Polo-like kinases (PLK) are eukaryotic regulators of cell cycle progression, mitosis and cytokinesis; PLK4 is a master regulator of centriole duplication. Here, we demonstrate that the SCL/TAL1 interrupting locus (STIL) protein interacts via its coiled-coil region (STIL-CC) with PLK4 in vivo. STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region. Structure determination of free PLK4-PB3 and its STIL-CC complex via NMR and crystallography reveals a novel mode of Polo-box-peptide interaction mimicking coiled-coil formation. In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding. We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.
- Jon Pines, The Gurdon Institute, United Kingdom
© 2015, Arquint et al.
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Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers connected via a [4Fe–4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron–sulfur cluster domain: a ‘closed bridge’ and an ‘open bridge’ conformation, where a Zn2+ site may act as a ‘hinge’ allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.
Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.