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The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.

Recent structural studies show distinct morphologies for the fibrils of Aβ(1-42) and Aβ(1-40), which are believed not to co-fibrillize. We describe here a novel, structurally-uniform 1 : 1 mixed fibrillar species, which differs from both pure fibrils. It forms preferentially even when Aβ(1-42) : Aβ(1-40) peptides are mixed in a non-stoichiometric ratio.

HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.

The fully characterization of tumor associated antigens (TAAs) and of tumor associated carbohydrate antigens (TACAs) have opened the avenue of cancer immunotherapy. The intrinsic poor immunogenicity of TACAs, however, spotlighted the importance of multivalent presentation of the antigen(s) to trigger an immune response. Nanoparticles are excellent scaffolds for this purpose. Here we reported on the easy glycosylation of iron-based and biocompatible dextran-based nanoparticles with 1, a mimetic of the TnThr antigen. The multivalent presentation of 1 induced the induction of TNF-α and IL-6/IL10, respectively. The multivalent glycosylation of silica nanoparticles (GSiNPs) was also performed and saccharide loading qualitative assessed by solid state NMR. Our results offer the proof of concept that biomolecules coating can also be investigated on solid system by NMR.

The inhibition of the PD-1/PD-L1 axis by monoclonal antibodies has achieved remarkable success in treating a growing number of cancers. However, a novel class of small organic molecules, with BMS-202 (1) as the lead, is emerging as direct PD-L1 inhibitors. Herein, we report a series of 2,4,6-tri- and 2,4-disubstituted 1,3,5-triazines, which were synthesized and assayed for their PD-L1 binding by NMR and homogeneous time-resolved fluorescence. Among them, compound 10 demonstrated to strongly bind with the PD-L1 protein and challenged it in a co-culture of PD-L1 expressing cancer cells (PC9 and HCC827 cells) and peripheral blood mononuclear cells enhanced antitumor immune activity of the latter. Compound 10 significantly increased interferon γ release and apoptotic induction of cancer cells, with low cytotoxicity in healthy cells when compared to 1, thus paving the way for subsequent preclinical optimization and medical applications.

Biocompatible hydrogels for tissue regeneration/replacement and drug release with specific architectures can be obtained by three-dimensional bioprinting techniques. The preservation of the higher order structure of the proteins embedded in the hydrogels as drugs or modulators is critical for their biological activity. Solution nuclear magnetic resonance (NMR) experiments are currently used to investigate the higher order structure of biotherapeutics in comparability, similarity, and stability studies. However, the size of pores in the gel, protein–matrix interactions, and the size of the embedded proteins often prevent the use of this methodology. The recent advancements of solid-state NMR allow for the comparison of the higher order structure of the matrix-embedded and free isotopically enriched proteins, allowing for the evaluation of the functionality of the material in several steps of hydrogel development. Moreover, the structural information at atomic detail on the matrix–protein interactions paves the way for a structure-based design of these biomaterials.

The variety of magnetic properties exhibited by paramagnetic lanthanoids provides outstanding information in NMR-based structural biology and therefore can be a very useful tool for characterizing lanthanoid-binding proteins. Because of their dependence on the relative positions of the protein nuclei and of the lanthanoid ion, the paramagnetic restraints (PCS, PRDC and PRE) provide information on structure and dynamics of proteins. In this Chapter, we cover the use of lanthanoids in structural biology including protein sample preparation, NMR experiments and data interpretation.

Glioblastoma Multiforme (GBM) is a highly invasive primary brain tumour characterized by chemo- and radio-resistance and poor overall survival. GBM can present an aberrant functionality of p53, caused by the overexpression of the murine double minute 2 protein (MDM2) and its analogue MDM4, which may influence the response to conventional therapies. Moreover, tumour resistance/invasiveness has been recently attributed to an overexpression of the chemokine receptor CXCR4, identified as a pivotal mediator of glioma neovascularization. Notably, CXCR4 and MDM2-4 cooperate in promoting tumour invasion and progression. Although CXCR4 actively promotes MDM2 activation leading to p53 inactivation, MDM2-4 knockdown induces the downregulation of CXCR4 gene transcription. Our study aimed to assess if the CXCR4 signal blockade could enhance glioma cells' sensitivity to the inhibition of the p53-MDMs axis. Rationally designed inhibitors of MDM2/4 were combined with the CXCR4 antagonist, AMD3100, in human GBM cells and GBM stem-like cells (neurospheres), which are crucial for tumour recurrence and chemotherapy resistance. The dual MDM2/4 inhibitor RS3594 and the CXCR4 antagonist AMD3100 reduced GBM cell invasiveness and migration in single-agent treatment and mainly in combination. AMD3100 sensitized GBM cells to the antiproliferative activity of RS3594. It is noteworthy that these two compounds present synergic effects on cancer stem components: RS3594 inhibited the growth and formation of neurospheres, AMD3100 induced differentiation of neurospheres while enhancing RS3594 effectiveness preventing their proliferation/clonogenicity. These results confirm that blocking CXCR4/MDM2/4 represents a valuable strategy to reduce GBM proliferation and invasiveness, acting on the stem cell component too.

Mutating the side-chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half-life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6-Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4-disubstituted 1,2,3-triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2R/AT1R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross-peaks. The most potent analogue, compound 2, has been explored regarding its neurotrophic potential and resulted in an enhanced neurite growth with respect to the established agent C21. © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. ISSN:0947-6539 ISSN:1521-3765

Two orthogonal, metal free click reactions, enabled to glycosylate ubiquitin and its mutant A28C forming two protein scaffolds with high affinity for BambL, a lectin from the human pathogen Burkholderia ambifaria. A new fucoside analogue, with high affinity with BambL, firstly synthetized and co-crystallized with the protein target, provided the insights for sugar determinants grafting onto ubiquitin. Three ubiquitin-based glycosides were thus assembled. Fuc-Ub, presented several copies of the fucoside analogue, with proper geometry for multivalent effect; Rha-A28C, displayed one thio-rhamnose, known for its ability to tuning the immunological response; finally, Fuc-Rha-A28C, included both multiple fucoside analogs and the rhamnose residue. Fuc-Ub and Fuc-Rha-A28C ligands proved high affinity for BambL and unprecedented immune modulatory properties towards macrophages activation., Metal free click reactions used to glycosylate ubiquitin and its mutant A28C afforded two protein scaffolds with high affinity for Burkholderia ambifaria lectin (BambL).

The possibility of immobilizing a protein with antigenic properties on a solid support offers significant possibilities in the development of immunosensors and vaccine formulations. For both applications, the orientation of the antigen should ensure ready accessibility of the antibodies to the epitope. However, an experimental assessment of the orientational preferences necessarily proceeds through the preparation/isolation of the antigen, the immobilization on different surfaces and one or more biophysical characterization steps. To predict a priori whether favorable orientations can be achieved or not would allow one to select the most promising experimental routes, partly mitigating the time cost towards the final product. In this manuscript, we apply a simple computational model, based on united-residue modelling, to the prediction of the orientation of the receptor binding domain of the SARS-CoV-2 spike protein on surfaces commonly used in lateral-flow devices. These calculations can account for the experimental observation that direct immobilization on gold gives sufficient exposure of the epitope to obtain a response in immunochemical assays., Graphical abstract Unlabelled Image, Highlights • Protein immobilization is a crucial in preparing immunosensors. • Immobilization must ensure exposition of the epitope. • A simple computational model can help predicting the exposition.

Resonance assignment and structural characterization of pharmacologically relevant proteins promise to improve understanding and safety of these proteins by rational design. However, the PEG coating that is used to evade the immune system also causes these molecules to "evade" the standard structural biology methodologies. We here demonstrate that it is possible to obtain the resonance assignment and a reliable structural model of large PEGylated proteins through an integrated approach encompassing NMR and X-ray crystallography.

[Image: see text] Protein–protein interactions (PPIs) contribute to the onset and/or progression of several diseases, especially cancer, and this discovery has paved the way for considering disruption of the PPIs as an attractive anti-tumor strategy. In this regard, simple and efficient biophysical methods for detecting the interaction of the inhibitors with the protein counterpart are still in high demand. Herein, we describe a convenient NMR method for the screening of putative PPI inhibitors based on the use of “hot peptides” (HOPPI-NMR). As a case study, HOPPI-NMR was successful applied to the well-known p53/MDM2 system. Our outcomes highlight the main advantages of the method, including the use of a small amount of unlabeled proteins, the minimization of the risk of protein aggregation, and the ability to identify weak binders. The last leaves open the possibility for application of HOPPI-NMR in tandem with fragment-based drug discovery as a valid strategy for the identification of novel chemotypes acting as PPI inhibitors.

Partial symmetry, i.e., the presence of more than one molecule in the asymmetric unit of a crystal, is a relatively rare phenomenon in small-molecule crystallography, but is quite common in protein crystallography, where it is typically known as non-crystallographic symmetry (NCS). Several papers in literature propose molecular determinants such as crystal contacts, thermal factors, or TLS parameters as an explanation for the phenomenon of intrinsic asymmetry among molecules that are in principle equivalent. Nevertheless, are all of the above determinants the cause or are they rather the effect? In the general frame of the NCS often observed in crystals of biomolecules, this paper deals with nickel(II)-substituted human carbonic anhydrase(II) (hCAII) and its SAD structure determination at the nickel edge. The structure revealed two non-equivalent molecules in the asymmetric unit, the presence of a secondary nickel-binding site at the N-terminus of both molecules (which had never been found before in the nickel-substituted enzyme) and two different coordination geometries of the active site nickel (hexa-coordinated in one molecule and mainly penta-coordinated in the other). The above-mentioned standard molecular crystallographic determinants of this asymmetry are analyzed and presented in detail for this particular case. From these considerations, we speculate on the existence of a fundamental, although yet unknown, common cause for the partial symmetry that is so often encountered in X-ray structures of biomolecules.

Carbohydrate‐based vaccines are among the safest and most effective vaccines and represent potent tools for prevention of life‐threatening bacterial infectious diseases, like meningitis and pneumonia. The chemical conjugation of a weak antigen to protein as a source of T‐cell epitopes generates a glycoconjugate vaccine that results more immunogenic. Several methods have been used so far to characterize the resulting polysaccharide–protein conjugates. However, a reduced number of methodologies has been proposed for measuring the degree of saccharide conjugation at the possible protein sites. Here we show that detailed information on large proteins conjugated with large polysaccharides can be achieved by a combination of solution and solid‐state NMR spectroscopy. As a test case, a large protein assembly, l‐asparaginase II, has been conjugated with Neisseria meningitidis serogroup C capsular polysaccharide and the pattern and degree of conjugation were determined.

Solid-state NMR is becoming a powerful tool to detect atomic-level structural features of biomolecules even when they are bound to (or trapped in) solid systems that lack long-range three-dimensional order. We here demonstrate that it is possible to probe protein-ligand interactions from a protein-based perspective also when the protein is entrapped in silica, thus translating into biomolecular solid-state NMR all of the considerations that are usually made to understand the chemical nature of the interaction of a protein with its ligands. This work provides a proof of concept that also immobilized enzymes can be used for protein-based NMR protein-ligand interactions for drug discovery.

Solid-state NMR has become the method of choice for the assessment of protein structure for insoluble objects lacking long-range order. In this context, it is apparent that solid-state NMR is also perfectly poised toward the characterization of immobilized proteins. For these systems, it is possible to understand at the atomic level which perturbations, if any, are occurring as a result of the functionalization. Here we describe how it is possible to accomplish the NMR characterization of enzymes that have been immobilized through different approaches, and we introduce the reader to the choice of the experimental strategy that can be useful in different cases. An outlook on the level of information that can be attained is also given, in view of recent methodological advancements.

The heterogeneous composition of vaccine formulations and the relatively low concentration make the characterization of the protein antigens extremely challenging. Aluminum-containing adjuvants have been used to enhance the immune response of several antigens over the last 90 years and still remain the most commonly used. Here, we show that solid-state NMR and isotope labeling methods can be used to characterize the structural features of the protein antigen component of vaccines and to investigate the preservation of the folding state of proteins adsorbed on Alum hydroxide matrix, providing the way to identify the regions of the protein that are mainly affected by the presence of the inorganic matrix. l-Asparaginase from E. coli has been used as a pilot model of protein antigen. This methodology can find application in several steps of the vaccine development pipeline, from the antigen optimization, through the design of vaccine formulation, up to stability studies and manufacturing process., Quality control: Assessing how adjuvants affect vaccine antigens Aluminum salts are the most frequently used adjuvants in vaccines, but they may negatively affect the antigens in suspension via chemical interactions that could reduce antigen quality and stability. Marco Fragai from the University of Florence and colleagues now report a method based on solid-state nuclear magnetic resonance, to characterize the structure of the model antigen L-asparaginase when adsorbed to the adjuvant aluminum hydroxide in a vaccine suspension. The researchers identify the antigen motifs that interact with the adjuvant directly, and are able to shed light on the antigen’s folding when in complex with the aluminum adjuvant in suspension. This approach could help identify antigen misfolding as a consequence of interactions with chemical adjuvants and may provide useful information during multiple stages of vaccine manufacturing, from antigen optimization to the vaccine manufacturing process, to ultimately improve vaccine quality.

α-Synuclein (α-syn) is found to be naturally present in biofluids such as cerebrospinal fluid (CSF) and serum. Human serum albumin (HSA) is the most abundant protein found in these biofluids, which, beyond transporting hormones and drugs, also exerts a chaperone-like activity binding other proteins in blood and inhibiting their aggregation. Contrasting results are reported in the literature about the effects of albumin on α-syn aggregation. We characterized the binding region of HSA on α-syn by high-field solution NMR spectroscopy and the effect of HSA on α-syn aggregation by thioflavin-T (ThT) fluorescence under both low-ionic-strength and physiological conditions at the albumin concentration in serum and CSF. We found that HSA, at the concentration found in human serum, slows the aggregation of α-syn significantly. α-Syn interacts with HSA in an ionic strength- and pH-dependent manner. The binding is driven by hydrophobic interactions at the N-terminus under physiological experimental conditions and by electrostatic interactions at the C-terminus at low ionic strength. This work provides novel information about the proteostasis of α-syn in biofluids and supports the hypothesis of a chaperone-like behavior of HSA.

The design and synthesis of the Ln3+ complexes of a DOTA-containing (DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) inhibitor of matrix metalloproteinases are reported. The tight binding of the sulfonamide scaffold to the catalytic domain of the investigated matrix metalloproteinase is not impaired by the presence of the Ln3+ -DOTA moiety. The paramagnetic properties of the Ln3+ complex are exploited to obtain insights into the structural features of the ligand-protein interactions and to evaluate the influence of the linker length on the quality of the paramagnetic restraints.

Solid state NMR (SSNMR) has earned a substantial success in the characterization of paramagnetic systems over the last decades. Nowadays, the resolution and sensitivity of solid state NMR in biological molecules has improved significantly and these advancements can be translated into the study of paramagnetic biomolecules. However, the electronic properties of different metal centers affect the quality of their SSNMR spectra differently, and not all systems turn out to be equally easy to approach by this technique. In this review we will try to give an overview of the properties of different paramagnetic centers and how they can be used to increase the chances of experimental success.

FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies.

Protein glycosylation is the most complex post-translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l-asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact-Ar-OSO2 F) with the ϵ-NH2 group of lysine residues of the proteins. This metal-free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ-NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact-Ar-OSO2 F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact-Ar-OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2 -NH) linkage.

Active bioinspired materials are appealing biotechnological targets, and their study is gaining momentum. These materials, which comprise of an inorganic matrix and one or more biomolecules, are extremely variable and therefore may result difficult to characterize in their intimate structure. In this work we have prepared a hydroxyapatite–L-asparaginase composite, with the perspective of using it in acute leukemia treatment. We demonstrate that the use of electron microscopy and powder X-ray diffraction, combined with the atomic-resolution information coming from solid-state NMR, allows us to understand the topology of the material and how the different components interplay to obtain an active composite.

PEGylated proteins are widely used in biomedicine but, in spite of their importance, no atomic-level information is available since they are generally resistant to structural characterization approaches. PEGylated proteins are shown here to yield highly resolved solid-state NMR spectra, which allows assessment of the structural integrity of proteins when PEGylated for therapeutic or diagnostic use.

DNA-minor-groove-binding ligands are potent antineoplastic molecules. The antibiotic distamycin A is the prototype of one class of these DNA-interfering molecules that have been largely used in vitro. The affinity of distamycin A for DNA is well known, and the structural details of the complexes with some B-DNA and G-quadruplex-forming DNA sequences have been already elucidated. Here, we show that distamycin A binds S100β, a protein involved in the regulation of several cellular processes. The reported affinity of distamycin A for the calcium(II)-loaded S100β reinforces the idea that some biological activities of the DNA-minor-groove-binding ligands arise from the binding to cellular proteins. Copyright © 2015 John Wiley & Sons, Ltd.

The human antigen R (HuR) is an RNA-binding protein known to modulate the expression of target mRNA coding for proteins involved in inflammation, tumorigenesis, and stress responses and is a valuable drug target. We previously found that dihydrotanshinone-I (DHTS, 1) prevents the association of HuR with its RNA substrate, thus imparing its function. Herein, inspired by DHTS structure, we designed and synthesized an array of ortho-quinones (tanshinone mimics) using a function-oriented synthetic approach. Among others, compound 6a and 6n turned out to be more effective than 1, showing a nanomolar Ki and disrupting HuR binding to RNA in cells. A combined approach of NMR titration and molecular dynamics (MD) simulations suggests that 6a stabilizes HuR in a peculiar closed conformation, which is incompatible with RNA binding. Alpha screen and RNA-electrophoretic mobility shift assays (REMSA) data on newly synthesized compounds allowed, for the first time, the generation of structure activity relationships (SARs), thus providing a solid background for the generation of highly effective HuR disruptors.

In the fight against Glioblastoma Multiforme, recent literature data have highlighted that integrin alpha 5 beta 1 and p53 are part of convergent pathways in the control of glioma apoptosis. This observation prompted us to seek a molecule able to simultaneously modulate both target families. Analyzing the results of a previous virtual screening against murine double minute 2 protein (MDM2), we envisaged that Arg-Gly-Asp (RGD)-mimetic molecules could be inhibitors of MDM2/4. Herein, we present the discovery of compound 7, which inhibits both MDM2/4 and alpha 5 beta 1/alpha v beta 3 integrins. A lead optimization campaign was carried out on 7 with the aim to preserve the activities on integrins while improving those on MDM proteins. Compound 9 turned out to be a potent MDM2/4 and alpha 5 beta 1/alpha v beta 3 blocker. In p53 -wild type glioma cells, 9 arrested cell cycle and proliferation and strongly reduced cell invasiveness, emerging as the first molecule of a novel class of integrin/MDM inhibitors, which might be especially useful in subpopulations of patients with glioblastoma expressing a functional p53 concomitantly with a high level of alpha 5 beta 1 integrin.

We have recently proposed a novel drug discovery approach based on biophysical screening of focused positional scanning libraries in which each element of the library contained a common binding moiety for the given target or class of targets. In this Letter, we report on the implementation of this approach to target metal containing proteins. In our implementation, we first derived a focused positional scanning combinatorial library of peptide mimetics (of approximately 100,000 compounds) in which each element of the library contained the metal-chelating moiety hydroxamic acid at the C-terminal. Screening of this library by nuclear magnetic resonance spectroscopy in solution allowed the identification of a novel and selective compound series targeting MMP-12. The data supported that our general approach, perhaps applied using other metal chelating agents or other initial binding fragments, may result very effective in deriving novel and selective agents against metalloenzyme.

Catalysis of collagen degradation by matrix metalloproteinase 1 (MMP-1) has been proposed to critically rely on flexibility between the catalytic (CAT) and hemopexin-like (HPX) domains. A rigorous assessment of the most readily accessed conformations in solution is required to explain the onset of substrate recognition and collagenolysis. The present study utilized paramagnetic NMR spectroscopy and small angle x-ray scattering (SAXS) to calculate the maximum occurrence (MO) of MMP-1 conformations. The MMP-1 conformations with large MO values (up to 47%) are restricted into a relatively small conformational region. All conformations with high MO values differ largely from the closed MMP-1 structures obtained by x-ray crystallography. The MO of the latter is ∼20%, which represents the upper limit for the presence of this conformation in the ensemble sampled by the protein in solution. In all the high MO conformations, the CAT and HPX domains are not in tight contact, and the residues of the HPX domain reported to be responsible for the binding to the collagen triple-helix are solvent exposed. Thus, overall analysis of the highest MO conformations indicated that MMP-1 in solution was poised to interact with collagen and then could readily proceed along the steps of collagenolysis. Background: Matrix metalloproteinase-1 (MMP-1) collagenolysis relies on interdomain flexibility. Results: In all high maximum occurrence conformations, the MMP-1 hemopexin-like domain residues reported responsible for binding to the collagen triple-helix are solvent exposed. Conclusion: MMP-1 in solution is poised to interact with collagen and proceed along the steps of collagenolysis. Significance: The maximum occurrence approach can evaluate the predominant domain conformations for numerous multidomain enzymes.

A hydrolytically stable mimetic of the tumour antigen GM3 lactone is used to decorate multivalent scaffolds. Two of them positively interfere on melanoma cell adhesion, migration and resistance to apoptosis (anoikis). Notably, their ability to hamper melanoma-cells adhesion and reduce the metastatic potential is enhanced when the two scaffolds, presenting a different shape, are used in combination. © 2013 Elsevier Ltd.All rights reserved.

The function of p53 protein, also known as "genome guardian", might be impaired by the overexpression of its primary cellular inhibitor, the murine double minute 2 protein (MDM2). However, the recent finding that MDM2-selective inhibitors induce high levels of its homologue MDM4, prompt us to identify, through a receptor-based virtual screening on an in house database, dual MDM2/MDM4 binders. Compound 1 turned out to possess an IC50 of 93.7 and of 4.6 nM on MDM2 and MDM4, respectively. A series of compounds were synthesized to optimize its activity on MDM2. As a result, compound 12 showed low nanomolar IC50 for both targets. NMR studies confirmed the pocket of binding of 12 as predicted by the Glide docking software. Notably, 12 was able to cause concentration-dependent inhibition of cell proliferation, yielding an IC50 value of 356 ± 21 nM in neuroblastoma SHSY5Y cells and proved even to efficiently block cancer stem cell growth. The function of p53 protein, also known as "genome guardian", might be impaired by the overexpression of its primary cellular inhibitor, the murine double minute 2 protein (MDM2). However, the recent finding that MDM2-selective inhibitors induce high levels of its homologue MDM4, prompt us to identify, through a receptor-based virtual screening on an in house database, dual MDM2/MDM4 binders. Compound 1 turned out to possess an IC50 of 93.7 and of 4.6 nM on MDM2 and MDM4, respectively. A series of compounds were synthesized to optimize its activity on MDM2. As a result, compound 12 showed low nanomolar IC50 for both targets. NMR studies confirmed the pocket of binding of 12 as predicted by the Glide docking software. Notably, 12 was able to cause concentration-dependent inhibition of cell proliferation, yielding an IC50 value of 356 ± 21 nM in neuroblastoma SHSY5Y cells and proved even to efficiently block cancer stem cell growth.

Human S100A14 is a member of the EF-hand calcium-binding protein family that has only recently been described in terms of its functional and pathological properties. The protein is overexpressed in a variety of tumor cells and it has been shown to trigger receptor for advanced glycation end products (RAGE)-dependent signaling in cell cultures. The solution structure of homodimeric S100A14 in the apo state has been solved at physiological temperature. It is shown that the protein does not bind calcium(II) ions and exhibits a "semi-open" conformation that thus represents the physiological structure of the S100A14. The lack of two ligands in the canonical EF-hand calcium(II)-binding site explains the negligible affinity for calcium(II) in solution, and the exposed cysteines and histidine account for the observed precipitation in the presence of zinc(II) or copper(II) ions.

Targeting the receptor for the advanced glycation endproducts (RAGE) signalling has a potential for the prevention and treatment of several pathologies. Extracellular activation of RAGE triggers the interactions of the RAGE cytoplasmic tail with intracellular protein partners. Here the cytoplasmic tail of RAGE has been investigated by NMR as part of the full-length protein, in the presence of a membrane-mimicking environment. The isolated cytoplasmic tail has also been studied for comparison. The NMR spectra of the whole receptor show that some but not all residues belonging to the C-terminal region of the cytoplasmic tail have a large flexibility, while the membrane proximal region seems to be rigidly connected to the trans-membrane domain and ectodomains. The analysis indicates that the behavior of the cytoplasmic tail is strongly affected by its being part of the whole receptor. These results provide new insight towards the understanding of signal transduction by RAGE.

Cell surface proteolysis is an integral yet poorly understood physiological process. The present study has examined how the pericellular collagenase membrane-type 1 matrix metalloproteinase (MT1-MMP) and membrane-mimicking environments interplay in substrate binding and processing. NMR derived structural models indicate that MT1-MMP transiently associates with bicelles and cells through distinct residues in blades III and IV of its hemopexin-like domain, while binding of collagen-like triple-helices occurs within blades I and II of this domain. Examination of simultaneous membrane interaction and triple-helix binding revealed a possible regulation of proteolysis due to steric effects of the membrane. At bicelle concentrations of 1%, enzymatic activity towards triple-helices was increased 1.5-fold. A single mutation in the putative membrane interaction region of MT1-MMP (Ser466Pro) resulted in lower enzyme activation by bicelles. An initial structural framework has thus been developed to define the role(s) of cell membranes in modulating proteolysis.

Among protein immobilization strategies, encapsulation in bioinspired silica is increasingly popular. Encapsulation offers high yields and the solid support is created through a protein-catalyzed polycondensation reaction that occurs under mild conditions. An integrated strategy is reported for the characterization of both the protein and bioinspired silica scaffold generated by the encapsulation of enzymes with an external silica-forming promoter or with the promoter expressed as a fusion to the enzyme. This strategy is applied to the catalytic domain of matrix metalloproteinase 12. Analysis reveals that the structure of the protein encapsulated by either method is not significantly altered with respect to the native form. The structural features of silica obtained by either strategy are also similar, but differ from those obtained by other approaches. In case of the covalently linked R5–enzyme construct, immobilization yields are higher. Encapsulation through a fusion protein, therefore, appears to be the method of choice.

Nucleic acids represent the alphabet of the cellular language and through their sequence and topology regulate vital cellular functions. In recent years, it has been found that many variations from the Watson–Crick duplex structure play key roles in many cellular processes. Examples are hairpins, cruciforms, parallel-stranded duplexes, triplexes, G-quadruplexes, and the i-motif. These structures can be formed by nucleotide sequences distributed throughout the whole human genome, their location is not random and often associated with human diseases. These complexes are formed from one to four strands, stabilized by base stacking and hydrogen bond interactions, with a variety of non-standard pairings. For instance, DNA triplexes can present G:G-C, A:A-T, C:G-C, and T:A-T pairings, with two strands in the standard Watson–Crick duplex structure (i.e. G-C and A-T) and the third one lying in the major groove of the duplex. In contrast, G-quadruplexes are four-stranded structures stabilized by stacking of two or more guanine tetrads (Figure 1). These examples highlight the structural polymorphism of DNA and suggest that other structures might exist, perhaps with specific cellular functions that are, to date, unknown. Herein, using metadynamics simulations, we have identified a stable folding intermediate of the thrombin binding aptamer (TBA) quadruplex. This intermediate is characterized by a “G-triplex” structure, having G:G:G triad planes stabilized by an array of Hoogsteen-like hydrogen-bonds (Figure 1). This kind of structure has been already hypothesized in other investigations on different DNA sequences, but never experimentally proven. Herein, for the first time, we have structurally and thermodynamically characterized this DNA structural motif, through a combination of biophysical experiments. Well-tempered metadynamics simulations have been used to study the folding of TBA, which is a 15-mer oligonucleotide (5’-dGGTTGGTGTGGTTGG-3’) organized in an anti-parallel monomolecular G-quadruplex with a chairlike structure (Figure 2a). This structure consists of two Gtetrads, able to coordinate a metal ion at the center, connected by two TT loops and a single TGT loop. Metadynamics accelerates the sampling, adding a bias on a few degrees of freedom of the system, called collective variables (CVs). In such a way, long time scale events, such as ligand/protein docking or protein/DNA folding, can be sampled in an affordable computational time and the free energy surface (FES) of the process can be computed. In the present case, the FES was calculated as a function of two CVs, the radius of gyration CV defined by the oxygen atoms of the guanines forming the G-tetrads and a second CV that counts the number of hydrogen bonds between these guanines (see Supporting Information). Looking at the FES obtained after approximately 80 ns of metadynamics simulation, three main energy minima can be identified (Figure 2b). The deepest one, basin A, corresponds to the experimental G-quadruplex structure of TBA. In the second minimum, basin B, TBA shows a partial opening of the 3’ end with residue G15

Pseudocontact shifts (pcs) and paramagnetic residual dipolar couplings (rdc) provide structural information that can be used to assess the adequacy of a crystallographic structure to represent the solution structure of a protein. This can be done by attaching a lanthanide binding tag to the protein. There are cases in which only local rearrangements are sufficient to match the NMR data and cases where significant secondary structure or domain rearrangements from the solid state to the solution state are needed. We show that the two cases are easily distinguishable. Whereas the use of solution restraints in the latter case is described in the literature, here we deal with how to obtain a better model of the solution structure in a case (the catalytic domain of the matrix metalloproteinase MMP-1) of the former class.

Paramagnetic NMR data can be profitably incorporated in structural refinement protocols of metalloproteins or metal-substituted proteins, mostly as distance or angle restraints. However, they could in principle provide much more information, because the magnetic susceptibility of a paramagnetic metal ion is largely determined by its coordination sphere. This information can in turn be used to evaluate changes occurring in the coordination sphere of the metal when ligands (e.g.: inhibitors) are bound to the protein. This gives an experimental handle on the molecular structure in the vicinity of the metal which falls in the so-called blind sphere. The magnetic susceptibility anisotropy tensors of cobalt(II) and nickel(II) ions bound to human carbonic anhydrase II in free and inhibited forms have been determined. The change of the magnetic susceptibility anisotropy is directly linked to the binding mode of different ligands in the active site of the enzyme. Indication about the metal coordination sphere in the presence of an inhibitor in pharmaceutically relevant proteins could be important in the design of selective drugs with a structure-based approach.

Proton-detection in solid-state NMR, enabled by high magnetic fields (>18 T) and fast magic angle spinning (>50 kHz), allows for the acquisition of traditional 1H-15N experiments on systems that are too big to be observed in solution. Among those, proteins entrapped in a bioinspired silica matrix are an attractive target that is receiving a large share of attention. We demonstrate that 1H-detected SSNMR provides a novel approach to the rapid assessment of structural integrity in proteins entrapped in bioinspired silica.