Multistep loading of a DNA sliding clamp onto DNA by replication factor C.

The DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded (ss)/double-stranded DNA (dsDNA) junctions harboring a recessed 3' end (3' ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3' ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and raising the question of whether PCNA ring closure is mechanistically coupled to ATP hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.

Mechanisms of loading and release of the 9-1-1 checkpoint clamp.

Single-stranded or double-stranded DNA junctions with recessed 5' ends serve as loading sites for the checkpoint clamp, 9-1-1, which mediates activation of the apical checkpoint kinase, ATRMec1. However, the basis for 9-1-1's recruitment to 5' junctions is unclear. Here, we present structures of the yeast checkpoint clamp loader, Rad24-replication factor C (RFC), in complex with 9-1-1 and a 5' junction and in a post-ATP-hydrolysis state. Unexpectedly, 9-1-1 adopts both closed and planar open states in the presence of Rad24-RFC and DNA. Moreover, Rad24-RFC associates with the DNA junction in the opposite orientation of processivity clamp loaders with Rad24 exclusively coordinating the double-stranded region. ATP hydrolysis stimulates conformational changes in Rad24-RFC, leading to disengagement of DNA-loaded 9-1-1. Together, these structures explain 9-1-1's recruitment to 5' junctions and reveal new principles of sliding clamp loading.

Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1.

The chloride-proton exchanger CLC-7 plays critical roles in lysosomal homeostasis and bone regeneration and its mutation can lead to osteopetrosis, lysosomal storage disease and neurological disorders. In lysosomes and the ruffled border of osteoclasts, CLC-7 requires a ß-subunit, OSTM1, for stability and activity. Here, we present electron cryomicroscopy structures of CLC-7 in occluded states by itself and in complex with OSTM1, determined at resolutions up to 2.8 Å. In the complex, the luminal surface of CLC-7 is entirely covered by a dimer of the heavily glycosylated and disulfide-bonded OSTM1, which serves to protect CLC-7 from the degradative environment of the lysosomal lumen. OSTM1 binding does not induce large-scale rearrangements of CLC-7, but does have minor effects on the conformation of the ion-conduction pathway, potentially contributing to its regulatory role. These studies provide insights into the role of OSTM1 and serve as a foundation for understanding the mechanisms of CLC-7 regulation.

Inside the cells of mammals, acidic compartments called lysosomes are responsible for breaking down large molecules and worn-out cells parts so their components can be used again. Similar to lysosomes, specialized cells called osteoclasts require an acidic environment to degrade tissues in the bone. Both osteoclasts and lysosomes rely on a two-component protein complex to help them digest molecules. Mutations in the genes for both proteins are directly linked to human diseases including neurodegeneration and osteopetrosis ­ a disease characterized by dense and brittle bones. For the main protein in this complex, called CLC-7, to remain stable and perform its roles, it requires an accessory subunit known as OSTM1. CLC-7 is a transporter that funnels electrically charged particles into and out of the lysosome, which helps to maintain the environment inside the lysosome compartment. However, due to the tight partnership between CLC-7 and OTSM1, how they influence each other is poorly understood. To determine the roles of CLC-7 and OSTM1, Schrecker et al. looked at the structure of the complex using a technique called single particle electron microscopy, which allows proteins to be visualized almost down to the individual atom. The analysis revealed that OSTM1 covers almost the entire inside surface of CLC-7, protecting it from the acidic environment inside the lysosome and contributing to its stability. When the two subunits are bound together, OSTM1 also slightly changes the structure of the pore formed by CLC-7, suggesting that OSTM1 may regulate CLC-7 activity. Schrecker et al. have laid the foundation for understanding more about the activity and regulation of CLC-7 and OSTM1 in lysosomes and osteoclasts. The structures described also help explain previous findings, including why OSTM1 is important for the stability of CLC-7.

A channel profile report of the unusual K+ channel KtrB.

KtrAB is a key player in bacterial K+ uptake required for K+ homeostasis and osmoadaptation. The system is unique in structure and function. It consists of the K+-translocating channel subunit KtrB, which forms a dimer in the membrane, and the soluble regulatory subunit KtrA, which attaches to the cytoplasmic side of the dimer as an octameric ring conferring Na+ and ATP dependency to the system. Unlike most K+ channels, KtrB lacks the highly conserved T(X)GYG selectivity filter sequence. Instead, only a single glycine residue is found in each pore loop, which raises the question of how selective the ion channel is. Here, we characterized the KtrB subunit from the Gram-negative pathogen Vibrio alginolyticus by isothermal titration calorimetry, solid-supported membrane-based electrophysiology, whole-cell K+ uptake, and ACMA-based transport assays. We found that, despite its simple selectivity filter, KtrB selectively binds K+ with micromolar affinity. Rb+ and Cs+ bind with millimolar affinities. However, only K+ and the poorly binding Na+ are efficiently translocated, based on size exclusion by the gating loop. Importantly, the physiologically required K+ over Na+ selectivity is provided by the channel's high affinity for potassium, which interestingly results from the presence of the sodium ions themselves. In the presence of the KtrA subunit, sodium ions further decrease the Michaelis-Menten constant for K+ uptake from milli- to micromolar concentrations and increase the Vmax, suggesting that Na+ also facilitates channel gating. In conclusion, high binding affinity and facilitated K+ gating allow KtrAB to function as a selective K+ channel.

How RCK domains regulate gating of K+ channels.

Potassium channels play a crucial role in the physiology of all living organisms. They maintain the membrane potential and are involved in electrical signaling, pH homeostasis, cell-cell communication and survival under osmotic stress. Many prokaryotic potassium channels and members of the eukaryotic Slo channels are regulated by tethered cytoplasmic domains or associated soluble proteins, which belong to the family of regulator of potassium conductance (RCK). RCK domains and subunits form octameric rings, which control ion gating. For years, a common regulatory mechanism was suggested: ligand-induced conformational changes in the octameric ring would pull open a gate in the pore via flexible linkers. Consistently, ligand-dependent conformational changes were described for various RCK gating rings. Yet, recent structural and functional data of complete ion channels uncovered that the following signal transduction to the pore domains is divers. The different RCK-regulated ion channels show remarkably heterogeneous mechanisms with neither the connection from the RCK domain to the pore nor the gate being conserved. Some channels even lack the flexible linkers, while in others the gate cannot easily be assigned. In this review we compare available structures of RCK-gated potassium channels, highlight the similarities and differences of channel gating, and delineate existing inconsistencies.