Identification of the binding surface on beta-lactamase for GroEL by limited proteolysis and MALDI-mass spectrometry.
Escherichia coli beta-lactamase, alone or as a complex with GroEL at 48 degreesC, was partially digested with trypsin, endoproteinase Glu-C, or thermolysin. Peptides were analyzed by matrix-assisted laser desorption and ionization mass spectrometry and aligned with the known sequence. From the protease cleavage sites which become protected upon binding and those which become newly accessible, a model of the complex is proposed in which the carboxy-terminal helix has melted, two loops form the binding interface and the large beta-sheet become partially uncovered by the slight dislocation of other structural elements. This explains how hydrophobic surface on the substrate protein can become accessible while scarcely disrupting the hydrogen bond network of the native structure. An analysis of the GroEL-bound peptides bound after digestion of the beta-lactamase showed no obvious sequence motifs, indicating that binding is provided by hydrophobic patches in the three-dimensional structure.
Biochemisches Institut der Universität Zürich, Switzerland.
P
P
Gervasoni
W
W
Staudenmann
P
P
James
A
A
Plückthun
D001665:Binding Sites; D018834:Chaperonin 60 / Q000302:isolation & purification / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006868:Hydrolysis; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism*
D018834:Chaperonin 60; D010446:Peptide Fragments; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D013820:Thermolysin; D001618:beta-Lactamases