title: Characterization of a double dockerin from the cellulosome of the anaerobic fungus Piromyces equi.
abstract: The assembly into supramolecular complexes of proteins having complementary activities is central to cellular function. One such complex of considerable biological and industrial significance is the plant cell wall-degrading apparatus of anaerobic microorganisms, termed the cellulosome. A central feature of bacterial cellulosomes is a large non-catalytic protein, the scaffoldin, which contains multiple cohesin domains. An array of digestive enzymes is incorporated into the cellulosome through the interaction of the dockerin domains, present in the catalytic subunits, with the cohesin domains that are present in the scaffoldin. By contrast, in anaerobic fungi, such as Piromyces equi, the dockerins of cellulosomal enzymes are often present in tandem copies; however, the identity of the cognate cohesin domains in these organisms is unclear, hindering further biotechnological development of the fungal cellulosome. Here, we characterise the solution structure and function of a double-dockerin construct from the P. equi endoglucanase Cel45A. We show that the two domains are connected by a flexible linker that is short enough to keep the binding sites of the two domains on adjacent surfaces, and allows the double-dockerin construct to bind more tightly to cellulosomes than a single domain and with greater coverage. The double dockerin binds to the GH3 beta-glucosidase component of the fungal cellulosome, which is thereby identified as a potential scaffoldin.
authors.affiliation: Institute for Cell and Molecular Biosciences, The University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
authors.forename: Tibor
authors.initials: T
authors.lastname: Nagy
authors.forename: Richard B
authors.initials: RB
authors.lastname: Tunnicliffe
authors.forename: Lee D
authors.initials: LD
authors.lastname: Higgins
authors.forename: Chris
authors.initials: C
authors.lastname: Walters
authors.forename: Harry J
authors.initials: HJ
authors.lastname: Gilbert
authors.forename: Mike P
authors.initials: MP
authors.lastname: Williamson
mesh_terms: D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry; D044603:Cellulosomes / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D020089:Piromyces / Q000254:growth & development / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides; D002480:Cellulase