Limited proteolysis of native proteins: the interaction between avidin and proteinase K.
Avidin is a tetramer of 16-kDa subunits that have a high affinity for biotin. Proteolysis of native apoavidin by proteinase K results in a limited attack at the loop between beta-strands 3 and 4, involving amino acids 38-43. Specifically, sites of proteolysis are at Thr 40-Ser 41 and Asn 42-Glu 43. The limited proteolysis results in an avidin product that remains otherwise intact and which has enhanced binding for 4'-hydroxyazobenzene-2-benzoic acid (HABA), a chromogenic reporter that can occupy the biotin-binding site. Saturation of the biotin-binding site with the natural ligand protects avidin from proteolysis, but saturation with HABA enhances the rate of proteolysis of the same site. Analysis of the three-dimensional structures of apoavidin and holoavidin reveals that the 3-4 loop is accessible to solvent and scores highly in an algorithm developed to identify sites of proteolytic attack. The structure of holoavidin is almost identical to the apoprotein. In particular, the 3-4 loop has the same structure in the apo and holo forms, yet there are marked differences in proteolytic susceptibility of this region. Evidence suggests that the 3-4 loop is rather mobile and flexible in the apoprotein, and that it becomes constrained upon ligand binding. In one crystal structure of the apoprotein, this loop appears constrained by contacts with symmetry-related molecules. Structural analyses suggest that the "lid" to the biotin-binding site, formed by the 3-4 loop, is displaced and made more accessible by HABA binding, thereby enhancing its proteolytic susceptibility.
Ellison
D
D
Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, United Kingdom.
Hinton
J
J
Hubbard
S J
SJ
Beynon
R J
RJ
D000595:Amino Acid Sequence; D001360:Avidin / Q000737:chemistry / Q000378:metabolism*; D001391:Azo Compounds / Q000378:metabolism / Q000494:pharmacology; D001665:Binding Sites; D001710:Biotin / Q000378:metabolism / Q000494:pharmacology; D002863:Chromogenic Compounds; D019286:Endopeptidase K; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000378:metabolism*
D001391:Azo Compounds; D002863:Chromogenic Compounds; D001360:Avidin; C002437:2-(4'-hydroxyazobenzene)benzoic acid; D001710:Biotin; D012697:Serine Endopeptidases; D019286:Endopeptidase K