5 A 180 subunit complex of a lumazine synthase mutant violates quasi-equivalence in
5.2 Conclusions
are no similarities between the subunit interfaces among the 3 subunits within the icosahedral asymmetric unit (Figure 8 in paper V).
Modeling studies showed that by a slight expansion of the capsid, the widened LSAQ-IDEA pentamers can be fitted into a wild type T = 1 icosahedral capsid without serious clashes. Recent EM studies have confirmed that the wild-type enzyme fromB. subtilis forms an enlarged T = 1 icosahedral capsid with an outer diameter of 186 Å instead of 156 Å (L. Xing, unpublished results). Pentamers in this enlarged T = 1 capsid are also widened and their conformation is similar to that of the pentamers in the LSAQ-IDEA mutant. It is therefore suggested, that the expanded pentameric structure, observed in this study, may serve as a model for an alternative conformation of the wild type LS pentamer as it could also be formed during the catalytic process in the form of local conformational fluctuations (Figure 5-3).
Adopting the opened conformation, the product could be released. Furthermore, there would be no steric hindrance for a substrate or product analog, e.g. 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidine-dione, to bind to the more exposed pyrimidine binding site. By this process, sufficient binding energy could be gained, which could trigger the conformational rearrangement leading in a ligand-driven reaction to the reconstitution of the closed pentamer structure, as it is seen in the native T = 1 capsids.10, 163
Figure 5-3. A hypothetical model of the catalytic cycle, in which the widening of the pentamer may play a role in binding of the substrates (S1 and S2) and in releasing of the products (lumazine and / or the phosphate ion). Note that the phosphate ion could either bind back to the original binding site or be released (paper II).
In the large capsid, the pentameric building blocks are widened compared to that of the wild-type enzyme. The widened pentamers may serve as a model for an alternative conformation of the wild-type enzyme playing a role in catalysis. It is shown that the opened form of the pentamer would exert no steric hindrance to substrate binding or product release. The energy gained by binding of the substrate could trigger the conformational rearrangement from the open enzymatically inactive form of the pentamer structure to the closed enzymatically active form, in accordance with ligand-driven reconstitution of T = 1 capsids from 180 subunit capsids. 10, 163
Although 180 LSAQ-IDEA subunits are assembled into a large icosahedral capsid and the number of subunits fits to the theory of quasi-equivalence assembly with T = 3, contact surface analysis has revealed no similar interactions at the interface between subunits within the icosahedral asymmetric unit or subunits around the icosahedral 2-fold, 3-fold, 5-fold and the quasi 2-fold, 3-fold and 6-fold. It is therefore concluded that the LSAQ-IDEA mutant doesn’t follow the principles of quasi-equivalence in capsid assembly.
6 ACKNOWLEDGEMENTS
The entire work and every single piece of it are largely due to the expert help of my supervisors. I would like to thank:
Prof. Rudolf Ladenstein, for offering me the opportunity to work in the X-ray group, for guiding me through the entire project, for his endless support, constant
encouragement and kindest understanding.
Dr. Winfried Meining, for those valuable advices and fruitful discussions on both theoretical and practical issues, for sharing great ideas on the project and many other scientific topics also for the great company during the past years.
Thanks to all my collaborators:
Prof. Adelbert Bacher, Dr. Markus Fischer, Dr. Ilka Haase, Prof. Mark Cushman, Dr.
Dmitri I. Svergun, Dr. Petr V. Konarev, Prof. Holland Cheng, Dr. Li Xing, Josefina Nilsson and Wit Suphamungmee for their contributions, which were indispensable for the project.
Thanks to:
Dr. Assen Koumanov and Mikael Karlström for their enormous help on studying, working and living in Sweden and for their great friendship, which I have always been enjoying very much.
Current and former colleagues in X-ray group: Dr. Andrey Karshikoff, Dr. Bin Ren, Dr. Jordi Benach, Dr. Wei Liu, Dr. Ekaterina Morgunova, Gudrun Tibbelin, Dr. Petras Kundrotas, Linda Arnfors, Dr. Heiko Bönisch, Dr. Joyce Lebbink, Niklas Hellgren, Dr.
Stefan Knapp, Dr. Thijs Kaper and Dr. Ida Helene Steen for their help and pleasant company.
Colleagues in CSB: Prof. Kurt Berndt, Prof. Lenart Nilsson, Prof. Hans Hebert, Ana Caballero-Herrera, Dr. Jan Norberg, Dr. Jianxin Duan, Cesar Santiago, Tobias Elgán, Johan Sagemark, Dr. Philip Koeck, Dr. Yijing Xian, Lars Haag, Leif Bergman and other colleagues, whose help and company have been very important for me during that time.
I greatly appreciate:
The IT and service group for their great support, especially Erik Lundgren, for his expert help with computing at CSB.
The administration board, especially Kristina Bergholm and Ingwar Lennerfors for their help throughout these years.
My dearest friends in Sweden:
Jiang Wu, Kejun Li, Shujing Zhang, Li Lan, Roger Eriksson, Xin Ma, Yuli Cao, Yu Shi, Zhong He, Yaofeng Zhao, Jinjing Pei, Wei Jia, Yintong Xue, Yongtao Xue and Hui Zhang for friendship and supports.
I shall also thank Prof. Yonggeng Hou and Prof. Lipu Li who introduced me to the field of X-ray crystallography and have been encouraging me all the time.
I am deeply grateful to my wife, Hailin Wang, for her understanding, encouragement and love.
I appreciate my parents and brother, who have given me so much.
This work was supported by Karolinska Institutet, Södertörns Högskola and the Swedish Natural Science Research Council.
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