Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica EstruturalThe microbial plant cell wall degradation is one of the most important processes in the global turnover of atmospheric carbon dioxide. The work presented in this thesis addressed the cellulosomes of Clostridium thermocellum and Bacteroides cellulosolvens, essential to the process of cellulose degradation, and aimed to study some of the components involved in their architecture (cohesins and dockerins) and efficiency (Carbohydrate-Binding Modules - CBMs). For this I used a combination of Nuclear Magnetic Resonance (NMR), X-ray crystallography and computer modeling techniques. My objective was to help rationalize the molecular determinants of specificity of CBMs, including the CtCBMs of families 11, 30 and 44, and the mechanisms of molecular recognition between cohesins and dockerins. In Chapter I, I present a general introduction to the theme of degradation of plant cell walls, with special attention to the cellulosome and its components. In Chapter II, I discuss the structural characteristics of the CtCBM11 based on the structures obtained by NMR at 25 and 50 °C and the structure obtained by crystallography. I found that although similar, the structures show some differences, particularly regarding the binding cleft area, which explains the negative results obtained by co-crystallization. In Chapter III and IV I study the molecular determinants of specificity in modules CtCBM11, 30 and 44, based on NMR and computer modeling data. I found that the atoms of the cellooligosaccharides most important for binding are the ones at positions 2 and 6 of the central units of the ligands. Moreover, I characterized the mechanisms responsible for selection and binding of these modules to various substrates. I established that binding occurs by a mechanism for conformational selection, where the topology of the residues of the protein, the conformation of the ligand and the number of glucose units, play a fundamental role. Chapters V and VI reveal the determination of the 3D structure of the cohesin-module X-dockerin complex of C. thermocellum and the cohesin-dockerin complex of B. cellulosolvens, respectively. Both complexes belong to the type II and their analysis allowed obtaining important information about the structural features that define the cohesin-dockerin interaction. The structure belonging to C. thermocellum revealed that the module X is essential for the stability of the complex. Moreover, for the first time the 3D structure of a cohesin-dockerin complex from B. cellulosolvens was determined. In this complex the dockerin is rotated 180º when compared to other complexes. This gives the cellulosome plasticity. In the final chapters, I present the NMR and X-ray crystallography techniques I used throughout the study. Finally, I draw some general conclusions about all the work done.Fundação para a Ciência e Tecnologia - SFRH/BD/35992/2007, and projects PTDC/QUI/68286/2006, PTDC/QUI-BIQ/100359/2008 and PTDC/BIA-PRO/103980/200

The architecture of the 10-23 DNAzyme and its implications for DNA-mediated catalysis

Funding Information: The authors acknowledge access to the Jülich‐Düsseldorf Biomolecular NMR Center. HG is grateful for computational support and infrastructure provided by the ‘Zentrum für Informations‐ und Medientechnologie’ (ZIM) at the Heinrich Heine University Düsseldorf and the John von Neumann Institute for Computing (NIC) (user ID: HKF7, VSK33). We thank Hannah Rosenbach for providing activity data. This work was supported by the German Research Foundation (DFG) (103/4‐1, ET 103/4‐3, and the Heisenberg grant ET 103/5‐1) to ME, the Volkswagen Foundation to ME and HG (project no. 9B798) and the European Union‘s Horizon 2020 research and innovation program under the Marie Skłodowska‐Curie grant agreement no. 660258 to AV. Open Access funding enabled and organized by Projekt DEAL. Publisher Copyright: © 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.Understanding the molecular features of catalytically active DNA sequences, so-called DNAzymes, is essential not only for our understanding of the fundamental properties of catalytic nucleic acids in general, but may well be the key to unravelling their full potential via tailored modifications. Our recent findings contributed to the endeavour to assemble a mechanistic picture of DNA-mediated catalysis by providing high-resolution structural insights into the 10-23 DNAzyme (Dz) and exposing a complex interplay between the Dz's unique molecular architecture, conformational plasticity, and dynamic modulation by metal ions as central elements of the DNA catalyst. Here, we discuss key features of our findings and compare them to other studies on similar systems.publishersversionpublishe

Reconstitution and NMR Characterization of the Ion-Channel Accessory Subunit Barttin in Detergents and Lipid-Bilayer Nanodiscs

Barttin is an accessory subunit of ClC-K chloride channels expressed in the kidney and the inner ear. Main functions of ClC-K/barttin channels are the generation of the cortico-medullary osmotic gradients in the kidney and the endocochlear potential in the inner ear. Mutations in the gene encoding barttin, BSND, result in impaired urinary concentration and sensory deafness. Barttin is predicted to be a two helical integral membrane protein that directly interacts with its ion channel in the membrane bilayer where it stabilizes the channel complex, promotes its incorporation into the surface membrane and leads to channel activation. It therefore is an attractive target to address fundamental questions of intermolecular communication within the membrane. However, so far inherent challenges in protein expression and stabilization prevented comprehensive in vitro studies and structural characterization. Here we demonstrate that cell-free expression enables production of sufficient quantities of an isotope-labeled barttin variant (I72X Barttin, capable to promote surface membrane insertion and channel activation) for NMR-based structural studies. Additionally, we established purification protocols as well as reconstitution strategies in detergent micelles and phospholipid bilayer nanodiscs. Stability, folding, and NMR data quality are reported as well as a suitable assignment strategy, paving the way to its structural characterization

A dual cohesin-dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin- dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B. cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B. cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin-dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dualbinding mode interactions seems to play a pivotal role in the assembly of B. cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.publishersversionpublishe

Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - An NMR, X-ray crystallography and computational chemistry approach

12 pags, 6 figs, 1 tabThe direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies. © 2008 The Authors.The authors would like to thank the research network REQUIMTE (Project Reqmol), as well as the Portuguese Science and Technology Foundation (FCT-MCTES), for financial support through projectPTDC⁄QUI⁄68286⁄2006 and scholarships SFRH⁄BPD⁄27237⁄2006 and SFRH⁄BD⁄31359⁄200

Is prnt a pseudogene? identification of ram prt in testis and ejaculated spermatozoa

A hallmark of prion diseases or transmissible spongiform encephalopaties is the conversion of the cellular prion protein (PrPC), expressed by the prion gene (prnp), into an abnormally folded isoform (PrPSc) with amyloid-like features that causes scrapie in sheep among other diseases. prnp together with prnd (which encodes a prion-like protein designated as Doppel), and prnt (that encodes the prion protein testis specific - Prt) with sprn (shadow of prion protein gene, that encodes Shadoo or Sho) genes, constitute the "prion gene complex". Whereas a role for prnd in the proper functioning of male reproductive system has been confirmed, the function of prnt, a recently discovered prion family gene, comprises a conundrum leading to the assumption that ruminant prnt is a pseudogene with no protein expression. The main objective of the present study was to identify Prt localization in the ram reproductive system and simultaneously to elucidate if ovine prnt gene is transcribed into protein-coding RNA. Moreover, as Prt is a prnp-related protein, the amyloid propensity was also tested for ovine and caprine Prt. Recombinant Prt was used to immunize BALB/c mice, and the anti-Prt polyclonal antibody (APPA) immune response was evaluated by ELISA and Western Blot. When tested by indirect immunofluorescence, APPA showed high avidity to the ram sperm head apical ridge subdomain, before and after induced capacitation, but did not show the same behavior against goat spermatozoa, suggesting high antibody specificity against ovine-Prt. Prt was also found in the testis when assayed by immunohistochemistry during ram spermatogenesis, where spermatogonia, spermatocytes, spermatids and spermatozoa, stained positive. These observations strongly suggest ovine prnt to be a translated protein-coding gene, pointing to a role for Prt protein in the ram reproductive physiology. Besides, caprine Prt appears to exhibit a higher amyloid propensity than ovine Prt, mostly associated with its phenylalanine residue.publishersversionpublishe

Inhibition of ovine in vitro fertilization by anti-Prt antibody: hypothetical model for Prt/ZP interaction

BACKGROUND: The impact of prion proteins in the rules that dictate biological reproduction is still poorly understood. Likewise, the role of prnt gene, encoding the prion-like protein testis specific (Prt), in ram reproductive physiology remains largely unknown. In this study, we assessed the effect of Prt in ovine fertilization by using an anti-Prt antibody (APPA) in fertilization medium incubated with spermatozoa and oocytes. Moreover, a computational model was constructed to infer how the results obtained could be related to a hypothetical role for Prt in sperm-zona pellucida (ZP) binding. METHODS: Mature ovine oocytes were transferred to fertilization medium alone (control) or supplemented with APPA, or pre-immune serum (CSerum). Oocytes were inseminated with ovine spermatozoa and after 18 h, presumptive zygotes (n = 142) were fixed to evaluate fertilization rates or transferred (n = 374) for embryo culture until D6-7. Predicted ovine Prt tertiary structure was compared with data obtained by circular dichroism spectroscopy (CD) and a protein-protein computational docking model was estimated for a hypothetical Prt/ZP interaction. RESULTS: The fertilizing rate was lower (P = 0.006) in APPA group (46.0+/−6.79%) when compared to control (78.5+/−7.47%) and CSerum (64.5+/−6.65%) groups. In addition, the cleavage rate was higher (P < 0.0001) in control (44.1+/−4.15%) than in APPA group (19.7+/−4.22%). Prt CD spectroscopy showed a 22% alpha-helical structure in 30% (m/v) aqueous trifluoroethanol (TFE) and 17% alpha in 0.6% (m/v) TFE. The predominant alpha-helical secondary structure detected correlates with the predicted three dimensional structure for ovine Prt, which was subsequently used to test Prt/ZP docking. Computational analyses predicted a favorable Prt-binding activity towards ZP domains. CONCLUSIONS: Our data indicates that the presence of APPA reduces the number of fertilized oocytes and of cleaved embryos. Moreover, the CD analysis data reinforces the predicted ovine Prt trend towards an alpha-helical structure. Predicted protein-protein docking suggests a possible interaction between Prt and ZP, thus supporting an important role for Prt in ovine fertilization

Diverse specificity of cellulosome attachment to the bacterial cell surface

This work was supported by the EU FP7 programme under the WallTraC project (grant No. 263916) and by projects PTDC/BIA-MIC/5947/2014, RECI/BBB-BEP/0124/2012 and EXPL/BIA-MIC/1176/2012 supported by Fundacao para a Ciencia e Tecnologia (FCT-MCTES). The Research Unit UCIBIO (Unidade de Ciencias Biomoleculares Aplicadas) is financed by national funds from FCT/MCTES EC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). We thank the European Synchrotron Radiation Facility (Grenoble, France), Soleil (Saint-Aubin, France) and Diamond Light Source (Harwell, UK) for data collection and the European Community's Seventh Framework Programme (FP7/2007-2013) under BioStruct-X (grant agreement No. 283570, proposal number: Biostruct-X_ 4399) for funding.During the course of evolution, the cellulosome, one of Nature's most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.publishersversionpublishe

The power, pitfalls and potential of the nanodisc system for NMR-based studies

Membrane proteins act as a central interface between the extracellular environment and the intracellular response and as such represent one of the most important classes of drug targets. The characterization of the molecular properties of integral membrane proteins, such as topology and interdomain interaction, is key to a fundamental understanding of their function. Atomic force microscopy (AFM) and force spectroscopy have the intrinsic capabilities of investigating these properties in a near-native setting. However, atomic force spectroscopy of membrane proteins is traditionally carried out in a crystalline setup. Alternatively, model membrane systems, such as tethered bilayer membranes, have been developed for surface-dependent techniques. While these setups can provide a more native environment, data analysis may be complicated by the normally found statistical orientation of the reconstituted protein in the model membrane. We have developed a model membrane system that enables the study of membrane proteins in a defined orientation by single-molecule force spectroscopy. Our approach is demonstrated using cell-free expressed bacteriorhodopsin coupled to a quartz glass surface in a defined orientation through a protein anchor and reconstituted inside an artificial membrane system. This approach offers an effective way to study membrane proteins in a planar lipid bilayer. It can be easily transferred to all membrane proteins that possess a suitable tag and can be reconstituted into a lipid bilayer. In this respect, we anticipate that this technique may contribute important information on structure, topology, and intra- and intermolecular interactions of other seven-transmembrane helical receptors