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Extracellular vesicles (EVs) have enormous potential for the implementation of liquid biopsy and as effective drug delivery means, but the fulfilment of these expectations requires overcoming at least two bottlenecks relative to their purification, namely the finalization of reliable and affordable protocols for: (i) EV sub-population selective isolation and (ii) the scalability of their production/isolation from complex biological fluids. In this work, we demonstrated that these objectives can be achieved by a conceptually new affinity chromatography platform composed of a macroporous epoxy monolith matrix functionalized with anti-CD63 nanobodies with afflux of samples and buffers regulated through a pump. Such a system successfully captured and released integral EVs from urine samples and showed negligible unspecific binding for circulating proteins. Additionally, size discrimination of eluted EVs was achieved by different elution approaches (competitive versus pH-dependent). The physical characteristics of monolith material and the inexpensive production of recombinant nanobodies make scaling-up the capture unit feasible and affordable. Additionally, the availability of nanobodies for further specific EV biomarkers will allow for the preparation of monolithic affinity filters selective for different EV subclasses.

Reliable diagnosis is critical to identify infections of SARS-CoV-2 as well as to evaluate the immune response to virus and vaccines. Consequently, it becomes crucial the isolation of sensitive antibodies to use as immunocapture elements of diagnostic tools. The final bottleneck to achieve these results is the availability of enough antigen of good quality. We have established a robust pipeline for the production of recombinant, functional SARS-CoV-2 Spike receptor binding domain (RBD) at high yield and low cost in culture flasks. RBD was expressed in transiently transfected ExpiCHO cells at 32 °C and 5% CO

The proliferating cell nuclear antigen (PCNA) sliding clamp lies at the heart of the accurate duplication of eukaryotic genomes. While the outer surface of the PCNA ring interacts with polymerases and other factors, the role of the inner wall facing the DNA is elusive. Recent evidence shows that conserved basic residues in the PCNA central channel create a specific surface that recognizes the DNA backbone and enables the clamp to slide by rotationally tracking the DNA helix. The sliding surface can be modulated (i) through lysine acetylation, which triggers PCNA degradation during nucleotide excision repair (NER) and stimulates repair by homologous recombination (HR) or (ii) through binding of the protein factor p15PAF, which turns off DNA lesion bypass. Thus, the inner surface of PCNA is unexpectedly highly regulated to control resistance to DNA damage. From a structural viewpoint, we reflect on these findings that open a new perspective on PCNA function and offer opportunities to develop tools to manipulate the DNA damage response in cancer treatment.

RecQ helicases are essential in the maintenance of genome stability. Five paralogues (RecQ1, Bloom, Werner, RecQ4, and RecQ5) are found in human cells, with distinct but overlapping roles. Mutations in human RecQ4 give rise to three distinct genetic disorders (Rothmund-Thomson, RAPADILINO, and Baller-Gerold syndromes), characterized by genetic instability, growth deficiency, and predisposition to cancer. Previous studies suggested that RecQ4 was unique because it did not seem to contain a RecQ C-terminal region (RQC) found in the other RecQ paralogues; such a region consists of a zinc domain and a winged helix domain and plays an important role in enzyme activity. However, our recent bioinformatic analysis identified in RecQ4 a putative RQC. To experimentally confirm this hypothesis, we report the purification and characterization of the catalytic core of human RecQ4. Inductively coupled plasma-atomic emission spectrometry detected the unusual presence of two zinc clusters within the zinc domain, consistent with the bioinformatic prediction. Analysis of site-directed mutants, targeting key RQC residues (putative zinc ligands and the aromatic residue predicted to be at the tip of the winged helix β-hairpin), showed a decrease in DNA binding, unwinding, and annealing, as expected for a functional RQC domain. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4.

The human zinc transporter 8 (hZnT8) plays important roles in the storage of insulin in the secretory vesicles of pancreatic &beta, cells. hZnT8 consists of a transmembrane domain, with its N- and C-termini protruding into the cytoplasm. Interestingly, the exchange of arginine to tryptophan at position 325 in the C-terminal domain (CTD) increases the risk of developing type 2 diabetes mellitus (T2D). In the present study, the CTDs of hZnT8 (the wild-type (WT) and its disease risk variant (R325W)) were expressed, purified, and characterized in their native forms by biophysical techniques. The data reveal that the CTDs form tetramers which are stabilized by zinc binding, and exhibit negligible differences in their secondary structure content and zinc-binding affinities in solution. These findings provide the basis for conducting further structural studies aimed at unravelling the molecular mechanism underlying the increased susceptibility to develop T2D, which is modulated by the disease risk variant.

Members of the tripartite motif family of E3 ubiquitin ligases are characterized by the presence of a conserved N-terminal module composed of a RING domain followed by one or two B-box domains, a coiled-coil and a variable C-terminal region. The RING and B-box are both Zn-binding domains but, while the RING is found in a large number of proteins, the B-box is exclusive to the tripartite motif (TRIM) family members in metazoans. Whereas the RING has been extensively characterized and shown to possess intrinsic E3 ligase catalytic activity, much less is known about the role of the B-box domains. In this study, we adopted an in vitro approach using recombinant point- and deletion-mutants to characterize the contribution of the TRIM32 Zn-binding domains to the activity of this E3 ligase that is altered in a genetic form of muscular dystrophy. We found that the RING domain is crucial for E3 ligase activity and E2 specificity, whereas a complete B-box domain is involved in chain assembly rate modulation. Further, in vitro, the RING domain is necessary to modulate TRIM32 oligomerization, whereas, in cells, both the RING and B-box cooperate to specify TRIM32 subcellular localization, which if altered may impact the pathogenesis of diseases.

Using our previously reported maps of the electrostatic surface of horse heart ferri- and ferro-cyt c, comparisons were made between the complementary electrostatic surfaces of three cyt c peroxidase-cyt c complexes and the photosynthetic reaction center-cyt c complex, considering both iron oxidation states. The results obtained were consistent with a sliding mechanism for the electron shuttle on the surface of the protein complexes, promoted by the change in iron oxidation state. This mechanism was found to be in agreement with theoretical and NMR studies reported in the literature. Importantly, the analysis also provided a rationale for recognition of nonproductive associations. As we have previously reported the same conclusion on examination of redox partners of cyt c in the mitochondrial respiratory pathway, our hypothesis is that the proposed mechanism could represent a general exit strategy of monoheme cyts c and c2 in electron transfer complexes.

GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.

Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a ‘cogwheel’ mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA–DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme., We thank M. Polentarutti and N. Demitri (Elettra Sincrotrone Trieste) for their assistance at the XRD1 beamline, N.G. Abrescia (CIC bioGUNE) and R. Steiner (King’s College, London) for their advice in the crystallographic analysis, and A. Costa (Francis Crick Institute) for his useful comments. This work was supported by the Italian Association for Cancer Research (AIRC iCARE Fellowship to A.De.B. and AIRC Grant IG14718 to S.O.), and the Spanish Ministry of Economy and Competitiveness (CTQ2014-56966-R grant to F.J.B.). S.B.-V. acknowledges a fellowship from the Spanish Ministry of Economy and Competitiveness (Ref. BES-2013-063991). We acknowledge CERIC-ERIC for the use of the XRD1 beamline at Elettra Sincrotrone Trieste. The authors thankfully acknowledge the computer resources, technical expertise and assistance provided by the Red Española de Supercomputación, the Barcelona Supercomputing Center and the Catalan CSUC.

The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred.

A new pseudopeptide epoxide inhibitor, designed for irreversible binding to HIV protease (HIV-PR), has been synthesized and characterized in solution and in the solid state. However, the crystal structure of the complex obtained by inhibitor–enzyme cocrystallization revealed that a minor isomer, with inverted configuration of the epoxide carbons, has been selected by HIV-PR during crystallization. The structural characterization of the well-ordered pseudopeptide, inserted in the catalytic channel with its epoxide group intact, provides deeper insights into inhibitor binding and HIV-PR stereoselectivity, which aids development of future epoxide-based HIV inhibitors.

Highly efficient unwinding of double-stranded DNA is an essential prerequisite for duplication of the genetic material. In eukaryotes this process is catalysed by the CMG (Cdc45-Mcm2–7-GINS) complex, which is composed by 11 proteins: the Cdc45 replication factor, the hetero-hexameric Mcm2–7 and the tetrameric GINS complex. Although the Mcm2–7 is the helicase motor, in the absence of the other components the purified Mcm complex displays only a weak and labile helicase activity; in contrast, a stable complex comprising Cdc45, Mcm2–7 and GINS can be co-expressed and/or reconstituted, and shows a robust helicase activity.

Significance Cyclic nucleotide-gated (CNG) channels underlie sensory transduction in photoreceptors and olfactory epithelium and share a high degree of homology with K + channels. However, these channels conduct Na + and K + differently: although K + channels discriminate with high accuracy Na + from K + , CNG channels do not discriminate among different cations. By combining electrophysiology, molecular dynamics simulations, and X-ray crystallography we found that the pore region exhibits a dynamic structure. We show that ( i ) the selectivity filter can adapt to large and small ions with a different geometry and ( ii ) the pore diameter critically depends on the ion within. We conclude that the pore of CNG channels is highly flexible and that this flexibility is at the basis of their poor ionic selectivity.

Multiheme cytochromes c (cyts c) are c-type cyts characterized by non-standard structural and spectroscopic properties. The relative disposition of the heme cofactors in the core of these proteins is conserved and they can be classified from their geometry in two main groups. In one group the porphyrin planes are arranged in a perpendicular fashion, while in the other they are parallel. Orientation of the heme groups is a key factor that regulates the intramolecular electron transfer pathway. A 16.5 kDa diheme cyt c, isolated from the bacterium Shewanella baltica OS155 (Sb-DHC), was cloned and expressed in E. coli and its structure was investigated by X-ray crystallography. Using high-resolution data (1.14 Å) collected at ELETTRA (Trieste), the crystal structure, with an orthorhombic cell (a = 40.81, b = 42.97, c = 82.07 Å), was solved using the homologous diheme from Rhodobacter sphaeroides (Rs-DHC) as the initial model. The electron density map of the refined structure (Rfact of 13.8% and Rfree of 15.4%) shows a two domain structure connected by a central unstructured region (N72-G87). The Sb-DHC, like its homologue (Rs-DHC), folds into a new cyt c class: the N-terminal globular domain, with its three α-helices, belongs to class I of c-type cyts, while the C-terminal domain includes a rare π-helix. The metal centre of the c-type heme groups is axially coordinated by two His residues and it is covalently bound to the protein through two Cys bonds.

The interligand interactions in coordination compounds have been principally interpreted in terms of cis and trans influences and effects, which can be defined as the ability of a ligand X to affect the bond of another ligand, cis or trans to X, to the metal. This review analyzes these effects/influences in cobalamins (XCbl) and their simple models cobaloximes, LCo(chel)X. Important properties of these complexes, such as geometry, stability, and reactivity, can be rationalized in terms of steric and electronic factors of the ligands. Experimental evidence of normal and inverse trans influence is described in alkylcobaloximes for the first time. The study of simple B12 models has complemented that on the more complex cobalamins, with particular emphasis on the properties of the axial L-Co-X moiety. Some of the conclusions reached for the axial fragment of simple models have also been qualitatively detected in cobalamins and have furnished new insight into the as yet unestablished mechanism for the homolytic cleavage of the Cosingle bondC bond in the AdoCbl-based enzymes.

Background Our recent work revealed that some chronic myeloid leukemia (CML) patients failing imatinib therapy carry the BCR-ABL1 V304D mutation, and is associated with poor clinical outcomes. BCR-ABL1 V304D variant has rarely been described in clinical specimens, and its location within the BCR-ABL1 Kinase Domain (KD) appears to be in an area of the protein not involved in the direct binding with tyrosine kinase inhibitors (TKIs) such as Imatinib and Ponatinib. The present study integrates computational, structural, and molecular biology techniques to investigate the role of the BCR-ABL1 V304D mutation in kinase activity and TKI resistance. Materials and methods In silico experiments are used to probe whether, compared to the wild type (wt), the V304D mutation i) directly affects the kinase domain fold and/or thermodynamic stability and ii) if/how these effects might eventually interfere with BCR-ABL1 binding to the TKIs. Small angle X-ray scattering (SAXS), fluorescence, and circular dichroism spectroscopy are employed for structural characterization of the wt/mutated kinase domain and their interaction with TKIs. Cell proliferation, protein phosphorylation, and kinase activity assays are performed (with/without) the TKIs to assess the effect of this mutation on BCR-ABL1 KD, its enzymatic and transforming activities. Results Our preliminary results show that BCR-ABL1V304D does not transform Ba/F3 cells, retains minimal kinase activity, and fails to activate downstream pathways. At the same time, the V304D BCR-ABL1 isoform partially protects cells from cytokine withdrawal and exhibits imatinib resistance. The SAXS-based structural model of the kinase domain mutant in complex with Ponatinib does not reveal major structural perturbations compared to the wild type protein. Molecular simulations on the SH2-linker-SH3-KD construct suggest a possible rationale for the TKI resistance seen in the clinic. Conclusions The preliminary results obtained indicate that BCR-ABL1V304D results in kinase inactivation, pan-TKI resistance mediated by lower control of protein autoinhibition via perturbation of the SH3 binding domain and very poor prognosis. Citation Format: Sabrina Pricl, Erik Laurini, Maurizio Fermeglia, Paola Storici, Barbara Giabbai, Marta Semrau, Matteo De March, Nicholas J. Donato, Alfonso Quintas-Cardama. Bcr-Abl1 V304D mutation in CML: friend or foe? [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B61.

Using an engineered cyclic nucleotide-gated (CNG) channel mimic based on the bacterial non-selective NaK channel, we examined how different ionic species interact with the pore by means of electrophysiology, all-atoms molecular dynamics simulations and X-ray crystallography.Difference and anomalous difference electron-density maps of high-resolution crystal structures (up to 1.85A) obtained after soaking with different ionic species (Li+, Na+, K+, Rb+, Cs+, Methylammonium) clearly shows the presence of different peaks inside the pore. Depending on these ionic species Glu66 and Thr67, located at the extracellular entrance of the selectivity filter, adopt different rotameric states. In the presence of Methylammonium, Glu66 points towards Tyr55 of the same subunit, while in the presence of Na+, Glu66 forms H-bonds with Thr67 and Thr60 of a neighboring subunit (Derebe et al, 2011, PNAS). Moreover, large-scale molecular dynamics simulations in a hydrated lipid bilayer at 0 mV indicates that Glu66 and Thr67 side chains are capable of large structural fluctuations which are modulated by the nature of the ion residing in the pore. In particular, in presence of Rb+ and Cs+ Glu66 explores at least three different conformations, characterized by a significantly different distribution of the local charge. Single channel recordings from this chimeric ionic channel in symmetrical Rb+ and Cs+ conditions show a strong voltage dependency similar to what observed in the native CNGA1 channel (Marchesi et al, 2012, Nat. Communications). These results indicate that membrane voltage catalyzes a conformational change of these residues, especially of Glu66, perturbing the hydrogen bonding network behind the selectivity filter. Non-selective ionic permeation emerges as a robust feature of an intrinsic flexible pore and is a key microscopic factor controlling ionic selectivity in CNG and possibly in other non-selective channels.