The transition state of coupled folding and binding for a flexible β-finger.
Flexible and fully disordered protein regions that fold upon binding mediate numerous protein-protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a β-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the β-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the β-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the β-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.
Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden.
O Andreas
OA
Karlsson
Celestine N
CN
Chi
Ake
A
Engström
Per
P
Jemth
D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D054731:PDZ Domains; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding
C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C496320:NOS1 protein, human; D052248:Nitric Oxide Synthase Type I