title: Molecular determinants of ligand specificity in family 11 carbohydrate binding modules: an NMR, X-ray crystallography and computational chemistry approach.
abstract: The 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 A 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.
authors.affiliation: REQUIMTE-CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: Aldino
authors.initials: A
authors.lastname: Viegas
authors.forename: Natércia F
authors.initials: NF
authors.lastname: Brás
authors.forename: Nuno M F S A
authors.initials: NM
authors.lastname: Cerqueira
authors.forename: Pedro Alexandrino
authors.initials: PA
authors.lastname: Fernandes
authors.forename: José A M
authors.initials: JA
authors.lastname: Prates
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
authors.forename: Marta
authors.initials: M
authors.lastname: Bruix
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Romão
authors.forename: Ana Luísa
authors.initials: AL
authors.lastname: Carvalho
authors.forename: Maria João
authors.initials: MJ
authors.lastname: Ramos
authors.forename: Anjos L
authors.initials: AL
authors.lastname: Macedo
authors.forename: Eurico J
authors.initials: EJ
authors.lastname: Cabrita
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002475:Cellobiose / Q000737:chemistry / Q000378:metabolism*; D002480:Cellulase / Q000737:chemistry / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D013780:Tetroses / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; D009844:Oligosaccharides; D013780:Tetroses; C473132:cellulosome, Clostridium; D002475:Cellobiose; C099423:cellohexaose; C048468:cellotetraose; D002482:Cellulose; D002480:Cellulase