Cyclin-dependent kinase 5 (Cdk5) is a proline-directed Ser/ Thr kinase that plays important roles in various neuronal activities, including neuronal migration, synaptic activity, and neuronal cell death. Cdk5 is activated by association with a neuron-specific activator, p35 or its isoform p39, but little is known about the kinase activity of Cdk5-p39. In fact, kinaseactive Cdk5-p39 was not prepared from rat brain extracts nor from HEK293 cells expressing Cdk5 and p39 by immunoprecipitation in the presence of non-ionic detergent, under conditions with which active Cdk5-p35 could be isolated. p39 dissociated from Cdk5 in the presence of detergent, indicating that p39 has a lower binding affinity for Cdk5 than p35. We developed a method for purifying kinase-active Cdk5-p39 from Sf9 cells infected with baculovirus encoding Cdk5 and p39. The purified Cdk5-p39 complex showed similar substrate specificity to that of Cdk5-p35, but with opposite sensitivity to detergent. Cdk5-p39 was inactivated by Triton X-100, whereas Cdk5-p35 was activated. The N-terminal deletion from p35 and p39, the amino acid sequences of which are different, did not change the stability or substrate specificity of either Cdk5 complex. The different stability between Cdk5-p35 and Cdk5-p39 suggests their distinct roles under different regulation mechanisms in neurons.
The free-energy landscape of interaction between a medium-sized peptide, endothelin 1 (ET1), and its receptor, human endothelin type B receptor (hETB), was computed using multidimensional virtual-system coupled molecular dynamics, which controls the system’s motions by introducing multiple reaction coordinates. The hETB embedded in lipid bilayer was immersed in explicit solvent. All molecules were expressed as all-atom models. The resultant free-energy landscape had five ranges with decreasing ET1–hETB distance: completely dissociative, outside-gate, gate, binding pocket, and genuine-bound ranges. In the completely dissociative range, no ET1–hETB interaction appeared. In the outside-gate range, an ET1–hETB attractive interaction was the fly-casting mechanism. In the gate range, the ET1 orientational variety decreased rapidly. In the binding pocket range, ET1 was in a narrow pathway with a steep free-energy slope. In the genuine-bound range, ET1 was in a stable free-energy basin. A G-protein-coupled receptor (GPCR) might capture its ligand from a distant place.
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