Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase.

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation.

Accelerating Reaction Rates of Biomolecules by Using Shear Stress in Artificial Capillary Systems.

Biomimetics is a design principle within chemistry, biology, and engineering, but chemistry biomimetic approaches have been generally limited to emulating nature's chemical toolkit while emulation of nature's physical toolkit has remained largely unexplored. To begin to explore this, we designed biophysically mimetic microfluidic reactors with characteristic length scales and shear stresses observed within capillaries. We modeled the effect of shear with molecular dynamics studies and showed that this induces specific normally buried residues to become solvent accessible. We then showed using kinetics experiments that rates of reaction of these specific residues in fact increase in a shear-dependent fashion. We applied our results in the creation of a new microfluidic approach for the multidimensional study of cysteine biomarkers. Finally, we used our approach to establish dissociation of the therapeutic antibody trastuzumab in a reducing environment. Our results have implications for the efficacy of existing therapeutic antibodies in blood plasma as well as suggesting in general that biophysically mimetic chemistry is exploited in biology and should be explored as a research area.

Purification of Recombinant α-synuclein: A Comparison of Commonly Used Protocols.

The initial state of the intrinsically disordered protein α-synuclein (aSyn), e.g., the presence of oligomers and degradation products, or the presence of contaminants and adducts can greatly influence the aggregation kinetics and toxicity of the protein. Here, we compare four commonly used protocols for the isolation of recombinant aSyn from Escherichia coli: boiling, acid precipitation, ammonium sulfate precipitation, and periplasmic lysis followed by ion exchange chromatography and gel filtration. We identified, using nondenaturing electrospray ionization mass spectrometry, that aSyn isolated by acid precipitation and periplasmic lysis was the purest and yielded the highest percentage of monomeric protein, 100% and 96.5%, respectively. We then show that aSyn purified by the different protocols exerts different metabolic stresses in cells, with the more multimeric/degraded and least pure samples leading to a larger increase in cell vitality. However, the percentage of monomeric protein and the purity of the samples did not correlate with aSyn aggregation propensity. This study highlights the importance of characterizing monomeric aSyn after purification, as the choice of purification method can significantly influence the outcome of a subsequent study.

A Ctf4 trimer couples the CMG helicase to DNA polymerase α in the eukaryotic replisome.

Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks to avoid stalling of the replication machinery and consequent genomic instability. In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45-MCM-GINS (CMG) DNA helicase to DNA polymerase α (Pol α) within the replisome. We use X-ray crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a ß-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol α and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the amino-terminal tails of the catalytic subunit of Pol α and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol α and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol α to one CMG helicase within the replisome, providing a new model for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of Escherichia coli. The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.

Analysis of the natively unstructured RNA/protein-recognition core in the Escherichia coli RNA degradosome and its interactions with regulatory RNA/Hfq complexes.

The RNA degradosome is a multi-enzyme assembly that plays a central role in the RNA metabolism of Escherichia coli and numerous other bacterial species including pathogens. At the core of the assembly is the endoribonuclease RNase E, one of the largest E. coli proteins and also one that bears the greatest region predicted to be natively unstructured. This extensive unstructured region, situated in the C-terminal half of RNase E, is punctuated with conserved short linear motifs that recruit partner proteins, direct RNA interactions, and enable association with the cytoplasmic membrane. We have structurally characterized a subassembly of the degradosome-comprising a 248-residue segment of the natively unstructured part of RNase E, the DEAD-box helicase RhlB and the glycolytic enzyme enolase, and provide evidence that it serves as a flexible recognition centre that can co-recruit small regulatory RNA and the RNA chaperone Hfq. Our results support a model in which the degradosome captures substrates and regulatory RNAs through the recognition centre, facilitates pairing to cognate transcripts and presents the target to the ribonuclease active sites of the greater assembly for cooperative degradation or processing.

Crystal structure of the N-terminal domain of human Timeless and its interaction with Tipin.

Human Timeless is involved in replication fork stabilization, S-phase checkpoint activation and establishment of sister chromatid cohesion. In the cell, Timeless forms a constitutive heterodimeric complex with Tipin. Here we present the 1.85 Å crystal structure of a large N-terminal segment of human Timeless, spanning amino acids 1-463, and we show that this region of human Timeless harbours a partial binding site for Tipin. Furthermore, we identify minimal regions of the two proteins that are required for the formation of a stable Timeless-Tipin complex and provide evidence that the Timeless-Tipin interaction is based on a composite binding interface comprising different domains of Timeless.

Structural complexity of the co-chaperone SGTA: a conserved C-terminal region is implicated in dimerization and substrate quality control.

BACKGROUND: Protein quality control mechanisms are essential for cell health and involve delivery of proteins to specific cellular compartments for recycling or degradation. In particular, stray hydrophobic proteins are captured in the aqueous cytosol by a co-chaperone, the small glutamine-rich, tetratricopeptide repeat-containing protein alpha (SGTA), which facilitates the correct targeting of tail-anchored membrane proteins, as well as the sorting of membrane and secretory proteins that mislocalize to the cytosol and endoplasmic reticulum-associated degradation. Full-length SGTA has an unusual elongated dimeric structure that has, until now, evaded detailed structural analysis. The C-terminal region of SGTA plays a key role in binding a broad range of hydrophobic substrates, yet in contrast to the well-characterized N-terminal and TPR domains, there is a lack of structural information on the C-terminal domain. In this study, we present new insights into the conformation and organization of distinct domains of SGTA and show that the C-terminal domain possesses a conserved region essential for substrate processing in vivo. RESULTS: We show that the C-terminal domain region is characterized by α-helical propensity and an intrinsic ability to dimerize independently of the N-terminal domain. Based on the properties of different regions of SGTA that are revealed using cell biology, NMR, SAXS, Native MS, and EPR, we observe that its C-terminal domain can dimerize in the full-length protein and propose that this reflects a closed conformation of the substrate-binding domain. CONCLUSION: Our results provide novel insights into the structural complexity of SGTA and provide a new basis for mechanistic studies of substrate binding and release at the C-terminal region.

Mining 2:2 Complexes from 1:1 Stoichiometry: Formation of Cucurbit[8]uril-Diarylviologen Quaternary Complexes Favored by Electron-Donating Substituents.

A 1:1 binding stoichiometry of a host-guest complex need not consist of a single host and guest. Diarylviologens containing electron-donating substituents complexed with cucurbit[8]uril (CB[8]) in a 1:1 stoichiometry exhibit abnormally large binding enthalpies compared to typical enthalpy changes observed for 1:1 binary complexes. Here, several CB[8]-mediated host-guest complexes, which were previously reported as 1:1 binary complexes, are verified to be 2:2 quaternary complexes by a combination of isothermal titration calorimetry, 1H, NOESY, and ROESY NMR, and ion mobility mass spectrometry, clearly indicating a binding motif of two partially overlapping diarylviologens held in place with two CB[8] molecules. Formation of 2:2 quaternary complexes is favored by electron-donating substituents, while electron-withdrawing substituents typically result in 1:1 binary complexes. The stacking of two highly conjugated diarylviologens in one quaternary motif affords the complexes enhanced conductance when considered as a single-molecular conductor. Moreover, an additional conducting signal previously observed for this "supramolecular" conductor can be readily understood with our 2:2 complexation model, corresponding to a parallel conductance pathway. Therefore, a 2:2 quaternary complex model grants a greater understanding of such supramolecular complexes, enabling the design of engineered, hierarchical structures and functional materials.

Effect of DMSO on Protein Structure and Interactions Assessed by Collision-Induced Dissociation and Unfolding.

Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization-ion mobility-mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.

Sequential Release of Proteins from Structured Multishell Microcapsules.

In nature, a wide range of functional materials is based on proteins. Increasing attention is also turning to the use of proteins as artificial biomaterials in the form of films, gels, particles, and fibrils that offer great potential for applications in areas ranging from molecular medicine to materials science. To date, however, most such applications have been limited to single component materials despite the fact that their natural analogues are composed of multiple types of proteins with a variety of functionalities that are coassembled in a highly organized manner on the micrometer scale, a process that is currently challenging to achieve in the laboratory. Here, we demonstrate the fabrication of multicomponent protein microcapsules where the different components are positioned in a controlled manner. We use molecular self-assembly to generate multicomponent structures on the nanometer scale and droplet microfluidics to bring together the different components on the micrometer scale. Using this approach, we synthesize a wide range of multiprotein microcapsules containing three well-characterized proteins: glucagon, insulin, and lysozyme. The localization of each protein component in multishell microcapsules has been detected by labeling protein molecules with different fluorophores, and the final three-dimensional microcapsule structure has been resolved by using confocal microscopy together with image analysis techniques. In addition, we show that these structures can be used to tailor the release of such functional proteins in a sequential manner. Moreover, our observations demonstrate that the protein release mechanism from multishell capsules is driven by the kinetic control of mass transport of the cargo and by the dissolution of the shells. The ability to generate artificial materials that incorporate a variety of different proteins with distinct functionalities increases the breadth of the potential applications of artificial protein-based materials and provides opportunities to design more refined functional protein delivery systems.

Nanoscale click-reactive scaffolds from peptide self-assembly.

BACKGROUND: Due to their natural tendency to self-assemble, proteins and peptides are important components for organic nanotechnology. One particular class of peptides of recent interest is those that form amyloid fibrils, as this self-assembly results in extremely strong, stable quasi-one-dimensional structures which can be used to organise a wide range of cargo species including proteins and oligonucleotides. However, assembly of peptides already conjugated to proteins is limited to cargo species that do not interfere sterically with the assembly process or misfold under the harsh conditions often used for assembly. Therefore, a general method is needed to conjugate proteins and other molecules to amyloid fibrils after the fibrils have self-assembled. RESULTS: Here we have designed an amyloidogenic peptide based on the TTR105-115 fragment of transthyretin to form fibrils that display an alkyne functionality, important for bioorthogonal chemical reactions, on their surface. The fibrils were formed and reacted both with an azide-containing amino acid and with an azide-functionalised dye by the Huisgen cycloaddition, one of the class of "click" reactions. Mass spectrometry and total internal reflection fluorescence optical microscopy were used to show that peptides incorporated into the fibrils reacted with the azide while maintaining the structure of the fibril. These click-functionalised amyloid fibrils have a variety of potential uses in materials and as scaffolds for bionanotechnology. DISCUSSION: Although previous studies have produced peptides that can both form amyloid fibrils and undergo "click"-type reactions, this is the first example of amyloid fibrils that can undergo such a reaction after they have been formed. Our approach has the advantage that self-assembly takes place before click functionalization rather than pre-functionalised building blocks self-assembling. Therefore, the molecules used to functionalise the fibril do not themselves have to be exposed to harsh, amyloid-forming conditions. This means that a wider range of proteins can be used as ligands in this process. For instance, the fibrils can be functionalised with a green fluorescent protein that retains its fluorescence after it is attached to the fibrils, whereas this protein loses its fluorescence if it is exposed to the conditions used for aggregation.

Fragment Screening against the EthR-DNA Interaction by Native Mass Spectrometry.

Native nanoelectrospray ionization mass spectrometry is an underutilized technique for fragment screening. In this study, the first demonstration is provided of the use of native mass spectrometry for screening fragments against a protein-DNA interaction. EthR is a transcriptional repressor of EthA expression in Mycobacterium tuberculosis (Mtb) that reduces the efficacy of ethionamide, a second-line antitubercular drug used to combat multidrug-resistant Mtb strains. A small-scale fragment screening campaign was conducted against the EthR-DNA interaction using native mass spectrometry, and the results were compared with those from differential scanning fluorimetry, a commonly used primary screening technique. Hits were validated by surface plasmon resonance and X-ray crystallography. The screening campaign identified two new fragments that disrupt the EthR-DNA interaction in vitro (IC50 =460-610 µm) and bind to the hydrophobic channel of the EthR dimer.

Spontaneous CO release from Ru(II)(CO)2-protein complexes in aqueous solution, cells, and mice.

We demonstrate that Ru(II)(CO)2-protein complexes, formed by the reaction of the hydrolytic decomposition products of [fac-RuCl(κ(2)-H2NCH2CO2)(CO)3] (CORM-3) with histidine residues exposed on the surface of proteins, spontaneously release CO in aqueous solution, cells, and mice. CO release was detected by mass spectrometry (MS) and confocal microscopy using a CO-responsive turn-on fluorescent probe. These findings support our hypothesis that plasma proteins act as CO carriers after in vivo administration of CORM-3. CO released from a synthetic bovine serum albumin (BSA)-Ru(II)(CO)2 complex leads to downregulation of the cytokines interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α in cancer cells. Finally, administration of BSA-Ru(II)(CO)2 in mice bearing a colon carcinoma tumor results in enhanced CO accumulation at the tumor. Our data suggest the use of Ru(II)(CO)2-protein complexes as viable alternatives for the safe and spatially controlled delivery of therapeutic CO in vivo.

Multimeric complexes among ankyrin-repeat and SOCS-box protein 9 (ASB9), ElonginBC, and Cullin 5: insights into the structure and assembly of ECS-type Cullin-RING E3 ubiquitin ligases.

Proteins of the ankyrin-repeat and SOCS-box (ASB) family act as the substrate-recognition subunits of ECS-type (ElonginBC-Cullin-SOCS-box) Cullin RING E3 ubiquitin ligase (CRL) complexes that catalyze the specific polyubiquitination of cellular proteins to target them for degradation by the proteasome. Therefore, ASB multimeric complexes are involved in numerous cell processes and pathways; however, their interactions, assembly, and biological roles remain poorly understood. To enhance our understanding of ASB CRL systems, we investigated the structure, affinity, and assembly of the quaternary multisubunit complex formed by ASB9, Elongin B, Elongin C (EloBC), and Cullin 5. Here, we describe the application of several biophysical techniques including differential scanning fluorimetry, isothermal titration calorimetry (ITC), nanoelectrospray ionization, and ion-mobility mass spectrometry (IM-MS) to provide structural and thermodynamic information for a quaternary ASB CRL complex. We find that ASB9 is unstable alone but forms a stable ternary complex with EloBC that binds with high affinity to the Cullin 5 N-terminal domain (Cul5NTD) but not to Cul2NTD. The structure of the monomeric ASB9-EloBC-Cul5NTD quaternary complex is revealed by molecular modeling and is consistent with IM-MS and temperature-dependent ITC data. This is the first experimental study to validate structural information for the assembly of the quaternary N-terminal region of an ASB CRL complex. The results suggest that ASB E3 ligase complexes function and assemble in an analogous manner to that of other CRL systems and provide a platform for further molecular investigation of this important protein family. The data reported here will also be of use for the future development of chemical probes to examine the biological function and modulation of other ECS-type CRL systems.

Binding interactions between long noncoding RNA HOTAIR and PRC2 proteins.

Long noncoding RNAs (lncRNAs) play a key role in the epigenetic regulation of cells. Many of these lncRNAs function by interacting with histone repressive proteins of the Polycomb group (PcG) family, recruiting them to gene loci to facilitate silencing. Although there are now many RNAs known to interact with the PRC2 complex, little is known about the details of the molecular interactions. Here, we show that the PcG protein heterodimer EZH2-EED is necessary and sufficient for binding to the lncRNA HOTAIR. We also show that protein recognition occurs within a folded 89-mer domain of HOTAIR. This 89-mer represents a minimal binding motif, as further deletion of nucleotides results in substantial loss of affinity for PRC2. These findings provide molecular insights into an important system involved in epigenetic regulation.

Nanoelectrospray ionization mass spectrometric study of Mycobacterium tuberculosis CYP121-ligand interactions.

Nondenaturing nanoelectrospray ionization mass spectrometry (nanoESI MS) of intact protein complexes was used to study CYP121, one of the 20 cytochrome P450s in Mycobacterium tuberculosis (Mtb) and an enzyme that is essential for bacterial viability. The results shed new light on both ligand-free and ligand-bound states of CYP121. Isolated unbound CYP121 is a predominantly dimeric protein, with a minor monomeric form present. High affinity azoles cause the dissociation of dimeric CYP121 into monomer, whereas weaker azole binders induce partial dimer dissociation or do not significantly destabilize the dimer. Complexes of CYP121 with azoles were poorly detected by nanoESI MS, indicating kinetically labile complexes that are easily prone to gas-phase dissociation. Unlike with the azoles, CYP121 forms a stable complex with its natural substrate cYY that does not undergo gas-phase dissociation. In addition, a series of potential ligands from fragment-based studies were used as a test for nanoESI MS work against CYP121. Most of these ligands formed stable complexes with CYP121, and their binding did not promote dimer dissociation. On the basis of binding to the monomer and/or CYP121 dimer it was possible to determine the relative order of their CYP121 binding affinities. The top nanoESI MS screening hit was confirmed by heme absorbance shift assay to have a Kd of 40 µM.

Evidence for the assembly of a bacterial tripartite multidrug pump with a stoichiometry of 3:6:3.

The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.

Opening of the outer membrane protein channel in tripartite efflux pumps is induced by interaction with the membrane fusion partner.

The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.

Non-homologous end-joining partners in a helical dance: structural studies of XLF-XRCC4 interactions.

XRCC4 (X-ray cross-complementation group 4) and XLF (XRCC4-like factor) are two essential interacting proteins in the human NHEJ (non-homologous end-joining) pathway that repairs DNA DSBs (double-strand breaks). The individual crystal structures show that the dimeric proteins are homologues with protomers containing head domains and helical coiled-coil tails related by approximate two-fold symmetry. Biochemical, mutagenesis, biophysical and structural studies have identified the regions of interaction between the two proteins and suggested models for the XLF-XRCC4 complex. An 8.5 Å (1 Å = 0.1 nm) resolution crystal structure of XLF-XRCC4 solved by molecular replacement, together with gel filtration and nano-ESI (nano-electrospray ionization)-MS results, demonstrates that XLF and XRCC4 dimers interact through their head domains and form an alternating left-handed helical structure with polypeptide coiled coils and pseudo-dyads of individual XLF and XRCC4 dimers at right angles to the helical axis.