Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners

Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagens and elastin, which is the first step of the cross-linking of these extracellular matrix proteins. It is secreted as a proenzyme activated by bone morphogenetic protein-1, which releases the LOX catalytic domain and its bioactive N-terminal propeptide. We characterized the recombinant human propeptide by circular dichroism, dynamic light scattering, and small-angle X-ray scattering (SAXS), and showed that it is elongated, monomeric, disordered and flexible (Dmax: 11.7 nm, Rg: 3.7 nm). We generated 3D models of the propeptide by coarse-grained molecular dynamics simulations restrained by SAXS data, which were used for docking experiments. Furthermore, we have identified 17 new binding partners of the propeptide by label-free assays. They include four glycosaminoglycans (hyaluronan, chondroitin, dermatan and heparan sulfate), collagen I, cross-linking and proteolytic enzymes (lysyl oxidase-like 2, transglutaminase-2, matrix metalloproteinase-2), a proteoglycan (fibromodulin), one growth factor (Epidermal Growth Factor, EGF), and one membrane protein (tumor endothelial marker-8). This suggests new roles for the propeptide in EGF signaling pathway

Некоторые аспекты эффективности обучения при переходе от очной формы обучения к дистанционной

ВЫСШЕЕ МЕДИЦИНСКОЕ ОБРАЗОВАНИЕОБРАЗОВАНИЕ МЕДИЦИНСКОЕ /МЕТОДЫОЧНАЯ ФОРМА ОБУЧЕНИЯЗАОЧНАЯ ФОРМА ОБУЧЕНИЯДИСТАНЦИОННОЕ ОБУЧЕНИЕ /МЕТОДЫОБРАЗОВАТЕЛЬНЫЙ ПРОЦЕССХИМИЯ (ДИСЦИПЛИНА

Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA

15 p.-6 fig.An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin.National Science Centre [2012/04/A/NZ1/00048 to I.K.;2017/26/D/NZ1/00239 to K.W.]; Foundation for Polish Science [TEAM, POIR.04.04.00-00-5C75/17-00 to I.K.]; International Institute of Molecular and Cell Biology in Warsaw (to J.M.B.); Ministerio de Economía,Industria y Competitividad (MINECO/AEI) [BIO2012-30852, RTI2018-094549-B-I00 to R.G.]. Funding for open access charge: Foundation for Polish Science [TEAM,POIR.04.04.00-00-5C75/17-00].Peer reviewe

Docking software performance in protein-glycosaminoglycan systems

International audienceGlycosaminoglycans (GAGs) are a diverse group of linear anionic periodic polysaccharides that participate in many biological processes through the regulation of their protein partners activity. They are produced by many cell types and are found in the extracellular space as well as on cell surfaces, where they play an important role in mediation of cell-extracellular matrix interactions. Crystallization of protein-GAG complexes is difficult, therefore molecular docking can be a useful technique for predicting the binding conformation and understanding specific interactions in protein-GAG systems. At the same time, GAGs are also very challenging ligands for docking due to their high flexibility, periodicity and charged nature. Previously, we tested six different molecular docking software in terms of the performance on the protein-GAG complexes. In this study, we further performed docking simulations with other eight open access docking programs (Dock, rDock, ClusPro, PLANTS, HADDOCK, Hex, SwissDock and ATTRACT) for a dataset of 28 protein-GAG complexes with experimentally available structures, where a GAG ligand was longer than a trimer. Our results showed that Dock yielded the best prediction of a GAG binding pose, and its performance was independent of a GAG length. Overall, although the ligand binding poses could be correctly predicted in many cases by the tested docking programs, the ranks of the docking poses are often poorly assigned. Further comparison of the performance of fourteen docking programs, eight of which were analyzed in this study and six in the previous one, with the binding free energy components calculated for the corresponding experimental complexes allowed us to establish which binding free energy patterns define the success of each of these docking programs. Our work contributes to the evaluation of computational tools that could be used specifically to decipher protein-GAG interactions

Hydroxyethylene isosteres introduced in type II collagen fragments substantially alter the structure and dynamics of class II MHC A(q)/glycopeptide complexes

Class II major histocompatibility complex (MHC) proteins are involved in initiation of immune responses to foreign antigens via presentation of peptides to receptors of CD4(+) T-cells. An analogous presentation of self-peptides may lead to autoimmune diseases, such as rheumatoid arthritis (RA). The glycopeptide fragment CII259-273, derived from type II collagen, is presented by A(q) MHCII molecules in the mouse and has a key role in development of collagen induced arthritis (CIA), a validated model for RA. We have introduced hydroxyethylene amide bond isosteres at the Ala(261)-Gly(262) position of CII259-273. Biological evaluation showed that A(q) binding and T cell recognition were dramatically reduced for the modified glycopeptides, although static models predicted similar binding modes as the native type II collagen fragment. Molecular dynamics (MD) simulations demonstrated that introduction of the hydroxyethylene isosteres disturbed the entire hydrogen bond network between the glycopeptides and A(q). As a consequence the hydroxyethylene isosteric glycopeptides were prone to dissociation from A(q) and unfolding of the beta(1)-helix. Thus, the isostere induced adjustment of the hydrogen bond network altered the structure and dynamics of A(q)/glycopeptide complexes leading to the loss of A(q) affinity and subsequent T cell response

Hydroxyethylene isosteres introduced in type II collagen fragments substantially alter the structure and dynamics of class II MHC A(q)/glycopeptide complexes

Class II major histocompatibility complex (MHC) proteins are involved in initiation of immune responses to foreign antigens via presentation of peptides to receptors of CD4(+) T-cells. An analogous presentation of self-peptides may lead to autoimmune diseases, such as rheumatoid arthritis (RA). The glycopeptide fragment CII259-273, derived from type II collagen, is presented by A(q) MHCII molecules in the mouse and has a key role in development of collagen induced arthritis (CIA), a validated model for RA. We have introduced hydroxyethylene amide bond isosteres at the Ala(261)-Gly(262) position of CII259-273. Biological evaluation showed that A(q) binding and T cell recognition were dramatically reduced for the modified glycopeptides, although static models predicted similar binding modes as the native type II collagen fragment. Molecular dynamics (MD) simulations demonstrated that introduction of the hydroxyethylene isosteres disturbed the entire hydrogen bond network between the glycopeptides and A(q). As a consequence the hydroxyethylene isosteric glycopeptides were prone to dissociation from A(q) and unfolding of the beta(1)-helix. Thus, the isostere induced adjustment of the hydrogen bond network altered the structure and dynamics of A(q)/glycopeptide complexes leading to the loss of A(q) affinity and subsequent T cell response