Applications of NMR screening techniques to the pharmaceutical target Checkpoint kinase 1.

Ligand screening techniques based on NMR spectroscopy are not as sensitive as other commonly used methods like fluorescence, radiolabeling and surface plasmon resonance. However, using modern NMR instrumentation, they can achieve reliable screening under near physiological condition using as little as 4.6 nmol of receptor and 100 nmol of ligand. Additionally, these NMR methods can also provide valuable and specific information on the ligand under investigation such as the dissociation constant KD, the binding epitope and most importantly some structural information on the actual conformation in the bound state. In this manuscript, we describe the use of NMR based screening techniques ("Saturation Transfer Difference" (STD) and "Water Ligand Observed via Gradient SpectroscopY" (WaterLOGSY)) to detect small therapeutic molecules that interact with the DNA damage checkpoint enzyme Checkpoint kinase 1 (Chk1). After the identification of the most potent ligand, we used specific NMR experiments to perform the epitope mapping of this ligand ("Group epitope mapping-STD" (GEM-STD), "Difference of Inversion REcovery rate with and without Target IrradiatiON" (DIRECTION)) and to characterize its bound conformation ("Transferred-Nuclear Overhauser Effect SpectroscopY" (tr-NOESY), "Transferred-Rotating frame Overhauser Effect SpectroscopY" (tr-ROESY)). Finally, we used molecular docking procedures to position the ligand within the active site of Chk1. On the experimental level, a comparison between NMR studies performed in a 90%H2O/10%D2O buffer and a 100% D2O buffer is also presented and discussed.

Solution structure and backbone dynamics of the defunct domain of calcium vector protein.

CaVP (calcium vector protein) is a Ca(2+) sensor of the EF-hand protein family which is highly abundant in the muscle of Amphioxus. Its three-dimensional structure is not known, but according to the sequence analysis, the protein is composed of two domains, each containing a pair of EF-hand motifs. We determined recently the solution structure of the C-terminal domain (Trp81-Ser161) and characterized the large conformational and dynamic changes induced by Ca(2+) binding. In contrast, the N-terminal domain (Ala1-Asp86) has lost the capacity to bind the metal ion due to critical mutations and insertions in the two calcium loops. In this paper, we report the solution structure of the N-terminal domain and its backbone dynamics based on NMR spectroscopy, nuclear relaxation, and molecular modeling. The well-resolved three-dimensional structure is typical of a pair of EF-hand motifs, joined together by a short antiparallel beta-sheet. The tertiary arrangement of the two EF-hands results in a closed-type conformation, with near-antiparallel alpha-helices, similar to other EF-hand pairs in the absence of calcium ions. To characterize the internal dynamics of the protein, we measured the (15)N nuclear relaxation rates and the heteronuclear NOE effect in (15)N-labeled N-CaVP at a magnetic field of 11.74 T and 298 K. The domain is mainly monomeric in solution and undergoes an isotropic Brownian rotational diffusion with a correlation time of 7.1 ns, in good agreement with the fluorescence anisotropy decay measurements. Data analysis using a model-free procedure showed that the amide backbone groups in the alpha-helices and beta-strands undergo highly restricted movements on a picosecond to nanosecond time scale. The amide groups in Ca(2+) binding loops and in the linker fragment also display rapid fluctuations with slightly increased amplitudes.

Backbone dynamics of the regulatory domain of calcium vector protein, studied by (15)N relaxation at four fields, reveals unique mobility characteristics of the intermotif linker.

UNLABELLED: CaVP is a calcium-binding protein from amphioxus. It has a modular composition with two domains, but only the two EF-hand motifs localized in the C-terminal domain are functional. We recently determined the solution structure of this regulatory half (C-CaVP) in the Ca(2+)-saturated form and characterized the stepwise ion binding. This paper reports the (15)N nuclear relaxation rates of the Ca(2+)-saturated C-CaVP, measured at four different NMR fields (9.39, 11.74, 14.1, and 18.7 T), which were used to map the spectral density function for the majority of the amide H(N)-N vectors. Fitting the spectral density values at eight frequencies by a model-free approach, we obtained the microdynamic parameters characterizing the global and internal movements of the polypeptide backbone. The two EF-hand motifs, including the ion binding loops, behave like compact structural units with restricted mobility as reflected in the quite uniform order parameter and short internal correlation time (< 20 nsec). Comparative analysis of the two Ca(2+) binding sites shows that site III, having a larger affinity for the metal ion, is generally more rigid, and the amide vector in the second residue of each loop is significantly less restricted. The linker fragment is animated simultaneously by a larger amplitude fast motion and a slow conformational exchange on a microsecond to millisecond time scale. The backbone dynamics of C-CaVP characterized here is discussed in relation with other well-characterized Ca(2+)-binding proteins. SUPPLEMENTAL MATERIAL: See www.proteinscience.org

Sequential calcium binding to the regulatory domain of calcium vector protein reveals functional asymmetry and a novel mode of structural rearrangement.

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.