Crystal structure and functional characterization of a light-driven chloride pump having an NTQ motif

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 Å resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 fromDictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

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Structural and functional characterization of an Isd-type haem-degradation enzyme fromListeria monocytogenes

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713030794 ◽

2014 ◽

Vol 70 (3) ◽

pp. 615-626 ◽

Cited By ~ 13

Author(s):

Thao Duong ◽

Kwangsu Park ◽

Truc Kim ◽

Sung Wook Kang ◽

Myung Joon Hahn ◽

...

Keyword(s):

Crystal Structure ◽

Degradation Product ◽

Functional Characterization ◽

Human Pathogen ◽

Monooxygenase Activity ◽

Gram Positive Bacteria ◽

Functional Assays ◽

Terminal Domain ◽

Life Threatening

Bacterial pathogens have evolved diverse types of efficient machinery to acquire haem, the most abundant source of iron in the human body, and degrade it for the utilization of iron. Gram-positive bacteria commonly encode IsdG-family proteins as haem-degrading monooxygenases.Listeria monocytogenesis predicted to possess an IsdG-type protein (Lmo2213), but the residues involved in haem monooxygenase activity are not well conserved and there is an extra N-terminal domain in Lmo2213. Therefore, its function and mechanism of action cannot be predicted. In this study, the crystal structure of Lmo2213 was determined at 1.75 Å resolution and its haem-binding and haem-degradation activities were confirmed. Structure-based mutational and functional assays of this protein, designated as an Isd-typeL. monocytogeneshaem-degrading enzyme (Isd-LmHde), identified that Glu71, Tyr87 and Trp129 play important roles in haem degradation and that the N-terminal domain is also critical for its haem-degrading activity. The haem-degradation product of Isd-LmHde is verified to be biliverdin, which is also known to be the degradation product of other bacterial haem oxygenases. This study, the first structural and functional report of the haem-degradation system inL. monocytogenes, sheds light on the concealed haem-utilization system in this life-threatening human pathogen.

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