No abstract

No abstract

No abstract

Activated receptor tyrosine kinases bind the Shc adaptor protein through its N-terminal phosphotyrosine-binding (PTB) and C-terminal Src homology 2 (SH2) domains. After binding, Shc is phosphorylated within the central collagen-homology (CH) linker regionon Tyr-317, a residue remote to both the PTB and SH2 domains. Shc phosphorylation plays a pivotal role in the initiation of mitogenic signaling through the Ras/Raf/ MEK/ERK pathway, but it is unclear if Tyr-317 phosphorylation affects Shc-receptor interactions through the PTB and SH2 domains. To investigate the structural impact of Shc phosphorylation, molecular dynamics simulations were carried out using special-purpose Molecular Dynamics Machine-Grape computers. After a 1-nanosecond equilibration, atomic motions in the structures of unphosphorylated Shc and Shc phosphorylated on Tyr-317 were calculated during a 2-nanosecond period. The results reveal larger phosphotyrosine-binding domain fluctuations and more structural flexibility of unphosphorylated Shc compared with phosphorylated Shc. Collective motions between the PTB-SH2, PTB-CH, and CH-SH2 domains were highly correlated only in unphosphorylated Shc. Dramatic changes in domain coupling and structural rigidity, induced by Tyr-317 phosphorylation, may alter Shc function, bringing about marked differences in the association of unphosphorylated and phosphorylated Shc with its numerous partners, including activated membrane receptors.The Src homology and collagen domain protein (Shc) 1 is an adaptor protein that plays a pivotal role in the signal transfer from receptor tyrosine kinases (RTK) to downstream signaling pathways regulating cell survival, proliferation, and differentiation (1). After ligand-induced autophosphorylation, RTKs bind and phosphorylate Shc on selected tyrosine residues. The Shc protein consists of an amino-terminal phosphotyrosinebinding (PTB) domain, a central collagen-homology (CH) region, which contains the major site of tyrosine phosphorylation at Tyr-317 and a carboxyl-terminal Src homology 2 (SH2) domain (2). Phosphorylation of Tyr-317 results in the formation of a docking site for other signaling proteins, e.g. growth factor receptor binding protein 2 (Grb2).Current paradigms for signal transduction emphasize that large adaptor proteins, such as Shc, are organized as "beads on a string." A single bead is a domain, ranging in size from 50 to 120 amino acids; they are responsible for particular interactions with other proteins or phospholipids (3). A signaling protein containing several domains can simultaneously associate with two or more binding partners. For example, an adaptor protein can bind to RTKs using an SH2 or PTB domain and associate with a signaling partner by means of another domain. It may appear at first that the modular structure underlying signaling interactions implies that the phosphorylation of residues located outside specialized binding domains should not affect the structural flexibility and intramolecular motions of these domains. Consequently, it should n...

Molecular dynamics simulation was performed on class A β-lactamase binding penicillin G (pen G). The structure of the acyl enzyme intermediate (AEI) was derived from the crystallographic data of the clavulanic acid bound enzyme. To execute the simulation precisely, the AEI was solvated by nearly 8000 water molecules and the no-cutoff (NCO) method was applied to the calculation of the Coulomb term. The Coulomb term calculation was accelerated with MDGRAPE-2 hardware. In the first step of this study, the relability of the NCO method was confirmed by comparing experimental and computational B-factors. We confirmed that the NCO method is much more reliable than the particle mesh Ewald and generalized Born methods. Hence the NCO method was applied for the simulation on AEI. The integrated simulation time was 1.2 ns. It was found from the simulation that Ser130, Asn132, Ser235, Gly237, and Arg244 cooperatively restricted the mobility of pen G moiety by making salt bridges among the side chains of these residues and the C3-carboxyl or C6-amide group of the substrate. The oxyanion hole composed of N atom in the main chain of Ser70 and Gly237 was properly reproduced under aqueous condition. The simulation also shows that it is impossible for Glu166 to act as a general base in the acylation of pen G because the average distance between Glu166 carboxyl oxygens and Ser70Oγ is too far for direct proton transfer (5.2 and 5.5 Å, respectively) and there is no water molecule between Glu166 carboxylate and Ser70Oγ. Molecular dynamics simulation on the substrate free enzyme (SFE) was also carried out and compared with AEI. While no drastic change due to the substrate binding was observed in both the secondary structure and the positions of catalytic residues of the enzyme, the mobility of the catalytic water molecule was strongly restricted by the presence of the substrate.