Structural characterization of type II dockerin module from the cellulosome of Clostridium thermocellum: calcium-induced effects on conformation and target recognition.
The assembly of a functional cellulose-degrading complex termed the cellulosome involves two specific calcium-dependent cohesin-dockerin interactions: type I and type II. Extensive structural and mutagenesis studies have been performed on the type I modules and their interaction in an attempt to identify the underlying molecular determinants responsible for this specificity. However, very little structural information exists for the type II interaction. We have performed a variety of biophysical studies on the type II dockerin-X-module modular pair (DocX), which comprises the C-terminal region of cellulosomal scaffoldin subunit from Clostridium thermocellum, to determine the effect of calcium on its structure and interaction with type II cohesin. Our results indicate that calcium binding to type II dockerin occurs with an apparent dissociation constant (K(d)) of 7 microM, induces stable secondary and tertiary structure, and leads to the exposure of a hydrophobic surface. Calcium binding also results in the homodimerization of DocX. Analytical ultracentrifugation experiments indicate that the DocX homodimer has an elongated shape and a K(d) of approximately 40 microM. However, addition of the SdbA type II cohesin binding partner led to the dissociation of the DocX homodimer and to the formation of a 1:1 heterodimer. We propose that the exposed hydrophobic surface forms, at least in part, the type II cohesin-binding site, which in the absence of cohesin results in the dimerization of DocX.
Adams
Jarrett J
JJ
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Webb
Bradley A
BA
Spencer
Holly L
HL
Smith
Steven P
SP
D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002118:Calcium / Q000737:chemistry*; D018797:Cell Cycle Proteins; D044603:Cellulosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D002868:Chromosomal Proteins, Non-Histone; D002942:Circular Dichroism; D003001:Cloning, Molecular; D048013:Acetivibrio thermocellus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins; D005822:Genetic Vectors; D008027:Light; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D012996:Solutions; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D000097722:Cohesins
D001426:Bacterial Proteins; D002118:Calcium; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D005656:Fungal Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D021122:Protein Subunits; D012996:Solutions; D014364:Tryptophan; D000097722:Cohesins; C080369:CipA protein, Clostridium; C105449:SdbA protein, bacteria