Efficiently driving protein-based fragment screening and lead discovery using two-dimensional NMR.

Fragment-based drug discovery (FBDD) and validation of small molecule binders using NMR spectroscopy is an established and widely used method in the early stages of drug discovery. Starting from a library of small compounds, ligand- or protein-observed NMR methods are employed to detect binders, typically weak, that become the starting points for structure-activity relationships (SAR) by NMR. Unlike the more frequently used ligand-observed 1D NMR techniques, protein-observed 2D 1H-15N or 1H-13C heteronuclear correlation (HSQC or HMQC) methods offer insights that include the mechanism of ligand engagement on the target and direct binding affinity measurements in addition to routine screening. We hereby present the development of a set of software tools within the MestReNova (Mnova) package for analyzing 2D NMR for FBDD and hit validation purposes. The package covers three main tasks: (1) unsupervised profiling of raw data to identify outlier data points to exclude in subsequent analyses; (2) batch processing of single-point spectra to identify and rank binders based on chemical shift perturbations or spectral peak intensity changes; and (3) batch processing of multiple titration series to derive binding affinities (KD) by tracing the changes in peak locations or measuring global spectral changes. Toward this end, we implemented and evaluated a set of algorithms for automated peak tracing, spectral binning, and variance analysis by PCA, and a new tool for spectral data intensity comparison using ECHOS. The accuracy and speed of the tools are demonstrated on 2D NMR binding data collected on ligands used in the development of potential inhibitors of the anti-apoptotic MCL-1 protein.

Interdomain dynamics explored by paramagnetic NMR.

An ensemble-based approach is presented to explore the conformational space sampled by a multidomain protein showing moderate interdomain dynamics in terms of translational and rotational motions. The strategy was applied on a complex of calmodulin (CaM) with the IQ-recognition motif from the voltage-gated calcium channel Ca(v)1.2 (IQ), which adopts three different interdomain orientations in the crystal. The N60D mutant of calmodulin was used to collect pseudocontact shifts and paramagnetically induced residual dipolar couplings for six different lanthanide ions. Then, starting from the crystal structure, pools of conformations were generated by free MD. We found the three crystal conformations in solution, but four additional MD-derived conformations had to be included into the ensemble to fulfill all the paramagnetic data and cross-validate optimally against unused paramagnetic data. Alternative approaches led to similar ensembles. Our "ensemble" approach is a simple and efficient tool to probe and describe the interdomain dynamics and represents a general method that can be used to provide a proper ensemble description of multidomain proteins.

The FKBP38 catalytic domain binds to Bcl-2 via a charge-sensitive loop.

FKBP38 is a regulator of the prosurvival protein Bcl-2, but in the absence of detailed structural insights, the molecular mechanism of the underlying interaction has remained unknown. Here, we report the contact regions between Bcl-2 and the catalytic domain of FKBP38 derived by heteronuclear NMR spectroscopy. The data reveal that a previously identified charge-sensitive loop near the putative active site of FKBP38 is mainly responsible for Bcl-2 binding. The corresponding binding epitope of Bcl-2 could be identified via a peptide library-based membrane assay. Site-directed mutagenesis of the key residues verified the contact sites of this electrostatic protein/protein interaction. The derived structure model of the complex between Bcl-2 and the FKBP38 catalytic domain features both electrostatic and hydrophobic intermolecular contacts and provides a rationale for the regulation of the FKBP38/Bcl-2 interaction by Ca(2+).

Cys-Ph-TAHA: a lanthanide binding tag for RDC and PCS enhanced protein NMR.

Here we present Cys-Ph-TAHA, a new nonadentate lanthanide tag for the paramagnetic labelling of proteins. The tag can be easily synthesized and is stereochemically homogenous over a wide range of temperatures, yielding NMR spectra with a single set of peaks. Bound to ubiquitin, it induced large residual dipolar couplings and pseudocontact shifts that could be measured easily and agreed very well with the protein structure. We show that Cys-Ph-TAHA can be used to label large proteins that are biochemically challenging such as the Lac repressor in a 90 kDa ternary complex with DNA and inducer.

A charge-sensitive loop in the FKBP38 catalytic domain modulates Bcl-2 binding.

The Bcl-2 inhibitor FKBP38 is regulated by the Ca(2+)-sensor calmodulin (CaM). Here we show a hitherto unknown low-affinity cation-binding site in the FKBP domain of FKBP38, which may afford an additional level of regulation based on electrostatic interactions. Fluorescence titration experiments indicate that in particular the physiologically relevant Ca(2+) ion binds to this site. NMR-based chemical shift perturbation data locate this cation-interaction site within the ß5-α1 loop (Leu90-Ile96) of the FKBP domain, which contains the acidic Asp92 and Asp94 side-chains. Binding constants were subsequently determined for K(+), Mg(2+), Ca(2+), and La(3+), indicating that the net charge and the radius of the ion influences the binding interaction. X-ray diffraction data furthermore show that the conformation of the ß5-α1 loop is influenced by the presence of a positively charged guanidinium group belonging to a neighboring FKBP38 molecule in the crystal lattice. The position of the cation-binding site has been further elucidated based on pseudocontact shift data obtained by NMR via titration with Tb(3+). Elimination of the Ca(2+)-binding capacity by substitution of the respective aspartate residues in a D92N/D94N double-substituted variant reduces the Bcl-2 affinity of the FKBP38(35-153)/CaM complex to the same degree as the presence of Ca(2+) in the wild-type protein. Hence, this charge-sensitive site in the FKBP domain participates in the regulation of FKBP38 function by enabling electrostatic interactions with ligand proteins and/or salt ions such as Ca(2+).

New structural aspects of FKBP38 activation.

The human FK506-binding protein 38 (FKBP38) regulates Bcl-2 in neuronal apoptosis. To control Bcl-2 activity, FKBP38 requires a prior interaction with the Ca(2+)-sensor calmodulin (CaM). The resulting FKBP38/CaM complex is unique within the FKBP family. Here, we present novel insights into the structural arrangement of this complex. Chemical shift perturbation analyses of the individual protein domains revealed two separate interaction sites between FKBP38 and CaM. On the one hand, residues Glu303, Tyr307 and Leu311, belonging to the predicted CaM-binding site at the C-terminal end of FKBP38, become embedded in the hydrophobic target protein-binding cleft of the C-terminal CaM lobe. On the other hand, in a second binding interaction, the N-terminal end of the catalytic FKBP38 domain shows surface contacts to the AB and CD loops of CaM as well as the adjacent helices. Furthermore, a Glu-rich region at the non-structured FKBP38 N-terminus features additional contacts to CaM helix A. In combination with previous results, we thus conclude that the FKBP38/CaM complex is constituted by (i) a Ca(2+)-dependent interaction of the CaM-binding motif at the C-terminal end of FKBP38 with the C-terminal CaM lobe and (ii) a Ca(2+)-independent interaction between the N-terminal CaM lobe and the N-terminal region of the catalytic FKBP38 domain.

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short-term synaptic plasticity.

Ca(2+) signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca(2+)-CaM binds a conserved region in the priming proteins Munc13-1 and ubMunc13-2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca(2+) signals. We solved the structure of Ca(2+)(4)-CaM in complex with the CaM-binding domain of Munc13-1, which features a novel 1-5-8-26 CaM-binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13-2 isoform. The N-module can be dissociated with EGTA to form the half-loaded Munc13/Ca(2+)(2)-CaM complex. The Ca(2+) regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca(2+)-CaM interactions, where the C-module provides a high-affinity interaction activated at nanomolar [Ca(2+)](i), whereas the N-module acts as a sensor at micromolar [Ca(2+)](i). This Ca(2+)/CaM-binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca(2+)-dependent modulation of short-term synaptic plasticity.

Variation in protein C(alpha)-related one-bond J couplings.

Four types of polypeptide (1)J(C alpha X) couplings are examined, involving the main-chain carbon C(alpha) and either of four possible substituents. A total 3105 values of (1)J(C alpha H alpha), (1)J(C alpha C beta), (1)J(C alpha C'), and (1)J(C alpha N') were collected from six proteins, averaging 143.4 +/- 3.3, 34.9 +/- 2.5, 52.6 +/- 0.9, and 10.7 +/- 1.2 Hz, respectively. Analysis of variances (ANOVA) reveals a variety of factors impacting on (1)J and ranks their relative statistical significance and importance to biomolecular NMR structure refinement. Accordingly, the spread in the (1)J values is attributed, in equal proportions, to amino-acid specific substituent patterns and to polypeptide-chain geometry, specifically torsions phi, psi, and chi(1) circumjacent to C(alpha). The (1)J coupling constants correlate with protein secondary structure. For alpha-helical phi, psi combinations, (1)J(C alpha H alpha) is elevated by more than one standard deviation (147.8 Hz), while both (1)J(C alpha N') and (1)J(C alpha C beta) fall short of their grand means (9.5 and 33.7 Hz). Rare positive phi torsion angles in proteins exhibit concomitant small (1)J(C alpha H alpha) and (1)J(C alpha N') (138.4 and 9.6 Hz) and large (1)J(C alpha C beta) (39.9 Hz) values. The (1)J(C alpha N') coupling varies monotonously over the phi torsion range typical of beta-sheet secondary structure and is largest (13.3 Hz) for phi around -160 degrees. All four coupling types depend on psi and thus help determine a torsion that is notoriously difficult to assess by traditional approaches using (3)J. Influences on (1)J stemming from protein secondary structure and other factors, such as amino-acid composition, are largely independent.

A novel calmodulin-Ca2+ target recognition activates the Bcl-2 regulator FKBP38.

The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.