Structural Basis And Molecular Mechanism For Selective Inhibition Of Leishmanial GDP-Mannose Pyrophosphorylase

Leishmaniasis,an infectious disease caused by the protozoan parasite Leishmania infection,is mainly prevalent in tropics and subtropics.Nearly one million new cases of leishmaniasis and nearly 60,000 deaths from leishmaniasis are reported each year.To date,there is no available vaccine in clinical practice.The current therapies are limited to a poor selection of chemicals that demonstrate severe side-effects and drug resistance emergence.In this context,it is urgent to develop effective anti-leishmaniasis drugs with low toxicity.GDP-mannose pyrophosphorylase is an established therapeutic target for antileishmanial drug development.As structure-based drug design remains an important drug development strategy,lack of experimentally resolved structures of leishmanial GDP-MP has greatly impeded structure-based drug design.In this study,we aim to solve the structure and catalysis mechanism of GDP-MP from Leishmania donovani(LdGDP-MP).We employed the single-particle cryo-EM technique to investigate the high resolution structure of LdGDP-MP and its complexs with GTP or GDP-Man.We further revealed the molecular mechanism for catalysis and regulation of LdGDP-MP by structural analysis and biochemical experiments.These data will help the development of novel anti-leishmaniasis drugs based on the structure of GDP-MPsWe show here that LdGDP-MP exhibits a homohexameric structure,with each subsunit consisting of an N-terminal catalytic domain and a C-terminal organization domain.The N-terminal catalytic domain is a Rossmann-fold like domain,which contains a complete active pocket.We identified the key elements involved in catalysis,and subsequently revealed the molecular mechanism for catalysis of LdGDP-MP by a series of biochemical experiments.The C-terminal organization domain is a left-handedβ-helix domain.In the LdGDP-MP hexamer,each subunit is pinched by two neighboring subunits through two interacting regions established by C-terminal organization domains,namely,interface-1 and interface-2.Structural analysis shows that interface-1 stabilizes the conformation of signature motif which are necessary for catalysis.Disruption of the interface-1 contacts led to significant loss in activity and instability of the hexameric quaternary organization.These data in combination suggest that the hexameric quaternary structure is essential for the biological activity of LdGDP-MP,and the interface-1 plays a critical role in activity regulation of LdGDPMP.In addition,we show that although GDP-MPs from various species demonstrate distinct quaternary structures,the monomer structure of GDP-MPs,especially the catalytic active centers,are highly conserved during evolution.We further propose an evolutionary route in terms of regulatory machinery for this class of enzymes.In conclusion,we solved the atomic-resolution structures of leishmanial GDP-MP,and elucidated the catalytic mechanism.In particular,our studies highlight a previously uncharacterized interprotomer regulatory machinery that is unique to eukaryotic GDPMPs,and provide insights into a stepwise scenario that how the regulation machinery for this class of enzyme has evolutionarily advanced.We anticipate these structural insights,together with the regulatory machinery unveiled here,will help to enable structure-based drug development.