Structural Mechanism for Cargo Recognition by the Retromer Complex.

Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.

Evolutionary conservation of the structure and function of meiotic Rec114-Mei4 and Mer2 complexes.

Meiosis-specific Rec114-Mei4 and Mer2 complexes are thought to enable Spo11-mediated DNA double-strand break (DSB) formation through a mechanism that involves DNA-dependent condensation. However, the structure, molecular properties, and evolutionary conservation of Rec114-Mei4 and Mer2 are unclear. Here, we present AlphaFold models of Rec114-Mei4 and Mer2 complexes supported by nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), and mutagenesis. We show that dimers composed of the Rec114 C terminus form α-helical chains that cup an N-terminal Mei4 α helix, and that Mer2 forms a parallel homotetrameric coiled coil. Both Rec114-Mei4 and Mer2 bind preferentially to branched DNA substrates, indicative of multivalent protein-DNA interactions. Indeed, the Rec114-Mei4 interaction domain contains two DNA-binding sites that point in opposite directions and drive condensation. The Mer2 coiled-coil domain bridges coaligned DNA duplexes, likely through extensive electrostatic interactions along the length of the coiled coil. Finally, we show that the structures of Rec114-Mei4 and Mer2 are conserved across eukaryotes, while DNA-binding properties vary significantly. This work provides insights into the mechanism whereby Rec114-Mei4 and Mer2 complexes promote the assembly of the meiotic DSB machinery and suggests a model in which Mer2 condensation is the essential driver of assembly, with the DNA-binding activity of Rec114-Mei4 playing a supportive role.

Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.

Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM.

The structure and regulation of human muscle α-actinin.

The spectrin superfamily of proteins plays key roles in assembling the actin cytoskeleton in various cell types, crosslinks actin filaments, and acts as scaffolds for the assembly of large protein complexes involved in structural integrity and mechanosensation, as well as cell signaling. α-actinins in particular are the major actin crosslinkers in muscle Z-disks, focal adhesions, and actin stress fibers. We report a complete high-resolution structure of the 200 kDa α-actinin-2 dimer from striated muscle and explore its functional implications on the biochemical and cellular level. The structure provides insight into the phosphoinositide-based mechanism controlling its interaction with sarcomeric proteins such as titin, lays a foundation for studying the impact of pathogenic mutations at molecular resolution, and is likely to be broadly relevant for the regulation of spectrin-like proteins.

Hsp90-Tau complex reveals molecular basis for specificity in chaperone action.

Protein folding in the cell relies on the orchestrated action of conserved families of molecular chaperones, the Hsp70 and Hsp90 systems. Hsp70 acts early and Hsp90 late in the folding path, yet the molecular basis of this timing is enigmatic, mainly because the substrate specificity of Hsp90 is poorly understood. Here, we obtained a structural model of Hsp90 in complex with its natural disease-associated substrate, the intrinsically disordered Tau protein. Hsp90 binds to a broad region in Tau that includes the aggregation-prone repeats. Complementarily, a 106-Å-long substrate-binding interface in Hsp90 enables many low-affinity contacts. This allows recognition of scattered hydrophobic residues in late folding intermediates that remain after early burial of the Hsp70 sites. Our model resolves the paradox of how Hsp90 specifically selects for late folding intermediates but also for some intrinsically disordered proteins-through the eyes of Hsp90 they look the same.

Mechanism of activation and regulation of deubiquitinase activity in MINDY1 and MINDY2.

Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.

The bacterial DnaC helicase loader is a DnaB ring breaker.

Dedicated AAA+ ATPases deposit hexameric ring-shaped helicases onto DNA to promote replication in cellular organisms. To understand how loading occurs, we used electron microscopy and small angle X-ray scattering (SAXS) to determine the ATP-bound structure of the intact E. coli DnaB⋅DnaC helicase/loader complex. The 480 kDa dodecamer forms a three-tiered assembly, in which DnaC adopts a spiral configuration that remodels N-terminal scaffolding and C-terminal motor regions of DnaB to produce a clear break in the helicase ring. Surprisingly, DnaC's AAA+ fold is dispensable for ring remodeling because the DnaC isolated helicase-binding domain can both load DnaB onto DNA and increase the efficiency by which the helicase acts on substrates in vitro. Our data demonstrate that DnaC opens DnaB by a mechanism akin to that of polymerase clamp loaders and indicate that bacterial replicative helicases, like their eukaryotic counterparts, possess autoregulatory elements that influence how hexameric motor domains are loaded onto and unwind DNA.

An unusual topological structure of the HIV-1 Rev response element.

Nuclear export of unspliced and singly spliced viral mRNA is a critical step in the HIV life cycle. The structural basis by which the virus selects its own mRNA among more abundant host cellular RNAs for export has been a mystery for more than 25 years. Here, we describe an unusual topological structure that the virus uses to recognize its own mRNA. The viral Rev response element (RRE) adopts an "A"-like structure in which the two legs constitute two tracks of binding sites for the viral Rev protein and position the two primary known Rev-binding sites ~55 Å apart, matching the distance between the two RNA-binding motifs in the Rev dimer. Both the legs of the "A" and the separation between them are required for optimal RRE function. This structure accounts for the specificity of Rev for the RRE and thus the specific recognition of the viral RNA.

Cooperative tertiary interaction network guides RNA folding.

Noncoding RNAs form unique 3D structures, which perform many regulatory functions. To understand how RNAs fold uniquely despite a small number of tertiary interaction motifs, we mutated the major tertiary interactions in a group I ribozyme by single-base substitutions. The resulting perturbations to the folding energy landscape were measured using SAXS, ribozyme activity, hydroxyl radical footprinting, and native PAGE. Double- and triple-mutant cycles show that most tertiary interactions have a small effect on the stability of the native state. Instead, the formation of core and peripheral structural motifs is cooperatively linked in near-native folding intermediates, and this cooperativity depends on the native helix orientation. The emergence of a cooperative interaction network at an early stage of folding suppresses nonnative structures and guides the search for the native state. We suggest that cooperativity in noncoding RNAs arose from natural selection of architectures conducive to forming a unique, stable fold.

RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling.

Ubiquitin-dependent signaling during the DNA damage response (DDR) to double-strand breaks (DSBs) is initiated by two E3 ligases, RNF8 and RNF168, targeting histone H2A and H2AX. RNF8 is the first ligase recruited to the damage site, and RNF168 follows RNF8-dependent ubiquitination. This suggests that RNF8 initiates H2A/H2AX ubiquitination with K63-linked ubiquitin chains and RNF168 extends them. Here, we show that RNF8 is inactive toward nucleosomal H2A, whereas RNF168 catalyzes the monoubiquitination of the histones specifically on K13-15. Structure-based mutagenesis of RNF8 and RNF168 RING domains shows that a charged residue determines whether nucleosomal proteins are recognized. We find that K63 ubiquitin chains are conjugated to RNF168-dependent H2A/H2AX monoubiquitination at K13-15 and not on K118-119. Using a mutant of RNF168 unable to target histones but still catalyzing ubiquitin chains at DSBs, we show that ubiquitin chains per se are insufficient for signaling, but RNF168 target ubiquitination is required for DDR.

The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.

Teneurin4 dimer structures reveal a calcium-stabilized compact conformation supporting homomeric trans-interactions.

Establishment of correct synaptic connections is a crucial step during neural circuitry formation. The Teneurin family of neuronal transmembrane proteins promotes cell-cell adhesion via homophilic and heterophilic interactions, and is required for synaptic partner matching in the visual and hippocampal systems in vertebrates. It remains unclear how individual Teneurins form macromolecular cis- and trans-synaptic protein complexes. Here, we present a 2.7 Å cryo-EM structure of the dimeric ectodomain of human Teneurin4. The structure reveals a compact conformation of the dimer, stabilized by interactions mediated by the C-rich, YD-shell, and ABD domains. A 1.5 Å crystal structure of the C-rich domain shows three conserved calcium binding sites, and thermal unfolding assays and SAXS-based rigid-body modeling demonstrate that the compactness and stability of Teneurin4 dimers are calcium-dependent. Teneurin4 dimers form a more extended conformation in conditions that lack calcium. Cellular assays reveal that the compact cis-dimer is compatible with homomeric trans-interactions. Together, these findings support a role for teneurins as a scaffold for macromolecular complex assembly and the establishment of cis- and trans-synaptic interactions to construct functional neuronal circuits.

Crystal structure and allosteric activation of protein kinase C ßII.

Protein kinase C (PKC) isozymes are the paradigmatic effectors of lipid signaling. PKCs translocate to cell membranes and are allosterically activated upon binding of the lipid diacylglycerol to their C1A and C1B domains. The crystal structure of full-length protein kinase C ßII was determined at 4.0 Å, revealing the conformation of an unexpected intermediate in the activation pathway. Here, the kinase active site is accessible to substrate, yet the conformation of the active site corresponds to a low-activity state because the ATP-binding side chain of Phe629 of the conserved NFD motif is displaced. The C1B domain clamps the NFD helix in a low-activity conformation, which is reversed upon membrane binding. A low-resolution solution structure of the closed conformation of PKCßII was derived from small-angle X-ray scattering. Together, these results show how PKCßII is allosterically regulated in two steps, with the second step defining a novel protein kinase regulatory mechanism.

The Mre11:Rad50 structure shows an ATP-dependent molecular clamp in DNA double-strand break repair.

The MR (Mre11 nuclease and Rad50 ABC ATPase) complex is an evolutionarily conserved sensor for DNA double-strand breaks, highly genotoxic lesions linked to cancer development. MR can recognize and process DNA ends even if they are blocked and misfolded. To reveal its mechanism, we determined the crystal structure of the catalytic head of Thermotoga maritima MR and analyzed ATP-dependent conformational changes. MR adopts an open form with a central Mre11 nuclease dimer and two peripheral Rad50 molecules, a form suited for sensing obstructed breaks. The Mre11 C-terminal helix-loop-helix domain binds Rad50 and attaches flexibly to the nuclease domain, enabling large conformational changes. ATP binding to the two Rad50 subunits induces a rotation of the Mre11 helix-loop-helix and Rad50 coiled-coil domains, creating a clamp conformation with increased DNA-binding activity. The results suggest that MR is an ATP-controlled transient molecular clamp at DNA double-strand breaks.

Pressure pushes tRNALys3 into excited conformational states.

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

Design, synthesis, and characterization of protein origami based on self-assembly of a brick and staple artificial protein pair.

A versatile strategy to create an inducible protein assembly with predefined geometry is demonstrated. The assembly is triggered by a binding protein that staples two identical protein bricks together in a predictable spatial conformation. The brick and staple proteins are designed for mutual directional affinity and engineered by directed evolution from a synthetic modular repeat protein library. As a proof of concept, this article reports on the spontaneous, extremely fast and quantitative self-assembly of two designed alpha-repeat (αRep) brick and staple proteins into macroscopic tubular superhelices at room temperature. Small-angle X-ray scattering (SAXS) and transmission electron microscopy (TEM with staining agent and cryoTEM) elucidate the resulting superhelical arrangement that precisely matches the a priori intended 3D assembly. The highly ordered, macroscopic biomolecular construction sustains temperatures as high as 75 °C thanks to the robust αRep building blocks. Since the α-helices of the brick and staple proteins are highly programmable, their design allows encoding the geometry and chemical surfaces of the final supramolecular protein architecture. This work opens routes toward the design and fabrication of multiscale protein origami with arbitrarily programmed shapes and chemical functions.

Conformational switching and flexibility in cobalamin-dependent methionine synthase studied by small-angle X-ray scattering and cryoelectron microscopy.

Cobalamin-dependent methionine synthase (MetH) catalyzes the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate (CH3-H4folate) using the unique chemistry of its cofactor. In doing so, MetH links the cycling of S-adenosylmethionine with the folate cycle in one-carbon metabolism. Extensive biochemical and structural studies on Escherichia coli MetH have shown that this flexible, multidomain enzyme adopts two major conformations to prevent a futile cycle of methionine production and consumption. However, as MetH is highly dynamic as well as both a photosensitive and oxygen-sensitive metalloenzyme, it poses special challenges for structural studies, and existing structures have necessarily come from a "divide and conquer" approach. In this study, we investigate E. coli MetH and a thermophilic homolog from Thermus filiformis using small-angle X-ray scattering (SAXS), single-particle cryoelectron microscopy (cryo-EM), and extensive analysis of the AlphaFold2 database to present a structural description of the full-length MetH in its entirety. Using SAXS, we describe a common resting-state conformation shared by both active and inactive oxidation states of MetH and the roles of CH3-H4folate and flavodoxin in initiating turnover and reactivation. By combining SAXS with a 3.6-Å cryo-EM structure of the T. filiformis MetH, we show that the resting-state conformation consists of a stable arrangement of the catalytic domains that is linked to a highly mobile reactivation domain. Finally, by combining AlphaFold2-guided sequence analysis and our experimental findings, we propose a general model for functional switching in MetH.

The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation.

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.

Allostery and intrinsic disorder mediate transcription regulation by conditional cooperativity.

Regulation of the phd/doc toxin-antitoxin operon involves the toxin Doc as co- or derepressor depending on the ratio between Phd and Doc, a phenomenon known as conditional cooperativity. The mechanism underlying this observed behavior is not understood. Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites. The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain, illustrating allosteric coupling between highly disordered and highly unstable domains. This allosteric effect also couples Doc neutralization to the conditional regulation of transcription. In this way, higher levels of Doc tighten repression up to a point where the accumulation of toxin triggers the production of Phd to counteract its action. Our experiments provide the basis for understanding the mechanism of conditional cooperative regulation of transcription typical of toxin-antitoxin modules. This model may be applicable for the regulation of other biological systems.

Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC1.

Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.