title: Correlation of the amino-acid sequence and the 3D structure of the functional domain of EmaA from Aggregatibacter actinomycetemcomitans.
abstract: Adhesion to collagen is an important virulence determinant for the periodontal pathogen Aggregatibacter actinomycetemcomitans. Binding to collagen is mediated by the extracellular-matrix protein adhesin-A (EmaA). EmaA is a homotrimeric autotransporter protein that forms flexible antenna-like appendages on the bacterium surface. An ellipsoidal structure at the distal end of the appendage, composed of three subdomains, contains the functional domain of the molecule. A correlation between amino-acid sequence and subdomain structure (SI and SII) was proposed based on an analysis of the volume/molecular weight ratio. EmaA from three mutant strains (deletions of amino-acids 70-206 and 70-386 and a substitution mutation G162S) has been studied by electron microscopy to test this hypothesis. 3D structures were analyzed using single-axis tilt tomography of negatively stained preparations of bacteria combined with subvolume averaging. Additionally, a large number of 2D images of the apical domain of the adhesins from the mutants were extracted from micrographs of the bacterial surface, aligned and classified. The combined data showed that amino-acids 70-206 localize to subdomain SI and 70-386 comprise subdomains SI and SII. Moreover, we showed that the substitution mutation G162S, which abolishes collagen binding activity, does not affect the overall structural integrity of the functional domain. However, the structure of subdomain SI in this mutant is slightly altered with respect to the wild-type strain. These data also have allowed us to interpret the architectural features of each subdomain of EmaA in more detail and to correlate the 3D structure of the functional domain of EmaA with the amino-acid sequence.
authors.affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405, USA.
authors.forename: Fereshteh
authors.initials: F
authors.lastname: Azari
authors.forename: Michael
authors.initials: M
authors.lastname: Radermacher
authors.forename: Keith P
authors.initials: KP
authors.lastname: Mintz
authors.forename: Teresa
authors.initials: T
authors.lastname: Ruiz
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D055032:Electron Microscope Tomography; D021621:Imaging, Three-Dimensional; D008958:Models, Molecular; D015661:Pasteurellaceae; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
chemical_list: D018829:Adhesins, Bacterial