Demonstration of long-range interactions in a PDZ domain by NMR, kinetics, and protein engineering.
Understanding the basis of communication within protein domains is a major challenge in structural biology. We present structural and dynamical evidence for allosteric effects in a PDZ domain, PDZ2 from the tyrosine phosphatase PTP-BL, upon binding to a target peptide. The NMR structures of its free and peptide-bound states differ in the orientation of helix alpha2 with respect to the remainder of the molecule, concomitant with a readjustment of the hydrophobic core. Using an ultrafast mixing instrument, we detected a deviation from simple bimolecular kinetics for the association with peptide that is consistent with a rate-limiting conformational change in the protein (k(obs) approximately 7 x 10(3) s(-1)) and an induced-fit model. Furthermore, the binding kinetics of 15 mutants revealed that binding is regulated by long-range interactions, which can be correlated with the structural rearrangements resulting from peptide binding. The homologous protein PSD-95 PDZ3 did not display a similar ligand-induced conformational change.
Gianni
Stefano
S
Istituto di Biologia e Patologia Molecolari del CNR, Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Università di Roma La Sapienza, Piazzale A. Moro 5, 00185 Rome, Italy.
Walma
Tine
T
Arcovito
Alessandro
A
Calosci
Nicoletta
N
Bellelli
Andrea
A
Engström
Ake
A
Travaglini-Allocatelli
Carlo
C
Brunori
Maurizio
M
Jemth
Per
P
Vuister
Geerten W
GW
D020816:Amino Acid Motifs; D001665:Binding Sites; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D017434:Protein Structure, Tertiary
D008024:Ligands; D010455:Peptides