The Hsp90 chaperone is an ATPase enzyme composed of two copies of a three-domain subunit. Hsp90 stabilizes and activates a diverse array of regulatory proteins. Substrates are bound and released by the middle domain through a clamping cycle involving conformational transitions between a dynamic open state and a compact conformationally restricted closed state. Intriguingly, the overall ATPase activity of dimeric Hsp90 can be asymmetrically enhanced through a single subunit when Hsp90 is bound to a cochaperone or when Hsp90 is composed of one active and one catalytically defunct subunit as a heterodimer. To explore the mechanism of asymmetric Hsp90 activation, we designed a subunit bearing N-terminal ATPase mutations that demonstrate increased intra- and interdomain dynamics. Using intact Hsp90 and various N-terminal and middle domain constructs, we blended 19F NMR spectroscopy, molecular dynamics (MD) simulations, and ATPase assays to show that within the context of heterodimeric Hsp90, the conformationally dynamic subunit stimulates the ATPase activity of the normal subunit. The contrasting dynamic properties of the subunits within heterodimeric Hsp90 provide a mechanistic framework to understand the molecular basis for asymmetric Hsp90 activation and its importance for the biological function of Hsp90.

Histidine kinases are key bacterial sensors that recognize diverse environmental stimuli. While mechanisms of phosphorylation and phosphotransfer by cytoplasmic kinase domains are relatively well-characterized, the ways in which extracytoplasmic sensor domains regulate activation remain mysterious. The Cpx envelope stress response is a conserved Gram-negative two-component system which is controlled by the sensor kinase CpxA. We report the structure of the Escherichia coli CpxA sensor domain (CpxA-SD) as a globular Per-ARNT-Sim (PAS)-like fold highly similar to that of Vibrio parahaemolyticus CpxA as determined by X-ray crystallography. Because sensor kinase dimerization is important for signaling, we used AlphaFold2 to model CpxA-SD in the context of its connected transmembrane domains, which yielded a novel dimer of PAS domains possessing a distinct dimer organization compared to previously characterized sensor domains. Gain of function cpxA* alleles map to the dimer interface, and mutation of other residues in this region also leads to constitutive activation. CpxA activation can be suppressed by mutations that restore inter-monomer interactions, suggesting that inhibitory interactions between CpxA-SD monomers are the major point of control for CpxA activation and signaling. Searching through hundreds of structural homologues revealed the sensor domain of Pseudomonas aeruginosa sensor kinase PfeS as the only PAS structure in the same novel dimer orientation as CpxA, suggesting that our dimer orientation may be utilized by other extracytoplasmic PAS domains. Overall, our findings provide insight into the diversity of the organization of PAS sensory domains and how they regulate sensor kinase activation.

The ProQ/FinO family of RNA binding proteins mediate sRNA-directed gene regulation throughout gram-negative bacteria. Here, we investigate the structural basis for RNA recognition by ProQ/FinO proteins, through the crystal structure of the ProQ/FinO domain of the Legionella pneumophila DNA uptake regulator, RocC, bound to the transcriptional terminator of its primary partner, the sRNA RocR. The structure reveals specific recognition of the 3' nucleotide of the terminator by a conserved pocket involving a β-turn-α-helix motif, while the hairpin portion of the terminator is recognized by a conserved α-helical N-cap motif. Structure-guided mutagenesis reveals key RNA contact residues that are critical for RocC/RocR to repress the uptake of environmental DNA in L. pneumophila. Structural analysis and RNA binding studies reveal that other ProQ/FinO domains also recognize related transcriptional terminators with different specificities for the length of the 3' ssRNA tail.

MDC1 is a key mediator of DNA-damage signaling. When DNA double-strand breaks (DSB) occur, the histone variant H2AX on the nucleosome is phosphorylated on its C-terminus at residue Ser139 to form the γH2AX nucleosome. This phosphorylated form is specifically recognized by the tandem BRCT repeats of MDC1. The MDC1-bound nucleosome serves as a docking platform to promote the localization of other DNA repair factors. To further characterize the nucleosome-BRCT interaction, we developed a time efficient two-step modified native chemical ligation protocol to prepare phosphorylated nucleosomes. Our binding studies show that BRCT interacts with the nucleosome with a higher affinity than the phosphorylated peptide. Using cryogenic electron microscopy (cryo-EM), we obtained structures of the γH2AX nucleosome revealing the structural basis for nucleosome-nucleosome stacking promoted by interactions of the H4 N-terminal of one nucleosome with its stacked partner. In contrast, we show that binding of the MDC1 BRCT domain disrupts this stacking, suggesting that histone/DNA dynamics are integral to DNA damage signaling.

The FinO family of proteins constitutes a group of RNA chaperones that interacts with small RNAs (sRNAs) to regulate gene expression in many bacterial species. Here we describe detailed protocols for the biochemical analysis of the RNA chaperone activity of these proteins. Methods are described for preparation of RNA, RNA 5' end labeling with radioisotope and modified EMSA protocols to test the ability of these proteins to catalyze RNA strand exchange and RNA duplex formation.

ASPP (apoptosis-stimulating proteins of p53) proteins bind PP-1c (protein phosphatase 1) and regulate p53 impacting cancer cell growth and apoptosis. Here we determine the crystal structure of the oncogenic ASPP protein, iASPP, bound to PP-1c. The structure reveals a 1:1 complex that relies on interactions of the iASPP SILK and RVxF motifs with PP-1c, plus interactions of the PP-1c PxxPxR motif with the iASPP SH3 domain. Small-angle X-ray scattering analyses suggest that the crystal structure undergoes slow interconversion with more extended conformations in solution. We show that iASPP, and the tumor suppressor ASPP2, enhance the catalytic activity of PP-1c against the small-molecule substrate, pNPP as well as p53. The combined results suggest that PxxPxR binding to iASPP SH3 domain is critical for complex formation, and that the modular ASPP-PP-1c interface provides dynamic flexibility that enables functional binding and dephosphorylation of p53 and other diverse protein substrates.

Topoisomerase IIβ binding protein 1 (TopBP1) is a critical protein-protein interaction hub in DNA replication checkpoint control. It was proposed that TopBP1 BRCT5 interacts with Bloom syndrome helicase (BLM) to regulate genome stability through either phospho-Ser304 or phospho-Ser338 of BLM. Here we show that TopBP1 BRCT5 specifically interacts with the BLM region surrounding pSer304, not pSer338. Our crystal structure of TopBP1 BRCT4/5 bound to BLM reveals recognition of pSer304 by a conserved pSer-binding pocket, and interactions between a FVPP motif N-terminal to pSer304 and a hydrophobic groove on BRCT5. This interaction utilizes the same surface of BRCT5 that recognizes the DNA damage mediator, MDC1, however the binding orientations of MDC1 and BLM are reversed. While the MDC1 interactions are largely electrostatic, the interaction with BLM has higher affinity and relies on a mix of electrostatics and hydrophobicity. We suggest similar evolutionarily conserved interactions may govern interactions between TopBP1 and 53BP1.

Topoisomerase IIβ binding protein 1 (TopBP1) is a critical protein-protein interaction hub in DNA replication checkpoint control. It was proposed that TopBP1 BRCT5 interacts with Bloom syndrome helicase (BLM) to regulate genome stability through either phospho-Ser304 or phospho-Ser338 of BLM. Here we show that TopBP1 BRCT5 specifically interacts with the BLM region surrounding pSer304, not pSer338. Our crystal structure of TopBP1 BRCT4/5 bound to BLM reveals recognition of pSer304 by a conserved pSer-binding pocket, and interactions between an FVPP motif N-terminal to pSer304 and a hydrophobic groove on BRCT5. This interaction utilizes the same surface of BRCT5 that recognizes the DNA damage mediator, MDC1; however the binding orientations of MDC1 and BLM are reversed. While the MDC1 interactions are largely electrostatic, the interaction with BLM has higher affinity and relies on a mix of electrostatics and hydrophobicity. We suggest that similar evolutionarily conserved interactions may govern interactions between TopBP1 and 53BP1.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.

A highly conserved DNA uptake system allows many bacteria to actively import and integrate exogenous DNA. This process, called natural transformation, represents a major mechanism of horizontal gene transfer (HGT) involved in the acquisition of virulence and antibiotic resistance determinants. Despite evidence of HGT and the high level of conservation of the genes coding the DNA uptake system, most bacterial species appear non-transformable under laboratory conditions. In naturally transformable species, the DNA uptake system is only expressed when bacteria enter a physiological state called competence, which develops under specific conditions. Here, we investigated the mechanism that controls expression of the DNA uptake system in the human pathogen Legionella pneumophila We found that a repressor of this system displays a conserved ProQ/FinO domain and interacts with a newly characterized trans-acting sRNA, RocR. Together, they target mRNAs of the genes coding the DNA uptake system to control natural transformation. This RNA-based silencing represents a previously unknown regulatory means to control this major mechanism of HGT. Importantly, these findings also show that chromosome-encoded ProQ/FinO domain-containing proteins can assist trans-acting sRNAs and that this class of RNA chaperones could play key roles in post-transcriptional gene regulation throughout bacterial species.

DNA double strand break (DSB) responses depend on the sequential actions of the E3 ubiquitin ligases RNF8 and RNF168 plus E2 ubiquitin-conjugating enzyme Ubc13 to specifically generate histone Lys-63-linked ubiquitin chains in DSB signaling. Here, we defined the activated RNF8-Ubc13∼ubiquitin complex by x-ray crystallography and its functional solution conformations by x-ray scattering, as tested by separation-of-function mutations imaged in cells by immunofluorescence. The collective results show that the RING E3 RNF8 targets E2 Ubc13 to DSB sites and plays a critical role in damage signaling by stimulating polyubiquitination through modulating conformations of ubiquitin covalently linked to the Ubc13 active site. Structure-guided separation-of-function mutations show that the RNF8 E2 stimulating activity is essential for DSB signaling in mammalian cells and is necessary for downstream recruitment of 53BP1 and BRCA1. Chromatin-targeted RNF168 rescues 53BP1 recruitment involved in non-homologous end joining but not BRCA1 recruitment for homologous recombination. These findings suggest an allosteric approach to targeting the ubiquitin-docking cleft at the E2-E3 interface for possible interventions in cancer and chronic inflammation, and moreover, they establish an independent RNF8 role in BRCA1 recruitment.

Many intracellular protein-protein interactions are mediated by the phosphorylation of serine, and phosphoserine-containing peptides can inhibit these interactions. However, hydrolysis of the phosphate by phosphatases, and the poor cell permeability associated with phosphorylated peptides has limited their utility in cellular and in vivo contexts. Compounding the problem, strategies to replace phosphoserine in peptide inhibitors with easily accessible mimetics (such as Glu or Asp) routinely fail. Here, we present an in vitro selection strategy for replacement of phosphoserine. Using mRNA display, we created a 10 trillion member structurally diverse unnatural peptide library. From this library, we found a peptide that specifically binds to the C-terminal domain (BRCT)2 of breast cancer associated protein 1 (BRCA1) with an affinity comparable to phosphorylated peptides. A crystal structure of the peptide bound reveals that the pSer-x-x-Phe motif normally found in BRCA1 (BRCT)2 binding partners is replaced by a Glu-x-x-4-fluoroPhe and that the peptide picks up additional contacts on the protein surface not observed in cognate phosphopeptide binding. Expression of the peptide in human cells led to defects in DNA repair by homologous recombination, a process BRCA1 is known to coordinate. Overall, this work validates a new in vitro selection approach for the development of inhibitors of protein-protein interactions mediated by serine phosphorylation.

Ubc13 is an E2 ubiquitin conjugating enzyme that functions in nuclear DNA damage signaling and cytoplasmic NF-κB signaling. Here, we present the structures of complexes of Ubc13 with two inhibitors, NSC697923 and BAY 11-7082, which inhibit DNA damage and NF-κB signaling in human cells. NSC697923 and BAY 11-7082 both inhibit Ubc13 by covalent adduct formation through a Michael addition at the Ubc13 active site cysteine. The resulting adducts of both compounds exploit a binding groove unique to Ubc13. We developed a Ubc13 mutant which resists NSC697923 inhibition and, using this mutant, we show that the inhibition of cellular DNA damage and NF-κB signaling by NSC697923 is largely due to specific Ubc13 inhibition. We propose that unique structural features near the Ubc13 active site could provide a basis for the rational development and design of specific Ubc13 inhibitors.

Antisense RNAs have long been known to regulate diverse aspects of plasmid biology. Here we review the FinOP system that modulates F plasmid gene expression through regulation of the F plasmid transcription factor, TraJ. FinOP is a two component system composed of an antisense RNA, FinP, which represses TraJ translation, and a protein, FinO, which is required to stabilize FinP and facilitate its interactions with its traJ mRNA target. We review the evidence that FinO acts as an RNA chaperone to bind and destabilize internal stem-loop structures within the individual RNAs that would otherwise block intermolecular RNA duplexing. Recent structural studies have provided mechanistic insights into how FinO may facilitate interactions between FinP and traJ mRNA. We also review recent findings that two other proteins, Escherichia coli ProQ and Neisseria meningitidis NMB1681, may represent FinO-like RNA chaperones.

The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon–helix–helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH β-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.

Activation of the DNA replication checkpoint by the ATR kinase requires protein interactions mediated by the ATR-activating protein, TopBP1. Accumulation of TopBP1 at stalled replication forks requires the interaction of TopBP1 BRCT5 with the phosphorylated SDT repeats of the adaptor protein MDC1. Here, we present the X-ray crystal structures of the tandem BRCT4/5 domains of TopBP1 free and in complex with a MDC1 consensus pSDpT phosphopeptide. TopBP1 BRCT4/5 adopts a variant BRCT-BRCT packing interface and recognizes its target peptide in a manner distinct from that observed in previous tandem BRCT- peptide structures. The phosphate-binding pocket and positively charged residues in a variant loop in BRCT5 present an extended binding surface for the negatively charged MDC1 phosphopeptide. Mutations in this surface reduce binding affinity and recruitment of TopBP1 to γH2AX foci in cells. These studies reveal a different mode of phosphopeptide binding by BRCT domains in the DNA damage response.

The repair of DNA double strand breaks by homologous recombination relies on the unique topology of the chains formed by Lys-63 ubiquitylation of chromatin to recruit repair factors such as breast cancer 1 (BRCA1) to sites of DNA damage. The human RING finger (RNF) E3 ubiquitin ligases, RNF8 and RNF168, with the E2 ubiquitin-conjugating complex Ubc13/Mms2, perform the majority of Lys-63 ubiquitylation in homologous recombination. Here, we show that RNF8 dimerizes and binds to Ubc13/Mms2, thereby stimulating formation of Lys-63 ubiquitin chains, whereas the related RNF168 RING domain is a monomer and does not catalyze Lys-63 polyubiquitylation. The crystal structure of the RNF8/Ubc13/Mms2 ternary complex reveals the structural basis for the interaction between Ubc13 and the RNF8 RING and that an extended RNF8 coiled-coil is responsible for its dimerization. Mutations that disrupt the RNF8/Ubc13 binding surfaces, or that truncate the RNF8 coiled-coil, reduce RNF8-catalyzed ubiquitylation. These findings support the hypothesis that RNF8 is responsible for the initiation of Lys-63-linked ubiquitylation in the DNA damage response, which is subsequently amplified by RNF168.

Retromer is a peripheral membrane protein complex that plays a critical role in a broad range of physiological, developmental and pathological processes by mediating retrograde transport of proteins from endosomes to the trans-Golgi (TGN) network.Wnt signalling, toxin transport and amyloid production in Alzheimer's disease are just some of the processes known to be regulated by retromer-mediated trafficking.Mammalian retromer consists of a core heterotrimeric cargorecognition subcomplex associated with a membrane-targeting dimer of sorting nexins.The core subcomplex consists of vacuolar protein sorting (VPS)26, VPS29 and VPS35 subunits that play different roles in complex stabilisation and in binding to transport cargo and regulatory proteins.The composition of the membrane-binding subcomplex is inconclusive but consists of a homo-or heterodimer of sorting nexins, SNX1, SNX2, SNX5 and SNX6.These BAR (Bin/Amphiphysin/Rvs)domain proteins can induce the formation of high curvature membrane tubules through the formation of a polymerised helical coat.We have used small-angle X-ray scattering (SAXS), X-ray crystallography, nuclear magnetic resonance (NMR) and isothermal titration calorimetry to elucidate a qualitative and quantitative model of retromer assembly [1].In our proposed model, VPS35 forms an extended, gently curved structure composed of alternating HEAT-like helical repeats.VPS26 and VPS29 bind to distal ends through N-and C-terminal regions of VPS35, respectively, to form a stable trimeric core assembly.Results from thermodynamics experiments have shown that VPS29 and VPS26 bind to VPS35 completely independently of each other, confirming that VPS35 plays the role of central scaffold and VPS29 and VPS26 do not form any contact with each other.Intriguingly, the core trimeric complex is able to form a symmetric dimer, which may have implications for functional interactions in vivo.Solution structures of SNX1 and SNX2 homodimers have been determined using SAXS.The SNX dimer associates with cellular membranes enriched in phosphatidylinositol 3-phosphate and 3,5diphosphate and is believed to polymerise into a helical protein coat.NMR studies have confirmed that VPS29 coordinates the binding of the core subcomplex to this membrane remodelling complex.This coupling of cargo binding and membrane tubulating events enable retromer to recruit cargo molecules into a tubular endosome-to-Golgi transport carrier.