Background: HLTF is responsible for template-switching of DNA damage tolerance; HLTF has a novel DNA-binding HIRAN domain, but its function is unknown. Results: The structure of HIRAN domain bound to DNA reveals that the domain recognizes 3Ј-end of DNA. Conclusion: HLTF is recruited to a damaged site via interaction of the HIRAN domain with 3Ј-end. Significance: The structure provides a structural basis for the mechanism of template-switching.

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Membrane-bound proteases play several important roles in protein quality control and regulation. In the genome of the hyperthermophilic archaebacterium Pyrococcus horikoshii, the open reading frames PH1510 and PH1511 are homologous to the genes nfed (nodulation formation efficiency D) and stomatin, respectively, and probably form an operon. The nfed proteins are putative membrane proteins, and the N-terminal region shows homology to ClpP-type serine proteases. Stomatin is one of the major integral membrane proteins of human erythrocytes, and its absence is associated with the hemolytic anemia known as hereditary stomatocytosis. Thus, the N-terminal region of PH1510 (1510-N, residues 16 -236) was expressed and purified. From activity staining and SDS-PAGE analysis using fluorescein isothiocyanate-casein, 1510-N was identified as a thermostable endo-type protease. From site-directed mutagenesis, the conserved Ser-97 and Lys-138 are involved in proteolysis and, therefore, PH1510 is probably a serine protease with a catalytic Ser-Lys dyad. The sites of cleavage by 1510-N are rich in hydrophobic residues. The site P1 (position ؊1 relative to the cleavage site) is mainly leucine. P4 and P4 are mainly hydrophobic residues. Interestingly, the 1510-N protease cleaves the Cterminal hydrophobic region of PH1511. From this result and the probability of an operon, PH1510 probably functions in cooperation with PH1511. It is hypothesized that the cleavage of the stomatin-homolog PH1511 by the PH1510 protease causes an ion channel to open.

Kinesin spindle protein Eg5 is a target for anticancer therapies, and small molecule inhibitors of its ATPase activity have been developed. We herein report for the first time the crystal structure of and biochemical studies on the Eg5 motor domain in complex with a new type of allosteric inhibitor. The biphenyl-type inhibitor PVZB1194 binds to the α4/α6 allosteric pocket 15 Å from the ATP-binding pocket, which differs from conventional allosteric inhibitors that bind to the allosteric L5/α2/α3 pocket of Eg5. Binding of the inhibitor is involved in the neck-linker conformation and also causes conformational changes around the ATP-binding pocket through Tyr104 to affect the interaction of ATP with the pocket. This structure provides useful information for the development of novel types of allosteric drugs as well as a novel insight into the molecular mechanism responsible for regulating the motor activity of kinesins.

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