find this recognition pattern. As shown in Figure 4, the GAT domain has a structure that recognizes the bumps and dips of the ARF protein very well.

Importance of 3-dimentional structural research of carrier protein
The intracellular transport of substances is the origin of maintaining cell activities and elucidation of the role of the protein that controls intracellular transport is critical in order to understand cell activities. It is well known that abnormalities of proteins involved in intracellular transport and subsequent protein transport to wrong sites cause a number of diseases. Thus, the research to understand the protein structure and function involved in intracellular transport is not only of biological interest but also may be very important in the medical field. In the present study, the structures of all 3 domains of carrier proteins GGA have been determined and it was shown that each domain performs each role successfully. Moreover, the binding manner of the carrier protein via the other go-between protein has been clarified for the first time from the structure of the complex with ARF. The research to elucidate protein function that supports our life activities through the protein 3-D structure determination using synchrotron radiation produced by the accelerator is also important work of KEK.



Figure 1
This exhibits the interaction between GGA1 protein anda the other protein involved in protein transport. GGA1 protein is composed of 3 domains as shown in the gray box. The GAT domain of GGA1 and the complex between the N-terminal of the GAT domain and ArF, as shown by the red dotted circle, has been solved this time.
High magnification (42 KB)




Figure 2.
These pictures are for a stereo-vision of GAT domain of GGA1 (a) and complex of the N-terminal region of the GAT domain and ARF1 (b). Use your left eye for the left picture, right eye for the right picture and try to merge them together. This will give you a 3-D view of the protein. If you don't succeed, please print the PDF file and then do the same thing.
When unbound, the GAT domain is composed of 3 alpha helices and the N-terminal domain is thought to have no fixed structure. In the complex with ARF1, the N-terminal region of the GAT domain forms the structure of 2 alpha helices and interacts with ARF1.
High magnification (48 KB)




Figure 3
The comparison of the GAT domain structures of GGA1 solved by 3 groups independently at almost the same time. Blue indicates the structure by the KEK group, green by the group of Cambridge University, the UK, and red by the NIH group, the US. Except for the dot circled part, all 3 structures are almost identical.
High magnification (21 KB)




Figure 4
Interaction of the GAT domain and ARF1 when you look down from above the membrane. The ARF1 molecule is colored as follows: surface, translucent gray; the side chains of amino acids that are interacting, light blue; GAT domain, red; the side chains of amino acid residue involved in the interaction, yellow. This describes that the GAT domain recognizes bumps and dips of the ARF1 very well.
High magnification (51 KB)

For further information
Photon Factory (PF) Homepage
http://pfwww.kek.jp/eng/

Related articles
Carrier Protein in Cells -Capturing the moment of taking cargo-
Investigating Proteins by X-ray -Synchrotron radiation will open the way for structural biology-
Ear of Carrier Protein -Determination of 3-D structure-
Grand Project of Protein Research -Three thousand kinds of proteins in 5 years-
Strategy of Natural Protein -Its 3-D structure-
Research of Protein Structure -New purpose-built beam line-

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