List of Publications

    2019

    • "Buezo Montero S., Gabrieli P., Severini F., Picci L., Di Luca M., Forneris F., Facchinelli L., Ponzi M., Lombardo F., Arcà B."
      Analysis in a murine model points to IgG responses against the 34k2 salivary proteins from Aedes albopictus and Aedes aegypti as novel promising candidate markers of host exposure to Aedes mosquitoes
      PLoS Neglected Tropical Diseases, 13, e0007806. (2019) - PubMed
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      "BACKGROUND- Taking advantage of several factors, as worldwide trading, climatic changes and urbanization, Aedes mosquitoes are impressively expanding their geographic distribution. A paradigm is provided by the rapid global spreading of Aedes albopictus, a species that is a competent vector of several arboviral diseases (e.g. dengue, Zika, chikungunya) and has been responsible of quite a few outbreaks in the last decade. Historically, vector control always played a pivotal role for the containment of arthropod-borne diseases, and this appears especially crucial for arboviral diseases for which no effective vaccines or specific medications are available. Currently, host exposure to mosquitoes is indirectly evaluated by entomological methods; however, exploitation of human immune responses to mosquito salivary proteins is emerging as a relevant additional tool, with important epidemiological implications for the evaluation of mosquito-borne disease risk. This study provides preliminary but solid indications that the 34k2 salivary proteins from Ae. albopictus and Aedes aegypti may be suitable candidates for the development of serological assays to evaluate spatial and/or temporal variation of human exposure to Aedes vectors. Combined to the presently available tools to assess arboviral exposure/infection, this may be of great help for the development of a serological toolbox allowing for the simultaneous determination of human exposure to Aedes vectors and to the pathogens they transmit."


    • Ewans LJ, Colley A, Gaston-Massuet C, Gualtieri A, Cowley MJ, McCabe MJ, Anand D, Lachke S, Scietti L, Forneris F, Zhu Y, Ying K, Walsh C, Kirk EP, Miller D, Giunta C, Sillence D, Dinger M, Buckley M, Roscioli T.
      Pathogenic variants in PLOD3 result in a Stickler syndrome-like connective tissue disorder with vascular complications
      Journal of Medical Genetics, 56, 629-638. (2019) - PubMed
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      "BACKGROUND- Pathogenic PLOD3 variants cause a connective tissue disorder (CTD) that has been described rarely. We further characterise this CTD and propose a clinical diagnostic label to improve recognition and diagnosis of PLOD3-related disease.
      METHODS- Reported PLOD3 phenotypes were compared with known CTDs utilising data from three further individuals from a consanguineous family with a homozygous PLOD3 c.809C>T; p.(Pro270Leu) variant. PLOD3 mRNA expression in the developing embryo was analysed for tissue-specific localisation. Mouse microarray expression data were assessed for phylogenetic gene expression similarities across CTDs with overlapping clinical features.
      RESULTS- Key clinical features included ocular abnormalities with risk for retinal detachment, sensorineural hearing loss, reduced palmar creases, finger contractures, prominent knees, scoliosis, low bone mineral density, recognisable craniofacial dysmorphisms, developmental delay and risk for vascular dissection. Collated clinical features showed most overlap with Stickler syndrome with variable features of Ehlers-Danlos syndrome (EDS) and epidermolysis bullosa (EB). Human lysyl hydroxylase 3/PLOD3 expression was localised to the developing cochlea, eyes, skin, forelimbs, heart and cartilage, mirroring the clinical phenotype of this disorder.
      CONCLUSION- These data are consistent with pathogenic variants in PLOD3 resulting in a clinically distinct Stickler-like syndrome with vascular complications and variable features of EDS and EB. Early identification of PLOD3 variants would improve monitoring for comorbidities and may avoid serious adverse ocular and vascular outcomes."


    • Marabelli C.*, Marrocco B.*, Pilotto S:*, Chittori S.*, Picaud S., Marchese S., Ciossani G., Forneris F., Filippakopoulos P., Schoehn G., Rhodes D., Subramaniam S.**, Mattevi A.**
      A Tail-Based Mechanism Drives Nucleosome Demethylation by the LSD2/NPAC Multimeric Complex
      Cell Reports, 27, 387-399. (2019) *Equal Contribution - **Shared Corresponding Authors - PubMed
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      LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.


    • Angiolini F.*, Belloni E.*, Giordano M.*, Campioni M., Forneris F., Paronetto M.P., Lupia, M. Brandas C., Pradella D., Di Matteo A., Giampietro C., Jodice G., Luise C., Bertalot G., Freddi S., Malinverno M., Irimia M., Moulton J., Summerton J., Chiapparino A., Ghilardi C., Giavazzi R., Nyqvist D., Gabellini D., Dejana E., Cavallaro U., Ghigna C.
      A Novel L1 Isoform with Angiogenic Activity Generated by NOVA2-mediated Alternative Splicing
      eLife, 8, e44305. (2019) *Equal Contribution - PubMed

    • Canciani A., Catucci G., Forneris F.
      Structural characterization of the third scavenger receptor cysteine-rich domain of murine Neurotrypsin
      Protein Science, 28, 746-755. (2019) - PubMed
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      Neurotrypsin (NT) is a multi-domain serine protease of the nervous system with only one known substrate: the large proteoglycan Agrin. NT has seen to be involved in the maintenance/turnover of neuromuscular junctions and in processes of synaptic plasticity in the central nervous system. Roles which have been tied to its enzymatic activity, localized in the C-terminal serine-protease (SP) domain. However the purpose of NT's remaining 3-4 scavenger receptor cysteine-rich (SRCR) domains is still unclear. We have determined the crystal structure of the third SRCR domain of murine NT (mmNT-SRCR3), immediately preceding the SP domain and performed a comparative structural analysis using homologous SRCR structures. Our data and the elevated degree of structural conservation with homologous domains highlight possible functional roles for NT SRCRs. Computational and experimental analyses suggest the identification of a putative binding region for Ca2+ ions, known to regulate NT enzymatic activity. Furthermore, sequence and structure comparisons allow to single out regions of interest that, in future studies, might be implicated in Agrin recognition/binding or in interactions with as of yet undiscovered NT partners.


    • Scietti L.*, Campioni M.*, Forneris F.
      SiMPLOD, a structure-integrated database of collagen lysyl hydroxylase (LH/PLOD) enzyme variants
      Journal of Bone and Mineral Research, 34, 1376-1382. (2019) *Equal Contribution - Explore SiMPLOD - PubMed
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      PLOD genes encode for procollagen lysyl hydroxylase enzymes (LH/PLOD), a family of proteins essential for collagen biosynthesis. Several mutations affect these genes causing severe disorders, such as Ehlers-Danlos and Bruck syndrome, as well a connective tissue disease with phenotype resembling osteogenesis imperfecta caused by lack of LH3/PLOD3 functions. The recently determined three-dimensional structures of the full-length human LH3/PLOD3 isoform, together with the structure of a fragment of a viral LH/PLOD homolog, are now allowing molecular mapping of the numerous disease-causing mutations, providing insights often suitable for the interpretation of the resulting disease phenotypes. However, the added value of molecular structure interpretation is affected by the limited accessibility of complex molecular data to scientific communities lacking direct expertise in structural biology. In this work, we present SiMPLOD (Structurally-integrated database for Mutations of PLOD genes), a publicly-available manually-curated online database with an embedded molecular viewer interface for the visualization and interpretation of LH/PLOD mutations on available molecular models. Each SiMPLOD entry is accompanied by manual annotations extrapolated from literature references and comments about the localization of the amino acid variants on the molecular structure. Additional links to the appropriate online resources for clinically-relevant as well as biochemical data are also provided in a standardized format. The web application is available at http://fornerislab.unipv.it/SiMPLOD.


    • Falchetto M.*, Ciossani G.*, Scolari F., Di Cosimo A., Nenci S., Field L.M., Mattevi A., Zhou J.-J., Gasperi G.**, Forneris F.**
      Structural and biochemical evaluation of Ceratitis capitata OBP22 affinity for odorants involved in inter-sex communication
      Insect Molecular Biology, 28, 431-443. (2019) *Equal Contribution - **Shared Corresponding Authors - PubMed
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      In insects, odorant?binding proteins (OBPs) connect the peripheral sensory system to receptors of olfactory organs. Medfly Ceratitis capitata CcapObp22 shows 37% identity and close phylogenetic affinities with Drosophila melanogaster OBP69a/PB?PRP1 (pheromone?binding protein related protein 1). The CcapObp22 gene is transcribed in the antennae and maxillary palps, suggesting an active role in olfaction. Here, we recombinantly produced CcapObp22, obtaining a 13.5 kDa protein capable of binding multiple strongly hydrophobic terpene compounds, including medfly male pheromone components. The highest binding affinity (EC50 0.48 ?M) was to (E,E)???farnesene, one of the most abundant compounds in the male pheromone blend. This odorant was used in co?crystallization experiments, yielding the structure of CcapOBP22. The monomeric structure shows the typical OBP folding, constituted by six ??helical elements interconnected by three disulfide bridges. A C?terminal seventh ??helix constitutes the wall of a deep, L?shaped hydrophobic cavity. Analysis of the electron density in this cavity suggested trapping of farnesene in the crystal structure, although with partial occupancy. Superposition of the CcapOBP22 structure with related 7?helical OBPs highlights striking similarity in the organization of the C?terminal segment of these proteins. Collectively, our molecular and physiological data on medfly CcapOBP22 suggest its involvement in inter?sex olfactory communication.


    2018

    • Scietti L., Chiapparino A., De Giorgi F., Fumagalli M., Khoriauli L., Nergadze S., Basu S., Olieric V., Cucca L., Banushi B., Profumo A., Giulotto E., Gissen P., Forneris F.
      Molecular architecture of the multifunctional collagen lysyl hydroxylase and glycosyltransferase LH3
      Nature Communications, 9, 3163. (2018) - PubMed
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      Lysyl hydroxylases catalyze hydroxylation of collagen lysines, and sustain essential roles in extracellular matrix (ECM) maturation and remodeling. Malfunctions in these enzymes cause severe connective tissue disorders. Human lysyl hydroxylase 3 (LH3/PLOD3) bears multiple enzymatic activities, as it catalyzes collagen lysine hydroxylation and also their subsequent glycosylation. Our understanding of LH3 functions is currently hampered by lack of molecular structure information. Here, we present high resolution crystal structures of full-length human LH3 in complex with cofactors and donor substrates. The elongated homodimeric LH3 architecture shows two distinct catalytic sites at the N- and C-terminal boundaries of each monomer, separated by an accessory domain. The glycosyltransferase domain displays distinguishing features compared to other known glycosyltransferases. Known disease-related mutations map in close proximity to the catalytic sites. Collectively, our results provide a structural framework characterizing the multiple functions of LH3, and the molecular mechanisms of collagen-related diseases involving human lysyl hydroxylases.

      Press Release (in Italian) - Newspapers Articles (in Italian): 1, 2, 3, 4, 5


    • Buroni S.*, Scoffone V.C.*, Fumagalli M., Makarov V., Cagnone M., Trespidi G., De Rossi E., Forneris F., Riccardi G., Chiarelli L.R.
      Investigating the Mechanism of Action of Diketopiperazines Inhibitors of the Burkholderia cenocepacia Quorum Sensing Synthase CepI: A Site-Directed Mutagenesis Study
      Frontiers in Pharmacology, 9, 836. (2018) *Equal Contribution - PubMed
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      Quorum sensing (QS) is a bacterial intercellular communication process which controls the production of major virulence factors, such as proteases, siderophores, and toxins, as well as biofilm formation. Since the inhibition of this pathway reduces bacterial virulence, QS is considered a valuable candidate drug target, particularly for the treatment of opportunistic infections, such as those caused by Burkholderia cenocepacia in cystic fibrosis patients. Diketopiperazine inhibitors of the acyl homoserine lactone synthase CepI have been recently described. These compounds are able to impair the ability of B. cenocepacia to produce proteases, siderophores, and to form biofilm, being also active in a Caenorhabditis elegans infection model. However, the precise mechanism of action of the compounds, as well as their effect on the cell metabolism, fundamental for candidate drug optimization, are still not completely defined. Here, we performed a proteomic analysis of B. cenocepacia cells treated with one of these inhibitors, and compared it with a cepI deleted strain. Our results demonstrate that the effects of the compound are similar to the deletion of cepI, clearly confirming that these molecules function as inhibitors of the acyl homoserine lactone synthase. Moreover, to deepen our knowledge about the binding mechanisms of the compound to CepI, we exploited previously published in silico structural insights about this enzyme structure and validated different candidate binding pockets on the enzyme surface using site-directed mutagenesis and biochemical analyses. Our experiments identified a region near the predicted S-adenosylmethionine binding site critically involved in interactions with the inhibitor. These results could be useful for future structure-based optimization of these CepI inhibitors.


    2017

    2016

    • Gruber R., Rogerson C., Windpassinger C., Banushi B., Straatman-Iwanowska A., Hanley J., Forneris F., Strohal R., Ulz P., Crumrine D., Menon G.K., Blunder S., Schmuth M., Müller T., Smith H., Mills K., Kroisel P., Janecke A.R., Gissen P.
      Autosomal recessive Keratoderma-Ichthyosis-Deafness (ARKID) syndrome is caused by VPS33B mutations affecting Rab protein interaction and collagen modification
      Journal of Investigative Dermatology, 137, 32800-32807. (2016) - PubMed

    • Israyilova A., Buroni S., Forneris F., Scoffone V.C., Shixaliyev N.Q., Riccardi G., Chiarelli L.R.
      Biochemical characterization of Glutamate Racemase, a new candidate drug target against Burkholderia cenocepacia infections
      PLoS One, 11, e0167350. (2016) - PubMed

    • Speranzini V., Rotili D., Ciossani G., Pilotto S., Marrocco B., Forgione M., Lucidi A., Forneris F., Mehdipour P., Velankar S., Mai A., Mattevi A.
      Polymyxins and quinazolines are LSD1/KDM1A inhibitors with unusual structural features
      Science Advances, 2, e1601017. (2016) Watch the Movie - PubMed

    • Scoffone V.C.*, Chiarelli L.R.*, Makarov V.*, Brackman G., Israylova A., Azzalin A., Forneris F., Riabova O., Savina S., Coenye T., Riccardi G., Buroni S.
      Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo
      Scientific Reports, 6, 32487. (2016) *Equal Contribution - PubMed
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      Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia.


    • Palamini M., Canciani A., Forneris F.
      Identifying and visualizing macromolecular flexibility in structural biology
      Frontiers in Molecular Biosciences, 3, 47. (2016) - PubMed
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      Structural biology comprises a variety of tools to obtain atomic resolution data for the investigation of macromolecules. Conventional structural methodologies including crystallography, NMR and electron microscopy often do not provide sufficient details concerning flexibility and dynamics, even though these aspects are critical for the physiological functions of the systems under investigation. However, the increasing complexity of the molecules studied by structural biology (including large macromolecular assemblies, integral membrane proteins, intrinsically disordered systems, and folding intermediates) continuously demands in-depth analyses of the roles of flexibility and conformational specificity involved in interactions with ligands and inhibitors. The intrinsic difficulties in capturing often subtle but critical molecular motions in biological systems have restrained the investigation of flexible molecules into a small niche of structural biology. Introduction of massive technological developments over the recent years, which include time-resolved studies, solution X-ray scattering, and new detectors for cryo-electron microscopy, have pushed the limits of structural investigation of flexible systems far beyond traditional approaches of NMR analysis. By integrating these modern methods with powerful biophysical and computational approaches such as generation of ensembles of molecular models and selective particle picking in electron microscopy, more feasible investigations of dynamic systems are now possible. Using some prominent examples from recent literature, we review how current structural biology methods can contribute useful data to accurately visualize flexibility in macromolecular structures and understand its important roles in regulation of biological processes.


    • Banushi B., Forneris F.*, Straatman-Iwanowska A., Strange A., Lyne A., Rogerson C., Burden J.J., Heywood W.E., Hanley J., Doykov I., Straatman K.R., Smith H., Bem D., Kriston-Vizi J., Ariceta G., Risteli M., Wang C., Ardill R.E., Zaniew M., Latka-Grot J., Waddington S.N., Howe S.J., Ferraro F., Gjinovci A., Lawrence S., Marsh M., Girolami M., Bozec L., Mills K., Gissen P.*
      Regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis
      Nature Communications, 7, 12111. (2016) *Shared Corresponding Authors - PubMed
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      Post-translational modifications are necessary for collagen precursor molecules (procollagens) to acquire final shape and function. However, the mechanism and contribution of collagen modifications that occur outside endoplasmic reticulum and Golgi are not understood. We discovered that VIPAR, with its partner proteins, regulate sorting of Lysyl Hydroxylase 3 (LH3, also known as PLOD3) into newly-identified post-Golgi collagen IV carriers and that VIPAR-dependent sorting is essential for modification of lysines in multiple collagen types. Identification of structural and functional collagen abnormalities in cells and tissues from patients and murine models of the autosomal recessive multisystem disorder Arthrogryposis, Renal dysfunction and Cholestasis syndrome caused by VIPAR and VPS33B deficiencies confirmed our findings. Thus, regulation of post-Golgi LH3 trafficking is essential for collagen homeostasis and for the development and function of multiple organs and tissues.

      Press Release (in Italian) - Newspapers Articles (in Italian): 1, 2


    • Spadaro F.*, Scoffone V.C.*, Chiarelli L.R.**, Fumagalli M., Buroni S., Riccardi G., Forneris F.**
      The crystal structure of Burkholderia cenocepacia DfsA provides insights into substrate recognition and quorum sensing fatty acid biosynthesis
      Biochemistry, 55, 3241-3250. (2016) *Equal Contribution - **Shared Corresponding Authors - PubMed
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      Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes important for the bacterial virulence, such as biofilm formation or protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule into the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely-related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence.


    • Forneris F., Wu J., Xue X., Ricklin D., Lin Z., Sfyroera G., Tzekou A., Volokhina E., Granneman J.C.M., Hauhart R., Bertram P., Liszewski M.K., Atkinson J.P., Lambris J.D., Gros P.
      Regulators of complement activity mediate inhibitory mechanisms through a common C3b-binding mode
      The EMBO Journal, 35, 1133-1149. (2016) - PubMed
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      Regulators of complement activation (RCA) inhibit complement-induced immune responses on healthy host tissues. We present crystal structures of humanRCA(MCP, DAF, andCR1) and a smallpox virus homolog (SPICE) bound to complement component C3b. Our structural data reveal that up to four consecutive homologousCCPdomains (i-iv), responsible for inhibition, bind in the same orientation and extended arrangement at a shared binding platform on C3b. Large sequence variations inCCPdomains explain the diverse C3b-binding patterns, with limited or no contribution of some individual domains, while all regulators show extensive contacts with C3b for the domains at the third site. A variation of ~100° rotation around the longitudinal axis is observed for domains binding at the fourth site on C3b, without affecting the overall binding mode. The data suggest a common evolutionary origin for both inhibitory mechanisms, called decay acceleration and cofactor activity, with variable C3b binding through domains at sites ii, iii, and iv, and provide a framework for understanding RCA disease-related mutations and immune evasion.


      More info about Structural Biology of the Complement System


    • Savino S., Ferrandi E., Forneris F., Rovida S., Riva S., Monti D., Mattevi A.
      Structural and biochemical insights into 7b-hydroxysteroid dehydrogenase stereoselectivity
      Proteins, 84, 859-865. (2016) Cover Image - PubMed
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      Hydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo- and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7?-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP+, and the general architecture of the steroid binding site. Comparison with 7?-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the ?-selective enzyme, possibly leading to an inverted orientation of the bound substrate.

      An image from our paper was selected as cover image for this issue of proteins (Page C4):


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