Cdc42Hs, a member of the Ras superfamily of GTP-binding proteins, initiates a cascade that begins with the activation of several kinases, including p21-activated kinase (PAK). We have previously determined the structure of Cdc42Hs and found that the regions involved in effector (Switch I) and regulator (Switch II) actions are partially disordered [Feltham, J. L., et al. (1997) Biochemistry 36, 8755-8766]. Recently, we used a 46-amino acid fragment of PAK (PBD46) to define the binding surface on Cdc42Hs, which includes the beta2 strand and a portion of Switch I [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Here we describe the backbone dynamics of three constructs of [(15)N]Cdc42Hs (GDP-, GMPPCP-, and GMPPCP- and PBD46-bound) using (15)N-(1)H NMR measurements of T(1), T(1)(rho), and the steady-state NOE at three magnetic field strengths. Residue-specific values of the generalized order parameters (S(s)(2) and S(f)(2)), local correlation time (tau(e)), and exchange rate (R(ex)) were obtained using the Lipari-Szabo model-free formalism. Residues in Switch I were found to exhibit high-amplitude (low-order) motions on a nanosecond time scale, whereas those in Switch II experience low-amplitude motion on the nanosecond time scale and chemical (conformational) exchange on a millisecond time scale. The Insert region of Cdc42Hs-GDP exhibits high-order, nanosecond motions; the time scale of motion in the Insert is reduced in Cdc42Hs-GMPPCP and Cdc42Hs-PBD46. Overall, significant flexibility was observed mainly in the regions of Cdc42Hs that are involved in protein-protein interactions (Switch I, Switch II, and Insert), and flexibility was reduced upon interaction with a protein ligand. These results suggest that protein flexibility is important for high-affinity binding interactions.
Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission. Although the structure of the GluR2 binding domain (S1S2) is well known (agonist binding site between two lobes), little is known about the time scales of conformational transitions or the relationship between dynamics and function.19 F NMR ( 19 F-labeled tryptophan) spectroscopy was used to monitor motions in the S1S2 domain bound to ligands with varying efficacy and in the apo state. One tryptophan (Trp-671) undergoes chemical exchange in some but not all agonists, consistent with s-ms motion. The dynamics can be correlated to ligand affinity, and a likely source of the motion is a peptide bond capable of transiently forming hydrogen bonds across the lobe interface. Another tryptophan (Trp-767) appears to monitor motions of the relative positions of the lobes and suggests that the relative orientation in the apo-and antagonist-bound forms can exchange between at least two conformations on the ms time scale. Ionotropic glutamate receptors (GluRs)2 mediate the majority of excitatory synaptic transmission in the central nervous system of higher vertebrates (1) and play important roles in the formation of synaptic plasticity underlying higher order processes such as learning and memory as well as in neuronal development (2). In addition, ionotropic GluRs have been implicated in various neurodegenerative disorders such as Parkinson and Alzheimer diseases, Huntington chorea, and neurologic disorders including epilepsy and ischemic brain damage. Antagonists of glutamate receptors have been shown to limit tumor growth in a variety of human tumors and to inhibit tumor cell migration (3). In recent years many advances in characterizing the relationship between ionotropic GluR structure and function have been made. Ionotropic GluRs are membrane-bound receptor ion channels composed of multiple subunits arranged as a rosette, forming a central ion channel in which each subunit contributes to pore formation. Individual subunits are categorized by pharmacological properties, sequence, functionality, and biological roles into those that are sensitive 1) to the synthetic agonist N-methyl-D-aspartic acid (NR1, NR2A-D, NR3A-B), 2) to the synthetic agonist ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA; GluR1-4), and 3) to the naturally occurring neurotoxin kainate (GluR5-7, KA1,2).The structural analysis of glutamate receptors was dramatically advanced by the finding that the extracellular agonist binding domain (S1S2 domain) could be expressed in isolation as a soluble protein (4) and by the subsequent solution of the crystal structure bound to kainate (5). As predicted by homology models (6), the S1S2 domain was found to be a bilobed structure with the agonist binding pocket located between the two lobes. The first glimpse of the structural basis of channel activation was the finding by Armstrong and Gouaux (7) that the apo state exhibited a considerably larger angle between the two lobes than the full agonist-bound...
Cdc42Hs is a signal transduction protein that is involved in cytoskeletal growth and organization. We describe here the methyl side chain dynamics of three forms of (2)H,(13)C,(15)N-Cdc42Hs [GDP-bound (inactive), GMPPCP-bound (active), and GMPPCP/PBD46-bound (effector-bound)] from (13)C-(1)H NMR measurements of deuterium T(1) and T(1 rho) relaxation times. A wide variation in flexibility was observed throughout the protein, with methyl axis order parameters (S(2)(axis)) ranging from 0.2 to 0.4 (highly disordered) in regions near the PBD46 binding site to 0.8--1.0 (highly ordered) in some helices. The side chain dynamics of the GDP and GMPPCP forms are similar, with methyl groups on the PBD46 binding surface experiencing significantly greater mobility (lower S(2)(axis)) than those not on the binding surface. Binding of PBD46 results in a significant increase in the disorder and a corresponding increase in entropy for the majority of methyl groups. Many of the methyl groups that experience an increase in mobility are found in residues that are not part of the PBD46 binding interface. This entropy gain represents a favorable contribution to the overall entropy of effector binding and partially offsets unfavorable entropy losses such as those that occur in the backbone.
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