1,721,038 research outputs found
Expanding the structural biology toolbox with single-molecule holography
No full textThe discovery of the double-helical structure of DNA is generally considered to be the milestone discovery that led to the birth of molecular biology. Since then, the impact of structural studies on all aspects of biology, from the understanding of basic cellular processes to the development of drugs and vaccines, can be hardly overestimate
Enzymes Without Borders: Mobilizing Substrates, Delivering Products
No full textMany cellular reactions involve both hydrophobic and hydrophilic molecules that reside within the chemically distinct environments defined by the phospholipid-based membranes and the aqueous lumens of cytoplasm and organelles. Enzymes performing this type of reaction are required to access a lipophilic substrate located in the membranes and to catalyze its reaction with a polar, water-soluble compound. Here, we explore the different binding strategies and chemical tricks that enzymes have developed to overcome this problem. These reactions can be catalyzed by integral membrane proteins that channel a hydrophilic molecule into their active site, as well as by water-soluble enzymes that are able to capture a lipophilic substrate from the phospholipid bilayer. Many chemical and biological aspects of this type of enzymology remain to be investigated and will require the integration of protein chemistry with membrane biology
Modeling of Protein Complexes
No full text: The recent advances in structural biology, combined with continuously increasing computational capabilities and development of advanced softwares, have drastically simplified the workflow for protein homology modeling. Modeling of individual proteins is nowadays quick and straightforward for a large variety of protein targets, thanks to guided pipelines relying on advanced computational tools and user-friendly interfaces, which have extended and promoted the use of modeling also to scientists not focusing on molecular structures of proteins. Nevertheless, construction of models of multi-protein complexes remains quite challenging for the non-experts, often due to the usage of specific procedures depending on the system under investigation and the need for experimental validation approaches to strengthen the generated output.In this chapter, we provide a brief overview of the approaches enabling generation of multi-protein complex models starting from homology models of individual protein components. Using real-life examples, we include two examples to guide the reader in the generation of homomeric and heteromeric protein models
Full‐Length Human Collagen Lysyl Hydroxylases
No full textProcollagen lysyl hydroxylases and glycosyltransferases (LH, also known as procollagen lysyl‐2‐oxoglutarate dioxygenases (PLOD)) are essential biosynthesis enzymes present in all collagen‐containing organisms, from sponges to humans. Higher vertebrates present three separate PLOD genes encoding for distinct enzyme isoforms (LH1, LH2a/b, and LH3), sharing ∼70% amino acid sequence identity. The LH1 and LH2 isoforms exclusively display Fe2+, 2‐oxoglutarate‐dependent lysyl 5‐hydroxylase activity, whereas LH3 is a multifunctional enzyme, able to further catalyze the Mn2+‐dependent β‐(1,O)‐galactosylation and the subsequent α‐(1,2)‐glucosylation of 5‐hydroxylysines. Despite exclusive selectivity for lysine residues within collagenous polypeptides, little is known about the specificity of LH enzymes for different amino acid sequences in different collagen types: LH1 and LH3 isoforms act on collagen triple‐helical regions, whereas the LH2 isoform specifically hydroxylates collagen telopeptides, yet no consensus sequences, nor minimum sequence lengths, have been proposed as requirements for catalysis. Available crystal structures of full‐length human LH3 show an elongated homodimeric quaternary structure, with three aligned domains constituting each enzyme's polypeptide: the N‐terminal glycosyltransferase (GT) domain, a central noncatalytic accessory (AC) domain, and a C‐terminal lysyl hydroxylase (LH) domain. Dimerization occurs in the C‐terminal domain, in proximity to the LH catalytic site. Dimerization is indeed essential for LH activity, but is dispensable for the glycosyltransferase activities of LH3
Collagen hydroxylysine glycosylation: non-conventional substrates for atypical glycosyltransferase enzymes
No full textCollagen is a major constituent of the extracellular matrix (ECM) that confers fundamental mechanical properties to tissues. To allow proper folding in triple-helices and organization in quaternary super-structures, collagen molecules require essential post-translational modifications (PTMs), including hydroxylation of proline and lysine residues, and subsequent attachment of glycan moieties (galactose and glucose) to specific hydroxylysine residues on procollagen alpha chains. The resulting galactosyl-hydroxylysine (Gal-Hyl) and less abundant glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl) are amongst the simplest glycosylation patterns found in nature and are essential for collagen and ECM homeostasis. These collagen PTMs depend on the activity of specialized glycosyltransferase enzymes. Although their biochemical reactions have been widely studied, several key biological questions about the possible functions of these essential PTMs are still missing. In addition, the lack of three-dimensional structures of collagen glycosyltransferase enzymes hinders our understanding of the catalytic mechanisms producing this modification, as well as the impact of genetic mutations causing severe connective tissue pathologies. In this mini-review, we summarize the current knowledge on the biochemical features of the enzymes involved in the production of collagen glycosylations and the current state-of-the-art methods for the identification and characterization of this important PTM
Phasing protein structures using the group-subgroup relation
No full textDiffraction data from two non-isomorphous crystals (forms 1 and 2) of an artificial protein with a four-helix bundle motif, di-Co(II)-DF1-L13A, have been collected using synchrotron radiation. The phase of form 1 has been assigned using the group and minimal non-isomorphic supergroup relation between the space group of the previously determined di-Mn(II)-DF1-L13G structure and the space group of this form. This unconventional method of solving the phase problem has also been tested with form 2 using a reverse relation. The structure of the latter form has been solved using the group and maximal non-isomorphic subgroup relation with the space group of form 2 of the analogous dimanganese protein. This application has shown that this phasing method can be used for solving the protein structures of polymorphic crystals as an alternative to the molecular-replacement method
Identifying and Visualizing Macromolecular Flexibility in Structural Biology
Full text linkStructural 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
Structural characterization of the third scavenger receptor cysteine-rich domain of murine Neurotrypsin
Full text linkNeurotrypsin (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. This article is protected by copyright. All rights reserved
Proteolysis, complex formation and conformational changes drive the complement pathways
No full textThe complement system is an important part of the mammalian immune defense in blood and interstitial fluids. This set of ~30 plasma proteins and receptors enables the host to recognize and clear invading pathogens and altered host cells, while protecting healthy host cells and tissues. Over the last 7 years, we have resolved the structural details of the central components of this system, which is referred to as the Alternative Pathway of complement activation, and deduced the molecular mechanisms that underlie the amplification and regulation of this protein network. In short, we revealed that large domain-domain rearrangements of these multi-domain proteins, upon proteolysis and complex formation, determine the specificity that provides a local and brief burst to mark targets cells for immune clearance. Most recently, we and others have revealed structural details of the Terminal Pathway that leads to pore formation by Membrane-Attack-Complexes in cell membranes yielding lysis
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