Biology subjects

Klink, B. U.

Publications and source records attributed to Klink, B. U..

7 recordsLinked to original sources

bioRxiv2024

Structure and mechanism of the RalGAP tumor suppressor complex

The RalGAP (GTPase activating protein) complexes are negative regulators of the Ral GTPases and thus crucial components that counteract (oncogenic) Ras signaling. However, no structural information on the architecture of this tumor suppressor complex is available hampering a mechanistic understanding of its functionality. Here, we present a cryo-EM structure of RalGAP that reveals an extended 58 nm tetrameric architecture comprising two heterodimers of the RalGAP and RalGAP{beta} subunits. We show that the catalytic domain of RalGAP requires stabilization by a unique domain of RalGAP{beta}, providing the molecular basis for why RalGAP complexes are obligatory heterodimers. Formation of RalGAP tetramers is not required for activity in vitro, but essential for function of the complex in vivo. Structural analysis of RalGAP subunit variants reported in cancer patients suggests effects on complex formation and thus functional relevance in tumor development, emphasizing the significance of the obtained structural information for medical research.

biochemistry↗

bioRxiv2024

Molecular mechanism of α-latrotoxin action

The potent neurotoxic venom of the black widow spider contains a cocktail of seven phylum-specific latrotoxins (LTXs), but only one, -LTX, targets vertebrates. This 130 kDa toxin binds to receptors at presynaptic nerve terminals and triggers a massive release of neurotransmitters. It is widely accepted that LTXs tetramerize and insert into the presynaptic membrane, thereby forming Ca2+-conductive pores, but the underlying mechanism remains poorly understood. LTXs are homologous and consist of an N-terminal region with three distinct domains, along with a C-terminal domain containing up to 22 consecutive ankyrin repeats. Here we report the first high resolution structures of the vertebrate-specific -LTX tetramer in its prepore and pore state. Our structures, in combination with AlphaFold2-based structural modeling and molecular dynamics simulations, reveal dramatic conformational changes in the N-terminal region of the complex. Four distinct helical bundles synchronously rearrange to progressively form a highly stable 15 nm cation-impermeable coiled-coil stalk. This stalk, in turn, positions an N-terminal pair of helices within the membrane, thereby enabling the assembly of a cation-permeable channel. Taken together, these data unveil a unique mechanism for membrane insertion and channel formation, characteristic of the LTX family, and provide the necessary framework for advancing novel therapeutics and biotechnological applications.

biochemistry↗

bioRxiv2022

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate

The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available. Here, we report cryoEM structures of Pex1/Pex6 from Saccharomyces cerevisiae, with an endogenous protein substrate trapped in the central pore of the second ring (D2). Pairs of Pex1/Pex6(D2) subdomains engage the substrate via a staircase of pore-1 loops with distinct properties. The first ring (D1) is catalytically inactive but undergoes significant conformational changes resulting in alternate widening and narrowing of its pore. These events are fueled by ATP hydrolysis in the D2 ring and disengagement of a "twin-seam" Pex1/Pex6(D2) heterodimer from the staircase. Mechanical forces are propagated in a unique manner along Pex1/Pex6 interfaces that are not available in homo-oligomeric AAA-ATPases. Our structural analysis reveals the mechanisms of how Pex1 and Pex6 coordinate to achieve substrate translocation.

molecular biology↗

bioRxiv2022

Structural basis of actin filament assembly and aging

The dynamic turnover of actin filaments (F-actin) controls cellular motility in eukaryotes and is coupled to changes in the F-actin nucleotide state. It remains unclear how F-actin hydrolyzes ATP and subsequently undergoes subtle conformational rearrangements that ultimately lead to filament depolymerization by actin-binding proteins. Here, we present cryo-EM structures of F-actin in all nucleotide states, polymerized in the presence of Mg2+ or Ca2+, at resolutions ([~]2.2 [A]) that allow for the visualization of hundreds of water molecules. The structures reveal that the G- to F-actin transition induces the relocation of water molecules in the nucleotide binding pocket, activating one of them for the nucleophilic attack of ATP. Unexpectedly, the back door for the subsequent release of inorganic phosphate (Pi) is closed in all structures, indicating that the F-actin conformation that allows for Pi release occurs transiently. The small changes in the nucleotide-binding pocket after ATP hydrolysis and Pi release are sensed by a key amino acid, amplified and transmitted to the filament periphery. Furthermore, differences in the positions of waters in the nucleotide binding pocket explain why Ca2+-actin exhibits slower polymerization rates than Mg2+-actin. Our work elucidates the solvent-driven rearrangements that govern actin filament assembly and aging and lays the foundation for the rational design of drugs and small molecules for imaging and therapeutic applications.

biochemistry↗

bioRxiv2021

Structure of the human FERRY Rab5 effector complex

Long-range mRNA transport is crucial for the spatio-temporal regulation of gene expression, and its malfunction leads to neurological disorders. The pentameric FERRY Rab5 effector complex is the molecular link between mRNA and early endosomes in mRNA intracellular distribution. Here, we determine the cryo-EM structure of the human FERRY complex, composed of Fy-1 to Fy-5. The structure reveals a clamp-like architecture, where two arm-like appendages of Fy-2 and a Fy-5 dimer, protrude from the central Fy-4 dimer. The coiled-coil domains of Fy-2 are flexible and project into opposite directions from the core. While the Fy-2 C-terminal coiled-coil acts as binding region for Fy-1/3 and Rab5, both coiled-coils and Fy-5 concur to bind mRNA. Mutations causing truncations of Fy-2 in patients with neurological disorders impair Rab5 binding or FERRY complex assembly. Thus, Fy-2 serves as a binding hub connecting all five complex subunits and mediating the binding to mRNA and early endosomes via Rab5. The FERRY structure provides novel mechanistic insights into long-distance mRNA transport.

biochemistry↗

bioRxiv2019

Towards the molecular architecture of the peroxisomal receptor docking complex

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo. The mechanistic principles are however unclear, mainly because structures of higher order assemblies are still lacking. Here, using an integrative approach, we provide the first structural characterization of the major components of the peroxisomal docking complex Pex14p/Pex17p, in a native bilayer environment and reveal its subunit organization. Our data show that three copies of Pex14p and a single copy of Pex17p assemble to form a 20 nm rod-like particle. The different subunits are arranged in a parallel manner, showing interactions along their complete sequences and providing receptor binding-sites on both membrane sides. The long rod facing the cytosol is mainly formed by the predicted coiled-coil domains of Pex14p and Pex17p, possibly providing the necessary structural support for the formation of the import pore. Further implications of Pex14p/Pex17p for formation of the peroxisomal translocon are discussed.

molecular biology↗

bioRxiv2019

Structure of the human BBSome core complex in the open conformation

The BBSome is a heterooctameric protein complex that plays a central role in primary cilia homeostasis. Its malfunction causes the severe ciliopathy Bardet-Biedl syndrome (BBS). The complex acts as a cargo adapter that recognizes signaling proteins such as GPCRs and links them to the intraflagellar transport machinery. The underlying mechanism is poorly understood. Here we present a high-resolution cryo-EM structure of a human heterohexameric core subcomplex of the BBSome. The structure reveals the architecture of the complex in atomic detail. It explains how the subunits interact with each other and how disease-causing mutations hamper this interaction. The complex adopts a conformation that is open for binding to membrane-associated GTPase Arl6 and a large positively charged patch likely strengthens the interaction with the membrane. A prominent negatively charged cleft at the center of the complex is likely involved in binding of positively charged signaling sequences of cargo proteins.

molecular biology↗