Protein flexibility and ligand rigidity: a thermodynamic and kinetic study of ITAM-based ligand binding to Syk tandem SH2.
The Syk tandem Src homology 2 domain (Syk tSH2) constitutes a flexible protein module involved in the regulation of Syk kinase activity. The Syk tSH2 domain is assumed to function by adapting the distance between its two SH2 domains upon bivalent binding to diphosphotyrosine ligands. A thermodynamic and kinetic analysis of ligand binding was performed by using surface plasmon resonance (SPR). Furthermore, the effect of binding on the Syk tSH2 structural dynamics was probed by hydrogen/deuterium exchange and electrospray mass spectrometry (ESI-MS). Two ligands were studied: 1, a flexible peptide derived from the tSH2 recognition ITAM sequence at the gamma chain of the FcepsilonRI-receptor, and 2, a ligand in which the amino acids between the two SH2 binding motifs in ligand 1 have been replaced by a rigid linker of comparable length. Both ligands display comparable affinity for Syk tSH2 at 25 degrees C, yet a major difference in thermodynamics is observed. Upon binding of the rigid ligand, 2, the expected entropy advantage is not realized. On the contrary, 2 binds with a considerably higher entropy price of approximately 9 kcal mol-1, which is attributed to a further decrease in protein flexibility upon binding to this rigid ligand. The significant reduction in deuterium incorporation in the Syk tSH2 protein upon binding of either 1 or 2, as monitored by ESI-MS, indicates a major reduction in protein dynamics upon binding. The results are consistent with a two-step binding model: after an initial binding step, a rapid structural change of the protein occurs, followed by a second binding step. Such a bivalent binding model allows high affinity and fast dissociation kinetics, which are very important in transient signal-transduction processes.
de Mol
Nico J
NJ
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University (UIPS), Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. n.j.demol@pharm.uu.nl
Catalina
M Isabel
MI
Dekker
Frank J
FJ
Fischer
Marcel J E
MJ
Heck
Albert J R
AJ
Liskamp
Rob M J
RM
D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017455:Receptors, IgE / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D020349:Surface Plasmon Resonance; D000072377:Syk Kinase; D013816:Thermodynamics; D018909:src Homology Domains
D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D010446:Peptide Fragments; D017455:Receptors, IgE; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase