A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry [preprint]

Tailed bacteriophage use a DNA packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component acts as a molecular matchmaker by recognizing the viral genome as well as the main motor component, the large terminase (TerL). How TerS binds DNA and the TerL protein remains unclear. Here, we identify the TerS protein of the thermophilic bacteriophage P74-26. TerSP76-26 oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. Our cryo-EM structure shows that TerSP76-26 forms a ring with a wide central pore and radially arrayed helix-turn-helix (HTH) domains. These HTH domains, which are thought to bind DNA by wrapping the helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA binding domain imposes strong constraints on how TerSP76-26 can bind DNA. Finally, the TerSP76-26 structure lacks the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL, suggesting that a well-ordered C-terminal β-barrel domain is not necessary for TerS to carry out its function as a matchmaker

Structure of the human clamp loader bound to the sliding clamp: a further twist on AAA+ mechanism [preprint]

DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be actively opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader Replication Factor C (RFC) and sliding clamp PCNA are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryo-EM to an overall resolution of ~3.4 Å. The active sites of RFC are fully bound to ATP analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation prior to PCNA opening, with the clamp loader ATPase modules forming an over-twisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a ‘Limited Change/Induced Fit’ mechanism in which the clamp first opens, followed by DNA binding inducing opening of the loader to release auto-inhibition. The proposed change from an over-twisted to an active conformation reveals a novel regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health

Effective mismatch repair depends on timely control of PCNA retention on DNA by the Elg1 complex

ACKNOWLEDGEMENTS We thank Richard Kolodner and Eric Alani for strains and plasmids. We thank Anne Donaldson, Alexander Lorenz and Catherine Johnson from University of Aberdeen for careful reading of the manuscript. We thank Annabelle Duff and Veronika Petrova for assisting with the mutation rate assays, and Duru Cosar for assisting with crystal structure analysis. We appreciate assistance from staff of the Microscopy and Histology Core Facility and the qPCR facility at the University of Aberdeen. FUNDING Medical Research Council (MRC) Career Development Fellowship [L019698/1 to T.K.]; American Cancer Society Research Scholar Award [Grant #440685 to B.A.K.]; National Institute of General Medical Sciences [R01 GM127776 to B.A.K.]; National Institutes of Health grant [R01 GM106060 to V.L.]. Swiss National Science Foundation Postdoc Mobility Fellowship (to C.G.). Funding for open access charge: Medical Research Council via University of Aberdeen Open Access Fund.Peer reviewedPublisher PD

CaMKII binds both substrates and activators at the active site [preprint]

Ca2+/calmodulin dependent protein kinase II (CaMKII) is a signaling protein that is required for long-term memory formation. Ca2+/CaM activates CaMKII by binding to its regulatory segment, thereby freeing the substrate binding site. Despite having a large variety of interaction partners, the specificity of CaMKII interactions have not been structurally well-characterized. One exceptional feature of this kinase is that interaction with specific binding partners persistently activates CaMKII. To address the molecular details of this, we solved X-ray crystal structures of the CaMKII kinase domain bound to four different binding partners that modulate CaMKII activity in different ways. We show that all four partners bind in the same manner across the substrate binding site. We generated a sequence alignment based on our structural observations, which revealed conserved interactions. Using biochemistry and molecular dynamics simulations, we propose a mechanistic model that persistent CaMKII activity is facilitated by high affinity binding partners, which compete with the regulatory segment to allow substrate phosphorylation

Structural and functional characterization of rapamycin-resistant TORC2

TORC2 is nucleated by Target of Rapamycin (TOR), a protein kinase of the phosphatidylinositol 3-kinase-related kinase (PIKK) family. TOR kinases are validated drug targets which can be inhibited by rapamycin, facilitating elucidation of TORC1 signaling. TORC2 cannot be inhibited by rapamycin, and the lack of specific inhibitors has impeded progress in understanding the functions of this essential complex. The EM reconstruction revealed a rhomboid shape with C2 pseudo-symmetry and a prominent central cavity. We found that the TORC2-specific subunit Avo3 is proximal to the rapamycin binding domain of Tor2. Building on this observation, we successfully engineered a yeast strain in which TORC2, but not TORC1, is inhibited by rapamycin. We leveraged this unique tool to study TORC2 function and regulation, demonstrating that acute TORC2 inhibition abolishes actin polarization and leads to cell-cycle arrest

Amino acid signaling in high definition

In this issue of Structure, Zhang and colleagues present the structure of the Ego3 dimer, demonstrating that dimerization is an obligate prerequisite in amino acid-induced TORC1 activation

TORC2 Structure and Function

The target of rapamycin (TOR) kinase functions in two multiprotein complexes, TORC1 and TORC2. Although both complexes are evolutionarily conserved, only TORC1 is acutely inhibited by rapamycin. Consequently, only TORC1 signaling is relatively well understood; and, at present, only mammalian TORC1 is a validated drug target, pursued in immunosuppression and oncology. However, the knowledge void surrounding TORC2 is dissipating. Acute inhibition of TORC2 with small molecules is now possible and structural studies of both TORC1 and TORC2 have recently been reported. Here we review these recent advances as well as observations made from tissue-specific mTORC2 knockout mice. Together these studies help define TORC2 structure-function relationships and suggest that mammalian TORC2 may one day also become a bona fide clinical target

Cloning, expression, purification, and characterisation of the HEAT-repeat domain of TOR from the thermophilic eukaryote Chaetomium thermophilum

International audienc

Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2

The target of rapamycin (TOR) kinase assembles into two distinct multiprotein complexes, conserved across eukaryote evolution. In contrast to TOR complex 1 (TORC1), TORC2 kinase activity is not inhibited by the macrolide rapamycin. Here, we present the structure of Saccharomyces cerevisiae TORC2 determined by electron cryo-microscopy. TORC2 contains six subunits assembling into a 1.4 MDa rhombohedron. Tor2 and Lst8 form the common core of both TOR complexes. Avo3/Rictor is unique to TORC2, but interacts with the same HEAT repeats of Tor2 that are engaged by Kog1/Raptor in mammalian TORC1, explaining the mutual exclusivity of these two proteins. Density, which we conclude is Avo3, occludes the FKBP12-rapamycin-binding site of Tor2's FRB domain rendering TORC2 rapamycin insensitive and recessing the kinase active site. Although mobile, Avo1/hSin1 further restricts access to the active site as its conserved-region-in-the-middle (CRIM) domain is positioned along an edge of the TORC2 active-site-cleft, consistent with a role for CRIM in substrate recruitment