**4. Acknowledgment**

This work was supported by Grants-in-Aid for Scientific Research on Priority Areas and Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

#### **5. References**

344 Current Trends in X-Ray Crystallography

prepared and used for single anomalous dispersion (SAD) phasing. The crystal structure of Atg16(50-123) is composed of one α-helix of 90 Å length (Fujioka et al., 2010). The asymmetric unit contains six Atg16(50-123) molecules, which form three similar parallel coiled-coil dimers. These coiled-coil dimers expose two acidic residues (Asp101 and Glu102). By replacing these acidic residues with alanine, we succeeded in obtaining good crystals of full-length Atg16 and determined the structure by the molecular replacement method (Fujioka et al., 2010). The crystal structure of full-length Atg16 (D101A+E102A) is composed of one α-helix of 130 Å length. The structure lacked the electron density of the Nterminal region (residues 1-54), suggesting that the N-terminal region has various orientations relative to the C-terminal region. The asymmetric unit contained two Atg16 molecules, which formed a parallel coiled-coil dimer similar to the Atg16(50-123) dimer (Figure 6B). In both crystals, various interactions were observed between the coiled-coil dimers. In order to establish the oligomerization state of Atg16 in solution, we performed analytical ultracentrifugation experiments, which showed that Atg16 exists as a homodimer in solution (Fujioka et al., 2010) and that the Atg5-Atg16 complex exists as a 2:2 heterotetramer in solution. These data are consistent with the coiled-coil dimer structure of Atg16 observed in both crystals, suggesting that Atg16 forms a dimer but not a tetramer in

The obtained structural and biochemical information suggested that the overall architecture of the Atg5-Atg16 complex is composed of two sets of the N-terminal short α-helix of Atg16 bound to Atg5, the C-terminal parallel coiled-coil homodimer of Atg16, and flexible linkers connecting them, resulting in a 2:2 complex. Since Atg12 is conjugated to Lys149 of each Atg5, the Atg12―Atg5-Atg16 complex forms a 2:2:2 complex. The overall architecture of the Atg12―Atg5-Atg16 complex, which is quite unique compared with any structure-reported proteins including other E3 enzymes, will be one basis for studying the molecular functions

Obtaining good crystals is the rate-limiting step of X-ray crystallography. We have no versatile method for obtaining good crystals and thus have attempted the crystallization of each protein through trial and error. However, accumulated experience will increase the probability of obtaining good crystals and accelerate structural studies. We started a comprehensive structural study of Atg proteins about ten years ago, and have already obtained good crystals for more than ten Atg proteins that include not only those involved in conjugation reactions but also those involved in the construction of the preautophagosomal structure (Suzuki and Ohsumi, 2010). Continuous trials will surely provide us with good crystals for all the Atg proteins, and through the comprehensive structural study of these protein crystals, the molecular mechanism of autophagy will be established in

This work was supported by Grants-in-Aid for Scientific Research on Priority Areas and Targeted Proteins Research Program from the Ministry of Education, Culture, Sports,

solution.

**3. Conclusion** 

the near future.

**4. Acknowledgment** 

Science and Technology of Japan.

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**15** 

*1Italy 2Germany* 

**Knowledge Based Membrane Protein Structure** 

**Bioinformatics and Back to Molecular Biology** 

Life, or at least as we know it, would not exist without the ability of living organisms to communicate with their surroundings and respond to changes within them. Cells are able to capture and decode environmental stimuli into biologically signals. Indeed, communicating mechanisms, able to detect stimuli coming from the outside world are of fundamental importance for the survival of living beings. A deep understanding of the molecular mechanisms underlying signal transduction is thus needed for a complete characterization

Living cells are surrounded by a plasma membrane that forms a boundary between the cell interior and the external physical world. As a consequence, the cellular plasma membrane presents a major target for environmental stimuli acting upon a living cell. The membrane

Integral membrane proteins play a key role in detecting and conveying outside signals into cells, allowing them to interact and respond to their environment in a specific manner. They are involved as main players in several signaling pathways and therefore, the majority of drug targets are associated to the cell's membrane. The original human genome sequence project estimated 20% of the total gene count of 31,778 genes to code for membrane proteins (Almen et al., 2009). Thus membrane proteins constitute a very large set of yet-to becharacterized proteins mediating all the relevant life-related functions both in prokaryotes and eukaryotes. Moreover, the total amount of membrane proteins for which the threedimensional structure is known, is just about 842, corresponding to 298 unique proteins, as included in the Membrane Proteins with Known Structure database

This extremely low number of membrane proteins with known structure is due to the fact that membrane proteins are very difficult to study because they are inserted into lipid bilayers surrounding the cell and its sub-compartments. In these conditions they expose to the polar outer and inner environments portions of different sizes, completely changing the biophysics with respect to soluble proteins. Thus, when isolated from membranes, membrane proteins are

of the way our cells communicate with the rest of the world.

contains protein molecules that confer various functions on it.

(http://blanco.biomol.uci.edu/mpstruc/listAll/list).

**1. Introduction** 

**Prediction: From X-Ray Crystallography to** 

*1Applied Bioinformatics Group, Dept. of Biotechnology, University of Verona, 2German Research School for Simulation Sciences, Jülich Research Center and* 

Alejandro Giorgetti1,2 and Stefano Piccoli1

*RWTH-Aachen University, Jülich* 

carrier protein (E2) enzyme that mediates Atg8 lipidation. *J Biol Chem*, Vol.282, No.11, Mar 16, pp. 8036-8043, 0021-9258

Yamaguchi, M., Noda, N.N., Nakatogawa, H., Kumeta, H., Ohsumi, Y. & Inagaki, F. (2010). Autophagy-related protein 8 (Atg8) family interacting motif in Atg3 mediates the Atg3-Atg8 interaction and is crucial for the cytoplasm-to-vacuole targeting pathway. *J Biol Chem*, Vol.285, No.38, Sep 17, pp. 29599-29607, 0021-9258
