NMR Dynamics-Derived Insights into the Binding Properties of a Peptide Interacting with an SH2 Domain

Abstract

The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.

Binding of Cbl to a Phospholipase Cγ1-docking Site on Platelet-derived Growth Factor Receptor β Provides a Dual Mechanism of Negative Regulation

“…Although the previously identified Cbl TKB domain-binding motifs on other Cbl targets are apparently Cbl-selective, Tyr 1021 on PDGFR␤ is a known binding site for PLC␥1 SH2 domains (42,43,50). Potential interaction of Cbl, a negative regulator, and PLC␥1, a positive effector, with the same phosphotyrosine-containing motif predicted that (1), unlike other Cbl-regulated PTKs such as ZAP70 or Syk (29,58), Tyr 3 Phe mutation of Tyr 1021 may not render PDGFR hyperactive because of concurrent loss of interaction with PLC␥1, and (2) Cbl binding to a PLC␥1-binding motif on PDGFR␤ could provide an additional mechanism for negative regulation of receptor signaling distinct from Cbl-mediated PDGFR ubiquitinylation and lysosomal sorting.…”

“…Competition between Cbl TKB and PLC␥1 SH2 Domain Binding to Phosphorylated PDGFR␤-Binding of Cbl TKB domain to a known PLC␥1 SH2 domain-binding site (42,43,50,51) raised the possibility that these proteins may compete for binding to PDGFR. We carried out in vitro competition studies with purified recombinant proteins to explore this possibility.…”

Compensatory and Long-Range Changes in Picosecond–Nanosecond Main-Chain Dynamics upon Complex Formation: 15N Relaxation Analysis of the Free and Bound States of the Ubiquitin-like Domain of Human Plexin-B1 and the Small GTPase Rac1

“…Remarkably, R ex contributions are almost constant across several regions, for example, β1 and β5, the two central β-strands with values of 3-4 Hz. Two regions with the highest R ex values (4)(5)(6)(7)(8), the N-terminus of the α1-helix and the α2-helix, are also close to each other in space, although only α2 is directly involved in GTPase binding (Fig. 4b), as determined by NMR cross saturation.…”

Direct 13C-detection for carbonyl relaxation studies of protein dynamics

“…But, for those same proteins, our experiments can be particularly useful for studies of ligand mobility in complex with large proteins. Specifically, for labeled peptide ligands, then 13 C-labeled peptides can be complexed with highly deuterated protein [39,40] The 13 C-detected experiments then provide a unique means to profile residual mobility of the bound ligand. Work is in progress along these lines.…”