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ShanghaiTech Researchers Discover a New Electron Transfer Mechanism from a Mycobacterial Respiratory Supercomplex

On October 25, 2018, the Science published online the research article from the team led by ShanghaiTech Distinguished Professor-in-Residence Rao Zihe with the title ¡°An electron transfer path connects subunits of a mycobacterial respiratory supercomplex¡±.

On the strength of cryo-electron microscopy (cryo-EM) with high resolution (3.5 Å) structure of mycobacterial respiratory supercomplex, the team revealed a new electron transfer mechanism coupling quinone oxidation with oxygen reduction. Furthermore, superoxide dismutase (SOD) was discovered, for the first time, directly involved in the assembly of the supercomplex and work in concert. These new findings have laid a solid groundwork for the R&D of new drugs against tuberculosis that pose grave threats on human health.

The 3.5-Å resolution cryo-EM structure of mycobacterial respiratory supercomplex CIII2CIV2SOD2

Respiration is one of the most basic energy metabolisms in life, and helps energetic transform from the substances (e.g. sugar, amino acid and fatty acid) into the adenosine triphosphate (ATP) that could be immediately consumed by the life body. It is run mainly by five large transmembrane complexes on the microbial cytoplasmic membrane or the inner mitochondrial membrane: complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (quinone: cytochrome c oxidordeuctase), complex IV (cytochrome c oxidase) and complex V (ATP synthase), together with two carriers for electron transfer, quinone and cytochrome c, which therefore is called the respiratory chain. Among them, electron transfers inside and between the complexes I-IV through cascading with redox reaction, thus forming the electron transfer chain, coupling which the transmembrane proton gradient is created for driving the synthesis of ATP by complex V. Previous studies have shown that the components of respiratory chain could be further assembled into supercomplexes to promote tandem reaction among them. This is of great significance in the regulation of energy metabolic efficiency and various physiological processes. The disorder in supercomplex assembly would be closely related to the occurrence of diseases in higher animals; when interrupted in microorganisms, it would be an important strategy for developing new drugs to inhibit their growth and infection.

Respiratory chain of Actinobacteria, a phylum containing Mycobacteria

¡°It is common for the assembly of complex III and complex IV to form a supercomplex, which is particularly important in energy production for Actinobacteria, a phylum containing Mycobacterium tuberculosis and multiple pathogenic bacteria. On the other hand, there were no extensive and direct interactions between complex III and complex IV in the previous reported structures including mammalian respiratory supercomplex I1III2IV1. Meanwhile, the cytochrome c protein in mitochondria exists in free soluble form to mediate electron transfer between complex III and complex IV. So, it is still questionable on whether it shuttles inside the supercomplex or across multiple supercomplexes. In this work, we have revealed the whole electron transfer path from complex III to complex IV and the tandem reaction mechanism for the two complexes. Such revelation answers one of the widely concerned scientific questions that has long been unresolved in the respiratory field, following our first report of the crystal structure of the eukaryotic mitochondrial complex II in the Cell in 2005,¡± said Professor Rao.

As explained by Gong Hongri and Xu Ao, doctoral students of the Nankai University and co-first authors of this article, success was the fruit from years of preparation and technical accumulation by the team in the study of Mycobacterium tuberculosis. Their close cooperation runs from the purification and optimization of samples to activity validation, from preparation of cryo-EM samples to improvement of data processing methods and eventually to the resolve of near-atomic resolution structural model. Such cooperation coupled with harmonized efforts in multiple biochemical and biophysical methods, such as lipidomics analysis, atomic absorption spectrometry and electron paramagnetic resonance test, empowered the researchers to gain comprehensive intimate knowledge of the whole electron transfer path inside this particular supercomplex.

¡°Another unexpected discovery is superoxide dismutase (SOD). Despite in zymologic function it has long been regarded correlated with redox reaction of the respiratory chain by radicals scavenging and also with the host¡¯s immunoreactions during microbial infection, direct evidence is still absent. Additionally, the molecular mechanism of SOD involved in such connection is rather controversial. From the viewpoint of the structural biology for the first time, we confirmed the direct interaction in the mycobacterial periplasm between SOD and the respiratory chain complex as well as SOD¡¯s capability of scavenging the potential free radicals to drive redox reaction. Moreover, this finding implies an important mechanism of Actinomycetes represented by Mycobacterium tuberculosis to resist immunoreactions in the host¡¯s macrophage, thus bringing new revelation to further understanding of the interactions between Mycobacterium tuberculosis and its host,¡± said key members of the team Dr. Wang Quan and Research Associate Professor Li Jun.

SOD involved in the redox reaction inside mycobacterial respiratory supercomplex

The research bears great significance in the R&D of new drugs. The World Health Organization (WHO) reports that tuberculosis has become a leading infectious disease in the world. For decades, the long-term use of isoniazid, rifampicin and other drugs has resulted in increasingly acute drug resistance. The multiple-drug resistance tuberculosis and even extremely drug-resistant tuberculosis have become one of the grave challenges in treating tuberculosis. Studies of recent years indicated that targeting energy metabolic system can remarkably address the drug resistance, which has been increasingly attracting attention. In 2012, the FDA in U.S. expedited approval of the Bedaquiline, the first drug treating the multiple-drug resistance tuberculosis and it gained access to China in March 2018. The working principle of this drug is to inhibit the respiratory chain system from synthesizing ATP to wipe out Mycobacterium tuberculosis.

¡°The complex III under our research is a highly popular drug target. The pharmaceutical molecule Telacebec (Q203) in the phase II clinical trials is just suppressing the binding of natural substrate of the complex to block the aerobic respiration pathway of Mycobacterium tuberculosis, thus to play its pharmacological role. Our research will serve as a great powerhouse for further improvement of this drug and development of even new drugs with better efficacy,¡± highlighted by Professor Rao.

Binding site of substrate (MK) and the potential drug in mycobacterial complex III

The research team led by Professor Rao Zihe has long been committed to the research on the structural biology of emerging and reemerging infectious disease pathogens in China. This article is the second one published in the Science this year following publication of the assembly mechanism of herpes virus earlier this year. Multiple institutions have been contributed to this research with ShanghaiTech University being one of the three key initiators. Research Associate Professor Li Jun of the Shanghai Institute for Advanced Immunochemical Studies is the co-first author of this article and Professor Rao Zihe is the co-corresponding author. Professor Jiang Biao, the doctoral student Wang Shuhui and Research Associate Professor Yang Xiuna have also contributed to this work. In addition, the National Center for Protein Science (Shanghai) provided some of technical support for this work.



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