High-affinity tamoxifen analogues retain extensive positional disorder when bound to calmodulin

Using a combination of NMR and fluorescence measurements, we have investigated the structure and dynamics of the complexes formed between calcium-loaded calmodulin (CaM) and the potent breast cancer inhibitor idoxifene, a derivative of tamoxifen. High-affinity binding (Kd∼300 nM) saturates with a 2:1 idoxifene:CaM complex. The complex is an ensemble where each idoxifene molecule is predominantly in the vicinity of one of the two hydrophobic patches of CaM but, in contrast with the lower-affinity antagonists TFP, J-8, and W-7, does not substantially occupy the hydrophobic pocket. At least four idoxifene orientations per domain of CaM are necessary to satisfy the intermolecular nuclear Overhauser effect (NOE) restraints, and this requires that the idoxifene molecules switch rapidly between positions. The CaM molecule is predominantly in the form where the N and C-terminal domains are in close proximity, allowing for the idoxifene molecules to contact both domains simultaneously. Hence, the 2:1 idoxifene:CaM complex illustrates how high-affinity binding occurs without the loss of extensive positional dynamics

Why the energy landscape of barnase is hierarchical

We have used NMR and computational methods to characterize the dynamics of the ribonuclease barnase over a wide range of timescales in free and inhibitor-bound states. Using temperature- and denaturant-dependent measurements of chemical shift, we show that barnase undergoes frequent and highly populated hinge bending. Using relaxation dispersion, we characterize a slower and less populated motion with a rate of 750 ± 200 s-¹, involving residues around the lip of the active site, which occurs in both free and bound states and therefore suggests conformational selection. Normal mode calculations characterize correlated hinge bending motions on a very rapid timescale. These three measurements are combined with previous measurements and molecular dynamics calculations on barnase to characterize its dynamic landscape on timescales from picoseconds to milliseconds and length scales from 0.1 to 2.5 nm. We show that barnase has two different large-scale fluctuations: one on a timescale of 10-⁹-10-⁶ s that has no free energy barrier and is a hinge bending that is determined by the architecture of the protein; and one on a timescale of milliseconds (i.e., 750 s-¹) that has a significant free energy barrier and starts from a partially hinge-bent conformation. These two motions can be described as hierarchical, in that the more highly populated faster motion provides a platform for the slower (less probable) motion. The implications are discussed. The use of temperature and denaturant is suggested as a simple and general way to characterize motions on the intermediate ns-μs timescale

Structural effects of the highly protective V127 polymorphism on human prion protein

Prion diseases, a group of incurable, lethal neurodegenerative disorders of mammals including humans, are caused by prions, assemblies of misfolded host prion protein (PrP). A single point mutation (G127V) in human PrP prevents prion disease, however the structural basis for its protective effect remains unknown. Here we show that the mutation alters and constrains the PrP backbone conformation preceding the PrP β-sheet, stabilising PrP dimer interactions by increasing intermolecular hydrogen bonding. It also markedly changes the solution dynamics of the β2-α2 loop, a region of PrP structure implicated in prion transmission and cross-species susceptibility. Both of these structural changes may affect access to protein conformers susceptible to prion formation and explain its profound effect on prion disease